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  • Sustainable microbial cell nanofactory for zinc oxide nanoparticles production by zinc-tolerant probiotic Lactobacillus plantarum strain TA4
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-15
    Hidayat Mohd Yusof; Rosfarizan Mohamad; Uswatun Hasanah Zaidan; Nor’Aini Abdul Rahman

    The use of microorganisms in the biosynthesis of zinc oxide nanoparticles (ZnO NPs) has recently emerged as an alternative to chemical and physical methods due to its low-cost and eco-friendly method. Several lactic acid bacteria (LAB) have developed mechanisms in tolerating Zn2+ through prevention against their toxicity and the production of ZnO NPs. The LAB’s main resistance mechanism to Zn2+ is highly depended on the microorganisms’ ability to interact with Zn2+ either through biosorption or bioaccumulation processes. Besides the inadequate studies conducted on biosynthesis with the use of zinc-tolerant probiotics, the understanding regarding the mechanism involved in this process is not clear. Therefore, this study determines the features of probiotic LAB strain TA4 related to its resistance to Zn2+. It also attempts to illustrate its potential in creating a sustainable microbial cell nanofactory of ZnO NPs. A zinc-tolerant probiotic strain TA4, which was isolated from local fermented food, was selected based on the principal component analysis (PCA) with the highest score of probiotic attributes. Based on the 16S rRNA gene analysis, this strain was identified as Lactobacillus plantarum strain TA4, indicating its high resistance to Zn2+ at a maximum tolerable concentration (MTC) value of 500 mM and its capability of producing ZnO NPs. The UV–visible spectroscopy analysis proved the formations of ZnO NPs through the notable absorption peak at 380 nm. It was also found from the dynamic light scattering (DLS) analysis that the Z-average particle size amounted to 124.2 nm with monodisperse ZnO NPs. Studies on scanning electron microscope (SEM), energy-dispersive X-ray (EDX) spectroscopy, and Fourier-transform infrared spectroscopy (FT-IR) revealed that the main mechanisms in ZnO NPs biosynthesis were facilitated by the Zn2+ biosorption ability through the functional groups present on the cell surface of strain TA4. The strong ability of zinc-tolerant probiotic of L. plantarum strain TA4 to tolerate high Zn2+ concentration and to produce ZnO NPs highlights the unique properties of these bacteria as a natural microbial cell nanofactory for a more sustainable and eco-friendly practice of ZnO NPs biosynthesis.

    更新日期:2020-01-15
  • Enhancement of 1,3-propanediol production from industrial by-product by Lactobacillus reuteri CH53
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-13
    Jung-Hyun Ju; Dexin Wang; Sun-Yeon Heo; Min-Soo Kim; Jeong-Woo Seo; Young-Min Kim; Dae-Hyuk Kim; Soon-Ah Kang; Chul-Ho Kim; Baek-Rock Oh

    1,3-propanediol (1,3-PDO) is the most widely studied value-added product that can be produced by feeding glycerol to bacteria, including Lactobacillus sp. However, previous research reported that L. reuteri only produced small amounts and had low productivity of 1,3-PDO. It is urgent to develop procedures that improve the production and productivity of 1,3-PDO. We identified a novel L. reuteri CH53 isolate that efficiently converted glycerol into 1,3-PDO, and performed batch co-fermentation with glycerol and glucose to evaluate its production of 1,3-PDO and other products. We optimized the fermentation conditions and nitrogen sources to increase the productivity. Fed-batch fermentation using corn steep liquor (CSL) as a replacement for beef extract led to 1,3-PDO production (68.32 ± 0.84 g/L) and productivity (1.27 ± 0.02 g/L/h) at optimized conditions (unaerated and 100 rpm). When CSL was used as an alternative nitrogen source, the activity of the vitamin B12-dependent glycerol dehydratase (dhaB) and 1,3-propanediol oxidoreductase (dhaT) increased. Also, the productivity and yield of 1,3-PDO increased as well. These results showed the highest productivity in Lactobacillus species. In addition, hurdle to 1,3-PDO production in this strain were identified via analysis of the half-maximal inhibitory concentration for growth (IC50) of numerous substrates and metabolites. We used CSL as a low-cost nitrogen source to replace beef extract for 1,3-PDO production in L. reuteri CH53. These cells efficiently utilized crude glycerol and CSL to produce 1,3-PDO. This strain has great promise for the production of 1,3-PDO because it is generally recognized as safe (GRAS) and non-pathogenic. Also, this strain has high productivity and high conversion yield.

    更新日期:2020-01-13
  • Homogeneous production and characterization of recombinant N-GlcNAc-protein in Pichia pastoris
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-13
    Shengjun Wang; Yongheng Rong; Yaoguang Wang; Decai Kong; Peng George Wang; Min Chen; Yun Kong

    Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N-Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N-glycoproteins with defined N-glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N-glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N-glycan structures and the N-glycosylation microheterogeneities between batches. In this study, we constructed a Pichia pastoris (Komagataella phaffii) expression system producing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N-glycans and the N-GlcNAc modification on the glycosite Asn297 was confirmed via Mass Spectrometry. This strategy develops a simple glycoengineered yeast expression system to produce N-GlcNAc modified proteins, which could be further extended to different N-glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.

    更新日期:2020-01-13
  • Correction to: Enhancement of the catalytic activity of Isopentenyl diphosphate isomerase (IDI) from Saccharomyces cerevisiae through random and site-directed mutagenesis
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-13
    Hailin Chen; Meijie Li; Changqing Liu; Haibo Zhang; Mo Xian; Huizhou Liu

    The authors of this article [1] wish to draw the readers’ attention to their closely related paper, published in RSC Advances [2] which should have been cited in this article. The authors regret that there is unattributed overlap in text describing the construction of the plasmid coding for the biosynthetic pathway because of the commonly used research strategies between this article [1] and similar work presented in RSC Advances, although this does not affect the main scientific conclusion in this study.

    更新日期:2020-01-13
  • Effects of ccpA gene deficiency in Lactobacillus delbrueckii subsp. bulgaricus under aerobic conditions as assessed by proteomic analysis
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-13
    Guofang Zhang; Libo Liu; Chun Li

    Aerobic growth provides benefits in biomass yield and stress tolerance of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Catabolite control protein A (CcpA) is a master regulator involved in the aerobic and anaerobic growth, metabolic production and stress response in L. bulgaricus, but its potential molecular mechanisms remains unclear. The aim of this study is to elucidate the role of CcpA in L. bulgaricus in aerobic growth at the proteomic perspective. The differential proteomic analysis was performed on the L. bulgaricus ATCC11842 and its ccpA inactivated mutant strain using iTRAQ technology. A total of 132 differentially expressed proteins were obtained, among which 58 were up-regulated and 74 were down-regulated. These proteins were mainly involved in the cellular stress response, carbohydrate and energy metabolism, amino acid transport and protein synthesis, genetic information processing. Moreover, inactivation of ccpA negatively affected the expression of key enzymes involved in glycolysis pathway, while it enhanced the expression of proteins related to the pyruvate pathway, supporting the conclusion that CcpA mediated the shift from homolactic fermentation to mixed acid fermentation in L. bulgaricus. Overall, these results showed that the role of CcpA in L. bulgaricus as a pleiotropic regulator in aerobic metabolism and stress response. This proteomic analysis also provide new insights into the CcpA-mediated regulatory network of L. bulgaricus and potential strategies to improve the production of starter and probiotic cultures based on the metabolic engineering of global regulators.

    更新日期:2020-01-13
  • Engineering of Streptomyces lividans for heterologous expression of secondary metabolite gene clusters
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-09
    Yousra Ahmed; Yuriy Rebets; Marta Rodríguez Estévez; Josef Zapp; Maksym Myronovskyi; Andriy Luzhetskyy

    Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

    更新日期:2020-01-09
  • Mineralization of the herbicide swep by a two-strain consortium and characterization of a new amidase for hydrolyzing swep
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-07
    Long Zhang; Ping Hang; Xiyi Zhou; Chen Dai; Ziyi He; Jiandong Jiang

    Swep is an excellent carbamate herbicide that kills weeds by interfering with metabolic processes and inhibiting cell division at the growth point. Due to the large amount of use, swep residues in soil and water not only cause environmental pollution but also accumulate through the food chain, ultimately pose a threat to human health. This herbicide is degraded in soil mainly by microbial activity, but no studies on the biotransformation of swep have been reported. In this study, a consortium consisting of two bacterial strains, Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34, was enriched from a contaminated soil sample and shown to be capable of mineralizing swep. Swep was first transformed by Comamonas sp. SWP-3 to the intermediate 3,4-dichloroaniline (3,4-DCA), after which 3,4-DCA was mineralized by Alicycliphilus sp. PH-34. An amidase gene, designated as ppa, responsible for the transformation of swep into 3,4-DCA was cloned from strain SWP-3. The expressed Ppa protein efficiently hydrolyzed swep and a number of other structural analogues, such as propanil, chlorpropham and propham. Ppa shared less than 50% identity with previously reported arylamidases and displayed maximal activity at 30 °C and pH 8.6. Gly449 and Val266 were confirmed by sequential error prone PCR to be the key catalytic sites for Ppa in the conversion of swep. These results provide additional microbial resources for the potential remediation of swep-contaminated sites and add new insights into the catalytic mechanism of amidase in the hydrolysis of swep.

    更新日期:2020-01-07
  • Proteome analysis guided genetic engineering of Corynebacterium glutamicum S9114 for tween 40-triggered improvement in l-ornithine production
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-06
    Yan Jiang; Ming-Zhu Huang; Xue-Lan Chen; Bin Zhang

    l-ornithine is a valuable amino acid with a wide range of applications in the pharmaceutical and food industries. However, the production of l-ornithine by fermentation cannot compete with other methods, because of the low titers produced with this technique. Development of fermentation techniques that result in a high yield of l-ornithine and efficient strategies for improving l-ornithine production are essential. This study demonstrates that tween 40, a surfactant promoter of the production of glutamate and arginine, improves l-ornithine production titers in engineered C. glutamicum S9114. The intracellular metabolism under tween 40 triggered fermentation conditions was explored using a quantitative proteomic approach, identifying 48 up-regulated and 132 down-regulated proteins when compared with the control. Numerous proteins were identified as membrane proteins or functional proteins involved in the biosynthesis of the cell wall. Modulation of those genes revealed that the overexpression of CgS9114_09558 and the deletion of CgS9114_13845, CgS9114_02593, and CgS9114_02058 improved the production of l-ornithine in the engineered strain of C. glutamicum Orn8. The final strain with all the exploratory metabolic engineering manipulations produced 25.46 g/L of l-ornithine, and a yield of 0.303 g l-ornithine per g glucose, which was 30.6% higher than that produced by the original strain (19.5 g/L). These results clearly demonstrate the positive effect of tween 40 addition on l-ornithine accumulation. Proteome analysis was performed to examine the impact of tween 40 addition on the physiological changes in C. glutamicum Orn8 and the results showed several promising modulation targets for developing l-ornithine-producing strains.

    更新日期:2020-01-06
  • Biosynthetic engineering of the antifungal, anti-MRSA auroramycin
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-06
    Wan Lin Yeo; Elena Heng; Lee Ling Tan; Yi Wee Lim; Kuan Chieh Ching; De-Juin Tsai; Yi Wun Jhang; Tsai-Ling Lauderdale; Kak-Shan Shia; Huimin Zhao; Ee Lui Ang; Mingzi M. Zhang; Yee Hwee Lim; Fong T. Wong

    Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.

    更新日期:2020-01-06
  • Correction to: Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation
    Microb. Cell Fact. (IF 4.402) Pub Date : 2020-01-02
    Daniel Moog; Johanna Schmitt; Jana Senger; Jan Zarzycki; Karl-Heinz Rexer; Uwe Linne; Tobias J. Erb; Uwe G. Maier

    The author’s middle name is missed out in the original publication of the article [1]. The correct coauthor’s name is Tobias J. Erb.

    更新日期:2020-01-02
  • Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-29
    Robin Dorau; Lin Chen; Jianming Liu; Peter Ruhdal Jensen; Christian Solem

    Diacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, however, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Due to its bacteriostatic properties, it is difficult to achieve high levels of diacetyl by fermentation. Here we present a novel strategy for producing diacetyl based on whole-cell catalysis, which bypasses the toxic effects of diacetyl. By expressing a robust α-acetolactate synthase (ALS) in a metabolically optimized Lactococcus lactis strain we obtained a whole-cell biocatalyst that efficiently converted pyruvate into α-acetolactate. After process optimization, we achieved a titer for α-acetolactate of 172 ± 2 mM. Subsequently we used a two-stage production setup, where pyruvate was produced by an engineered L. lactis strain and subsequently used as the substrate for the biocatalyst. Using this approach, 122 ± 5 mM and 113 ± 3 mM α-acetolactate could be made from glucose or lactose in dairy waste, respectively. The whole-cell biocatalyst was robust and fully active in crude fermentation broth containing pyruvate. An efficient approach for converting sugar into α-acetolactate, via pyruvate, was developed and tested successfully. Due to the anaerobic conditions used for the biotransformation, little diacetyl was generated, and this allowed for efficient biotransformation of pyruvate into α-acetolactate, with the highest titers reported to date. The use of a two-step procedure for producing α-acetolactate, where non-toxic pyruvate first is formed, and subsequently converted into α-acetolactate, also simplified the process optimization. We conclude that whole cell catalysis is suitable for converting lactose in dairy waste into α-acetolactate, which favors resource utilization.

    更新日期:2019-12-30
  • Correction to: Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-29
    Maren Wehrs; Jan-Philip Prahl; Jadie Moon; Yuchen Li; Deepti Tanjore; Jay D. Keasling; Todd Pray; Aindrila Mukhopadhyay

    Following publication of the original article [1], the authors have noted that the standard curve in Additional file 1: Figure S7 is incorrect.

    更新日期:2019-12-30
  • Soluble versions of outer membrane cytochromes function as exporters for heterologously produced cargo proteins
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-23
    Helge M. Dietrich; Miriam Edel; Thea Bursac; Manfred Meier; Katrin Sturm-Richter; Johannes Gescher

    This study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway.

    更新日期:2019-12-25
  • Enhanced production of recombinant serratiopeptidase in Escherichia coli and its characterization as a potential biosimilar to native biotherapeutic counterpart
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-17
    Vishal Srivastava; Shivam Mishra; Tapan K. Chaudhuri

    Serratia marcescens, a Gram-negative nosocomial pathogen secretes a 50 kDa multi-domain zinc metalloprotease called serratiopeptidase. Broad substrate specificity of serratiopeptidase makes it suitable for detergent and food processing industries The protein shows potent anti-inflammatory, anti-edemic, analgesic, antibiofilm activity and sold as an individual or fixed-dose enteric-coated tablets combined with other drugs. Although controversial, serratiopeptidase as drug is used in the treatment of chronic sinusitis, carpal tunnel syndrome, sprains, torn ligaments, and postoperative inflammation. Since the native producer of serratiopeptidase is a pathogenic microorganism, the current production methods need to be replaced by alternative approaches. Heterologous expression of serratiopeptidase in E. coli was tried before but not found suitable due to the limited yield, and other expression related issues due to its inherent proteolytic activity such as cytotoxicity, cell death, no expression, minimal expression, or inactive protein accumulation. Recombinant expression of mature form serratiopeptidase in E. coli seems toxic and resulted in the failure of transformation and other expression related issues. Although E. coli C43(DE3) cells, express protein correctly, the yield was compromised severely. Optimization of protein expression process parameters such as nutrient composition, induction point, inducer concentration, post-induction duration, etc., caused significant enhancement in serratiopeptidase production (57.9 ± 0.73% of total cellular protein). Expressed protein formed insoluble, enzymatically inactive inclusion bodies, and gave 40–45 mg/l homogenous (> 98% purity) biologically active and conformationally similar serratiopeptidase to the commercial counterpart upon refolding and purification. Expression of mature serratiopeptidase in E. coli C43(DE3) cells eliminated the protein expression associated with toxicity issues. Further optimization of process parameters significantly enhanced the overexpression of protein resulting in the higher yield of pure and functionally active recombinant serratiopeptidase. The biological activity and conformational features of recombinant serratiopeptidase were very similar to the commercially available counterpart suggesting it-a potential biosimilar of therapeutic and industrial relevance.

    更新日期:2019-12-18
  • Improved utilization of soybean meal through fermentation with commensal Shewanella sp. MR-7 in turbot (Scophthalmus maximus L.)
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-16
    Chaoqun Li; Beili Zhang; Xin Wang; Xionge Pi; Xuan Wang; Huihui Zhou; Kangsen Mai; Gen He

    Increased inclusion of plant proteins in aquafeeds has become a common practice due to the high cost and limited supply of fish meal but generally leads to inferior growth performance and health problems of fish. Effective method is needed to improve the plant proteins utilization and eliminate their negative effects on fish. This study took a unique approach to improve the utilization of soybean meal (SBM) by fish through autochthonous plant-degrading microbe isolation and subsequent fermentation. A strain of Shewanella sp. MR-7 was isolated and identified as the leading microbe that could utilize SBM in the intestine of turbot. It was further optimized for SBM fermentation and able to improve the protein availability and degrade multiple anti-nutritional factors of SBM. The fishmeal was able to be replaced up to 45% by Shewanella sp. MR-7 fermented SBM compared to only up to 30% by SBM in experimental diets without adverse effects on growth and feed utilization of turbot after feeding trials. Further analyses showed that Shewanella sp. MR-7 fermentation significantly counteracted the SBM-induced adverse effects by increasing digestive enzymes activities, suppressing inflammatory responses, and alleviating microbiota dysbiosis in the intestine of turbot. This study demonstrated that plant protein utilization by fish could be significantly improved through pre-digestion with isolated plant-degrading host microbes. Further exploitation of autochthonous bacterial activities should be valuable for better performances of plant-based diets in aquaculture.

    更新日期:2019-12-17
  • ClC transporter activity modulates histidine catabolism in Lactobacillus reuteri by altering intracellular pH and membrane potential
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-12
    Anne E. Hall; Melinda A. Engevik; Numan Oezguen; Anthony Haag; James Versalovic

    Histamine is a key mediator of the anti-inflammatory activity conferred by the probiotic organism Lactobacillus reuteri ATCC PTA 6475 in animal models of colitis and colorectal cancer. In L. reuteri, histamine synthesis and secretion requires l-histidine decarboxylase and a l-histidine/histamine exchanger. Chloride channel (ClC)-family proton/chloride antiporters have been proposed to act as electrochemical shunts in conjunction with amino acid decarboxylase systems, correcting ion imbalances generated by decarboxylation through fixed ratio exchange of two chloride ions for one proton. This family is unique among transporters by facilitating ion flux in either direction. Here we examine the histidine decarboxylase system in relation to ClC antiporters in the probiotic organism Lactobacillus reuteri. In silico analyses reveal that L. reuteri possesses two ClC transporters, EriC and EriC2, as well as a complete histidine decarboxylase gene cluster (HDC) for the synthesis and export of histamine. When the transport activity of either proton/chloride antiporter is disrupted by genetic manipulation, bacterial histamine output is reduced. Using fluorescent reporter assays, we further show that ClC transporters affect histamine output by altering intracellular pH and membrane potential. ClC transport also alters the expression and activity of two key HDC genes: the histidine decarboxylase (hdcA) and the histidine/histamine exchanger (hdcP). Histamine production is a potentially beneficial feature for intestinal microbes by promoting long-term colonization and suppression of inflammation and host immune responses. ClC transporters may serve as tunable modulators for histamine production by L. reuteri and other gut microbes.

    更新日期:2019-12-13
  • Process engineering of pH tolerant Ustilago cynodontis for efficient itaconic acid production
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-12
    Hamed Hosseinpour Tehrani; Katharina Saur; Apilaasha Tharmasothirajan; Lars M. Blank; Nick Wierckx

    Ustilago cynodontis ranks among the relatively unknown itaconate production organisms. In comparison to the well-known and established organisms like Aspergillus terreus and Ustilago maydis, genetic engineering and first optimizations for itaconate production were only recently developed for U. cynodontis, enabling metabolic and morphological engineering of this acid-tolerant organism for efficient itaconate production. These engineered strains were so far mostly characterized in small scale shaken cultures. In pH-controlled fed-batch experiments an optimum pH of 3.6 could be determined for itaconate production in the morphology-engineered U. cynodontis Δfuz7. With U. cynodontis ∆fuz7r ∆cyp3r PetefmttA Pria1ria1, optimized for itaconate production through the deletion of an itaconate oxidase and overexpression of rate-limiting production steps, titers up to 82.9 ± 0.8 g L−1 were reached in a high-density pulsed fed-batch fermentation at this pH. The use of a constant glucose feed controlled by in-line glucose analysis increased the yield in the production phase to 0.61 gITA gGLC−1, which is 84% of the maximum theoretical pathway yield. Productivity could be improved to a maximum of 1.44 g L−1 h−1 and cell recycling was achieved by repeated-batch application. Here, we characterize engineered U. cynodontis strains in controlled bioreactors and optimize the fermentation process for itaconate production. The results obtained are discussed in a biotechnological context and show the great potential of U. cynodontis as an itaconate producing host.

    更新日期:2019-12-13
  • Genomic diversity and meiotic recombination among isolates of the biotech yeast Komagataella phaffii (Pichia pastoris)
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-04
    Stephanie Braun-Galleani; Julie A. Dias; Aisling Y. Coughlan; Adam P. Ryan; Kevin P. Byrne; Kenneth H. Wolfe

    Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were previously called Pichia pastoris. However, almost all laboratory work on K. phaffii has utilized strains derived from a single natural isolate, CBS7435. There is little information about the sequence diversity of K. phaffii or the genetic properties of this species. We sequenced the genomes of all the known isolates of K. phaffii. We made a genetic cross between derivatives of two isolates that differ at 44,000 single nucleotide polymorphism sites, and used this cross to analyze the rate and landscape of meiotic recombination. We conducted tetrad analysis by making use of the property that K. phaffii haploids do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetra-type ascus germinates. We found that only four distinct natural isolates of K. phaffii exist in public yeast culture collections. The meiotic recombination rate in K. phaffii is approximately 3.5 times lower than in Saccharomyces cerevisiae, with an average of 25 crossovers per meiosis. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our work lays a foundation for future quantitative trait locus analysis in K. phaffii.

    更新日期:2019-12-05
  • Green pyomelanin-mediated synthesis of gold nanoparticles: modelling and design, physico-chemical and biological characteristics
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-03
    Imen Ben Tahar; Patrick Fickers; Andrzej Dziedzic; Dariusz Płoch; Bartosz Skóra; Małgorzata Kus-Liśkiewicz

    Synthesis of nanoparticles (NPs) and their incorporation in materials are amongst the most studied topics in chemistry, physics and material science. Gold NPs have applications in medicine due to their antibacterial and anticancer activities, in biomedical imaging and diagnostic test. Despite chemical synthesis of NPs are well characterized and controlled, they rely on the utilization of harsh chemical conditions and organic solvent and generate toxic residues. Therefore, greener and more sustainable alternative methods for NPs synthesis have been developed recently. These methods use microorganisms, mainly yeast or yeast cell extract. NPs synthesis with culture supernatants are most of the time the preferred method since it facilitates the purification scheme for the recovery of the NPs. Extraction of NPs, formed within the cells or cell-wall, is laborious, time-consuming and are not cost effective. The bioactivities of NPs, namely antimicrobial and anticancer, are known to be related to NPs shape, size and size distribution. Herein, we reported on the green synthesis of gold nanoparticles (AuNPs) mediated by pyomelanin purified from the yeast Yarrowia lipolytica. A three levels four factorial Box–Behnken Design (BBD) was used to evaluate the influence of temperature, pH, gold salt and pyomelanin concentration on the nanoparticle size distribution. Based on the BBD, a quadratic model was established and was applied to predict the experimental parameters that yield to AuNPs with specific size. The synthesized nanoparticles with median size value of 104 nm were of nanocrystalline structure, mostly polygonal or spherical. They exhibited a high colloidal stability with zeta potential of − 28.96 mV and a moderate polydispersity index of 0.267. The absence of cytotoxicity of the AuNPs was investigated on two mammalian cell lines, namely mouse fibroblasts (NIH3T3) and human osteosarcoma cells (U2OS). Cell viability was only reduced at AuNPs concentration higher than 160 µg/mL. Moreover, they did not affect on the cell morphology. Our results indicate that different process parameters affect significantly nanoparticles size however with the mathematical model it is possible to define the size of AuNPs. Moreover, this melanin-based gold nanoparticles showed neither cytotoxicity effect nor altered cell morphology.

    更新日期:2019-12-04
  • Combined artificial high-silicate medium and LED illumination promote carotenoid accumulation in the marine diatom Phaeodactylum tricornutum
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-12-02
    Zhiqian Yi; Yixi Su; Paulina Cherek; David R. Nelson; Jianping Lin; Ottar Rolfsson; Hua Wu; Kourosh Salehi-Ashtiani; Sigurdur Brynjolfsson; Weiqi Fu

    Diatoms, which can accumulate large amounts of carotenoids, are a major group of microalgae and the dominant primary producer in marine environments. Phaeodactylum tricornutum, a model diatom species, acquires little silicon for its growth although silicon is known to contribute to gene regulation and play an important role in diatom intracellular metabolism. In this study, we explored the effects of artificial high-silicate medium (i.e. 3.0 mM sodium metasilicate) and LED illumination conditions on the growth rate and pigment accumulation in P. tricornutum, which is the only known species so far that can grow without silicate. It’s well known that light-emitting diodes (LEDs) as novel illuminants are emerging to be superior monochromatic light sources for algal cultivation with defined and efficient red and blue lights. Firstly, we cultivated P. tricornutum in a synthetic medium supplemented with either 0.3 mM or 3.0 mM silicate. The morphology and size of diatom cells were examined: the proportion of the oval and triradiate cells decreased while the fusiform cells increased with more silicate addition in high-silicate medium; the average length of fusiform cells also slightly changed from 14.33 µm in 0.3 mM silicate medium to 12.20 µm in 3.0 mM silicate medium. Then we cultivated P. tricornutum under various intensities of red light in combination with the two different levels of silicate in the medium. Higher biomass productivity also achieved in 3.0 mM silicate medium than in 0.3 mM silicate medium under red LED light irradiation at 128 μmol/m2/s or higher light intensity. Increasing silicate reversed the down-regulation of fucoxanthin and chlorophyll a under high red-light illumination (i.e. 255 μmol/m2/s). When doubling the light intensity, fucoxanthin content decreased under red light but increased under combined red and blue (50:50) lights while chlorophyll a content reduced under both conditions. Fucoxanthin accumulation and biomass productivity increased with enhanced red and blue (50:50) lights. High-silicate medium and blue light increased biomass and fucoxanthin production in P. tricornutum under high light conditions and this strategy may be beneficial for large-scale production of fucoxanthin in diatoms.

    更新日期:2019-12-03
  • Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-29
    Xueyun Zheng; Yimin Zhang; Xinying Zhang; Cheng Li; Xiaoxiao Liu; Ying Lin; Shuli Liang

    The methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins. The function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities. This study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

    更新日期:2019-11-30
  • Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-29
    Vijaydev Ganesan; Michael Spagnuolo; Ayushi Agrawal; Spencer Smith; Difeng Gao; Mark Blenner

    Yarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR–Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

    更新日期:2019-11-30
  • Phosphate regulator PhoP directly and indirectly controls transcription of the erythromycin biosynthesis genes in Saccharopolyspora erythraea
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-27
    Ya Xu; Di You; Li-li Yao; Xiaohe Chu; Bang-Ce Ye

    The choice of phosphate/nitrogen source and their concentrations have been shown to have great influences on antibiotic production. However, the underlying mechanisms responsible for this remain poorly understood. We show that nutrient-sensing regulator PhoP (phosphate regulator) binds to and upregulates most of genes (ery cluster genes) involved in erythromycin biosynthesis in Saccharopolyspora erythraea, resulting in increase of erythromycin yield. Furthermore, it was found that PhoP also directly interacted with the promoter region of bldD gene encoding an activator of erythromycin biosynthesis, and induced its transcription. Phosphate limitation and overexpression of phoP increased the transcript levels of ery genes to enhance the erythromycin production. The results are further supported by observation that an over-producing strain of S. erythraea expressed more PhoP than a wild-type strain. On the other hand, nitrogen signal exerts the regulatory effect on the erythromycin biosynthesis through GlnR negatively regulating the transcription of phoP gene. These findings provide evidence that PhoP mediates the interplay between phosphate/nitrogen metabolism and secondary metabolism by integrating phosphate/nitrogen signals to modulate the erythromycin biosynthesis. Our study reveals a molecular mechanism underlying antibiotic production, and suggests new possibilities for designing metabolic engineering and fermentation optimization strategies for increasing antibiotics yield.

    更新日期:2019-11-28
  • Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-25
    Ioannis Mougiakos; Enrico Orsi; Mohammad Rifqi Ghiffary; Wilbert Post; Alberto de Maria; Belén Adiego-Perez; Servé W. M. Kengen; Ruud A. Weusthuis; John van der Oost

    Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected. In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

    更新日期:2019-11-26
  • Integration of a multi-step heterologous pathway in Saccharomyces cerevisiae for the production of abscisic acid
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-25
    Maximilian Otto; Paulo Gonçalves Teixeira; Maria Isabel Vizcaino; Florian David; Verena Siewers

    The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. Recent studies show that ABA also exhibits a variety of pharmacological activities. Affordable and sustainable production will be required to utilize the compound in agriculture and as a potential pharmaceutical. Saccharomyces cerevisiae is an established workhorse for the biotechnological production of chemicals. In this study, we constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. Expression of the B. cinerea genes bcaba1, bcaba2, bcaba3 and bcaba4 was sufficient to establish ABA production in the heterologous host. We characterised the ABA-producing strain further by monitoring ABA production over time and, since the pathway contains two cytochrome P450 enzymes, by investigating the effects of overexpressing the native S. cerevisiae or the B. cinerea cytochrome P450 reductase. Both, overexpression of the native or heterologous cytochrome P450 reductase, led to increased ABA titres. We were able to show that ABA production was not affected by precursor or NADPH supply, which suggested that the heterologous enzymes were limiting the flux towards the product. The B. cinerea cytochrome P450 monooxygenases BcABA1 and BcABA2 were identified as pathway bottlenecks and balancing the expression levels of the pathway enzymes resulted in 4.1-fold increased ABA titres while reducing by-product formation. This work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid.

    更新日期:2019-11-26
  • Growth of the facultative chemolithoautotroph Ralstonia eutropha on organic waste materials: growth characteristics, redox regulation and hydrogenase activity
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-18
    Anna Poladyan; Syuzanna Blbulyan; Mayramik Sahakyan; Oliver Lenz; Armen Trchounian

    The chemolithoautotrophic β-proteobacterium Ralstonia eutropha H16 (Cupriavidus necator) is one of the most studied model organisms for growth on H2 and CO2. R. eutropha H16 is also a biologically significant bacterium capable of synthesizing O2-tolerant [NiFe]-hydrogenases (Hyds), which can be used as anode biocatalysts in enzyme fuel cells. For heterotrophic growth of R. eutropha, various sources of organic carbon and energy can be used. Growth, bioenergetic properties, and oxidation–reduction potential (ORP) kinetics were investigated during cultivation of R. eutropha H16 on fructose and glycerol or lignocellulose-containing brewery spent grain hydrolysate (BSGH). BSGH was used as carbon and energy source by R. eutropha H16, and the activities of the membrane-bound hydrogenase (MBH) and cytoplasmic, soluble hydrogenase (SH) were measured in different growth phases. Growth of R. eutropha H16 on optimized BSGH medium yielded ~ 0.7 g cell dry weight L−1 with 3.50 ± 0.02 (SH) and 2.3 ± 0.03 (MBH) U (mg protein)−1 activities. Upon growth on fructose and glycerol, a pH drop from 7.0 to 6.7 and a concomitant decrease of ORP was observed. During growth on BSGH, in contrast, the pH and ORP stayed constant. The growth rate was slightly stimulated through addition of 1 mM K3[Fe(CN)6], whereas temporarily reduced growth was observed upon addition of 3 mM dithiothreitol. The overall and N,N′-dicyclohexylcarbodiimide-sensitive ATPase activities of membrane vesicles were ~ 4- and ~ 2.5-fold lower, respectively, upon growth on fructose and glycerol (FGN) compared with only fructose utilization (FN). Compared to FN, ORP was lower upon bacterial growth on FGN, GFN, and BSGH. Our results suggest that reductive conditions and low ATPase activity might be signals for energy depletion, which, in turn, leads to increased hydrogenase biosynthesis to overcome this unfavorable situation. Addition of fructose or microelements have no, or a negative, influence on hydrogenase activity. Organic wastes (glycerol, BSGH) are promising carbon and energy sources for the formation of biomass harboring significant amounts of the biotechnologically relevant hydrogenases MBH and SH. The results are valuable for using microbial cells as producers of hydrogenase enzymes as catalysts in enzymatic fuel cells.

    更新日期:2019-11-18
  • Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-18
    Xun-Cheng Zong; Chuang Li; Yao-Hui Xu; Die Hu; Bo-Chun Hu; Jia Zang; Min-Chen Wu

    Epoxide hydrolase can regioselectively catalyze the oxirane ring-opening hydrolysis of rac-epoxides producing the corresponding chiral diols. In our laboratory, a gene named pveh1 encoding an EH from Phaseolus vulgaris was cloned. Although the directed modification of PvEH1 was carried out, the mutant PvEH1Y3 showed a limited degree of enantioconvergence towards racemic (rac-) m-chlorostyrene oxide (mCSO). PvEH1 and PvEH1Y3 were combinatively subjected to laboratory evolution to further enhance the enantioconvergence of PvEH1Y3 towards rac-mCSO. Firstly, the substrate-binding pocket of PvEH1 was identified using a CAVER 3.0 software, and divided into three zones. After all residues in zones 1 and 3 were subjected to leucine scanning, two E. coli transformants, E. coli/pveh1Y149L and /pveh1P184L, were selected, by which rac-mCSO was transformed into (R)-m-chlorophenyl-1,2-ethanediol (mCPED) having 55.1% and 27.2% eep. Secondly, two saturation mutagenesis libraries, E. coli/pveh1Y149X and /pveh1P184X (X: any one of 20 residues) were created at sites Y149 and P184 of PvEH1. Among all transformants, both E. coli/pveh1Y149L (65.8% αS and 55.1% eep) and /pveh1P184W (66.6% αS and 59.8% eep) possessed the highest enantioconvergences. Finally, the combinatorial mutagenesis was conducted by replacements of both Y149L and P184W in PvEH1Y3, constructing E. coli/pveh1Y3Z2, whose αS reached 97.5%, higher than that (75.3%) of E. coli/pveh1Y3. In addition, the enantioconvergent hydrolysis of 20 mM rac-mCSO was performed by E. coli/pveh1Y3Z2, giving (R)-mCPED with 95.2% eep and 97.2% yield. In summary, the enantioconvergence of PvEH1Y3Z2 was successfully improved by laboratory evolution, which was based on the study of substrate-binding pocket by leucine scanning. Our present work introduced an effective strategy for the directed modification of enantioconvergence of PvEH1.

    更新日期:2019-11-18
  • Synthetic control devices for gene regulation in Penicillium chrysogenum
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-18
    László Mózsik; Zsófia Büttel; Roel A. L. Bovenberg; Arnold J. M. Driessen; Yvonne Nygård

    Orthogonal, synthetic control devices were developed for Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster of Neurospora crassa is fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid upstream activating sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system. The control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, upstream activating sequences, and the affinity of the DNA binding domain of the transcription factor to the upstream activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin. The characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.

    更新日期:2019-11-18
  • Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-14
    Fulu Liu; Yating Zhang; Wanjin Qiao; Duolong Zhu; Haijin Xu; Per Erik Joakim Saris; Mingqiang Qiao

    After 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000. Plasmid pNZ5417 containing a visually selectable marker PnisZ-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis. Thus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.

    更新日期:2019-11-14
  • Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-14
    Silvia Donzella; Daniela Cucchetti; Claudia Capusoni; Aurora Rizzi; Silvia Galafassi; Gambaro Chiara; Concetta Compagno

    Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.

    更新日期:2019-11-14
  • Laccases: structure, function, and potential application in water bioremediation
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-14
    Leticia Arregui; Marcela Ayala; Ximena Gómez-Gil; Guadalupe Gutiérrez-Soto; Carlos Eduardo Hernández-Luna; Mayra Herrera de los Santos; Laura Levin; Arturo Rojo-Domínguez; Daniel Romero-Martínez; Mario C. N. Saparrat; Mauricio A. Trujillo-Roldán; Norma A. Valdez-Cruz

    The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

    更新日期:2019-11-14
  • Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-11
    Jenny Johansson Söderberg; Miriam Grgic; Erik Hjerde; Peik Haugen

    Heterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identification of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efficient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes. We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNA-sequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5′-fusion to stimulate protein production and solubility. Finally we tested production of a set of “difficult-to-produce” enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5′-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of cold-active enzymes when used as a 5′-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion. Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries.

    更新日期:2019-11-11
  • Redesigning the Aspergillus nidulans xylanase regulatory pathway to enhance cellulase production with xylose as the carbon and inducer source
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-07
    Patrick Ballmann; Jorge Lightfoot; Michael Müller; Stephan Dröge; Rolf Prade

    Biomass contains cellulose (C6-sugars), hemicellulose (C5-sugars) and lignin. Biomass ranks amongst the most abundant hydrocarbon resources on earth. However, biomass is recalcitrant to enzymatic digestion by cellulases. Physicochemical pretreatment methods make cellulose accessible but partially destroy hemicellulose, producing a C5-sugar-rich liquor. Typically, digestion of pretreated LCB is performed with commercial cellulase preparations, but C5-sugars could in principle be used for “on site” production of cellulases by genetically engineered microorganism, thereby reducing costs. Here we report a succession of genetic interventions in Aspergillus nidulans that redesign the natural regulatory circuitry of cellulase genes in such a way that recombinant strains use C5-sugar liquors (xylose) to grow a vegetative tissue and simultaneously accumulate large amounts of cellulases. Overexpression of XlnR showed that under xylose-induction conditions only xylanase C was produced. XlnR overexpression strains were constructed that use the xynCp promoter to drive the production of cellobiohydrolases, endoglucanases and β-glucosidase. All five cellulases accumulated at high levels when grown on xylose. Production of cellulases in the presence of pretreated-biomass C5-sugar liquors was investigated, and cellulases accumulated to much higher enzyme titers than those obtained for traditional fungal cell factories with cellulase-inducing substrates. By replacing expensive substrates with a cheap by-product carbon source, the use of C5-sugar liquors directly derived from LCB pretreatment processes not only reduces enzyme production costs, but also lowers operational costs by eliminating the need for off-site enzyme production, purification, concentration, transport and dilution.

    更新日期:2019-11-11
  • 5-Aminolevulinic acid fermentation using engineered Saccharomyces cerevisiae
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-07
    Kiyotaka Y. Hara; Masaru Saito; Hiroko Kato; Kana Morikawa; Hiroshi Kikukawa; Hironari Nomura; Takanori Fujimoto; Yoko Hirono-Hara; Shigeyuki Watanabe; Kengo Kanamaru; Akihiko Kondo

    5′-Aminolevulinic acid (ALA) is widely used in the pharmaceutical industry, healthcare, and food production, and is a substrate for the biosynthesis of heme, which is required for respiration and photosynthesis. Enhancement of ALA biosynthesis has never been developed in Saccharomyces cerevisiae, which is a well-known model microorganism used for bioproduction of many value-added compounds. We demonstrated that metabolic engineering significantly improved ALA production in S. cerevisiae. First, we found that overexpression of HEM1, which encodes ALA synthetase, increased ALA production. Furthermore, addition of an optimal amount of glycine, a substrate for ALA biosynthesis, or levulinic acid, an inhibitor of ALA dehydrogenase, effectively increased ALA production. Next, we developed an assay for multiple metabolites including ALA and found that aconitase, encoded by ACO1 and ACO2, is the rate-limiting enzyme of ALA biosynthesis when sufficient glycine is supplied. Overexpression of ACO2 further enhanced ALA production in S. cerevisiae overexpressing HEM1. In this study, ALA production in S. cerevisiae was enhanced by metabolic engineering. This study also shows a strategy to identify the rate-limiting step of a target synthetic pathway by assay for multiple metabolites alongside the target product. This strategy can be applied to improve production of other valuable products in the well-studied and well-industrialized microorganism S. cerevisiae.

    更新日期:2019-11-11
  • High production of valencene in Saccharomyces cerevisiae through metabolic engineering
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-07
    Hefeng Chen; Chaoyi Zhu; Muzi Zhu; Jinghui Xiong; Hao Ma; Min Zhuo; Shuang Li

    The biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive. Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield. This study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.

    更新日期:2019-11-11
  • Extracellular production of the engineered thermostable protease pernisine from Aeropyrum pernix K1 in Streptomyces rimosus
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-07
    Marko Šnajder; Andrés Felipe Carrillo Rincón; Vasilka Magdevska; Miha Bahun; Luka Kranjc; Maja Paš; Polona Juntes; Hrvoje Petković; Nataša Poklar Ulrih

    The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation. We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease. Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.

    更新日期:2019-11-11
  • Isolation, identification, and potential probiotic characterization of isolated lactic acid bacteria and in vitro investigation of the cytotoxicity, antioxidant, and antidiabetic activities in fermented sausage
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-05
    Nadia S. AlKalbani; Mark S. Turner; Mutamed M. Ayyash

    Probiotic bacteria can provide health benefits when delivered in functional foods. This study involved isolation of lactic acid bacteria (LAB) from traditionally dried and salted anchovy fish and characterization of their survival in simulated gastrointestinal digestion. Promising strains were used to prepare fermented fish sausages which were then evaluated for cytotoxicity activity against two cancer cell-lines, antidiabetic activity as determined by α-amylase and α-glucosidase inhibition, and antioxidant and proteolytic activities in vitro, as compared to non-fermented control sausages. Out of 85 LAB obtained, 13 isolates with high tolerance to simulated gastrointestinal digestion were obtained, which were identified as Enterococcus spp. Four E. faecium strains, one E. faecalis, and one E. durans were used separately to make fermented fish sausages. The α-amylase and α-glucosidase inhibition from fish sausages fermented by Enterococcus spp. ranged from 29.2 to 68.7% and 23.9 to 41.4%, respectively, during 21 days of storage. The cytotoxicity activities against Caco2 and MCF-7 cells of fish sausages fermented with Enterococcus spp. ranged from 18.0 to 24% and 13.9 to 27.9%, respectively. Cytotoxicity activities correlated positively with proteolysis and antioxidant activities, α-amylase and α-glucosidase inhibition activities, but negatively with the pH in fermented fish sausages. Strains also exhibited antimicrobial activity against foodborne pathogens and presented no significant concerns with regards to antibiotic resistance or virulence gene content. Fish sausages fermented by potential probiotic isolates of Enterococcus spp. from dried fish had valuable health-promoting benefits compared with non-fermented control sausages.

    更新日期:2019-11-06
  • Redirecting photosynthetic electron flux in the cyanobacterium Synechocystis sp. PCC 6803 by the deletion of flavodiiron protein Flv3
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-05
    Kati Thiel; Pekka Patrikainen; Csaba Nagy; Duncan Fitzpatrick; Nicolas Pope; Eva-Mari Aro; Pauli Kallio

    Oxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron transfer routes may compete with the solar-driven production of CO2-derived target chemicals in biotechnological systems under development. This work focused on engineered cyanobacterial Synechocystis sp. PCC 6803 strains, to explore possibilities to rescue excited electrons that would normally be lost to molecular oxygen by an alternative acceptor flavodiiron protein Flv1/3—an enzyme that is natively associated with transfer of electrons from PSI to O2, as part of an acclimation strategy towards varying environmental conditions. The effects of Flv1/3 inactivation by flv3 deletion were studied in respect to three alternative end-products, sucrose, polyhydroxybutyrate and glycogen, while the photosynthetic gas fluxes were monitored by Membrane Inlet Mass Spectrometry (MIMS) to acquire information on cellular carbon uptake, and the production and consumption of O2. The results demonstrated that a significant proportion of the excited electrons derived from photosynthetic water cleavage was lost to molecular oxygen via Flv1/3 in cells grown under high CO2, especially under high light intensities. In flv3 deletion strains these electrons could be re-routed to increase the relative metabolic flux towards the monitored target products, but the carbon distribution and the overall efficiency were determined by the light conditions and the genetic composition of the respective pathways. At the same time, the total photosynthetic capacity of the Δflv3 strains was systematically reduced, and accompanied by upregulation of oxidative glycolytic metabolism in respect to controls with the native Flv1/3 background. The observed metabolic changes and respective production profiles were proposedly linked with the lack of Flv1/3-mediated electron transfer, and the associated decrease in the intracellular ATP/NADPH ratio, which is bound to affect the metabolic carbon partitioning in the flv3-deficient cells. While the deletion of flv3 could offer a strategy for enhancing the photosynthetic production of desired chemicals in cyanobacteria under specified conditions, the engineered target pathways have to be carefully selected to align with the intracellular redox balance of the cells.

    更新日期:2019-11-06
  • Generic estimator of biomass concentration for Escherichia coli and Saccharomyces cerevisiae fed-batch cultures based on cumulative oxygen consumption rate
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-05
    Renaldas Urniezius; Arnas Survyla; Dziugas Paulauskas; Vladas Algirdas Bumelis; Vytautas Galvanauskas

    The focus of this study is online estimation of biomass concentration in fed-batch cultures. It describes a bioengineering software solution, which is explored for Escherichia coli and Saccharomyces cerevisiae fed-batch cultures. The experimental investigation of both cultures presents experimental validation results since the start of the bioprocess, i.e. since the injection of inoculant solution into bioreactor. In total, four strains were analyzed, and 21 experiments were performed under varying bioprocess conditions, out of which 7 experiments were carried out with dosed substrate feeding. Development of the microorganisms’ culture invariant generic estimator of biomass concentration was the main goal of this research. The results show that stoichiometric parameters provide acceptable knowledge on the state of biomass concentrations during the whole cultivation process, including the exponential growth phase of both E. coli and S. cerevisiae cultures. The cell culture stoichiometric parameters are estimated by a procedure based on the Luedeking/Piret-model and maximization of entropy. The main input signal of the approach is cumulative oxygen uptake rate at fed-batch cultivation processes. The developed noninvasive biomass estimation procedure was intentionally made to not depend on the selection of corresponding bioprocess/bioreactor parameters. The precision errors, since the bioprocess start, when inoculant was injected to a bioreactor, confirmed that the approach is relevant for online biomass state estimation. This included the lag and exponential growth phases for both E. coli and S. cerevisiae. The suggested estimation procedure is identical for both cultures. This approach improves the precision achieved by other authors without compromising the simplicity of the implementation. Moreover, the suggested approach is a candidate method to be the microorganisms’ culture invariant approach. It does not depend on any numeric initial optimization conditions, it does not require any of bioreactor parameters. No numeric stability issues of convergence occurred during multiple performance tests. All this makes this approach a potential candidate for industrial tasks with adaptive feeding control or automatic inoculations when substrate feeding profile and bioreactor parameters are not provided.

    更新日期:2019-11-06
  • Metabolic engineering and transcriptomic analysis of Saccharomyces cerevisiae producing p-coumaric acid from xylose
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-05
    Gheorghe M. Borja; Angelica Rodriguez; Kate Campbell; Irina Borodina; Yun Chen; Jens Nielsen

    Aromatic amino acids and their derivatives are valuable chemicals and are precursors for different industrially compounds. p-Coumaric acid is the main building block for complex secondary metabolites in commercial demand, such as flavonoids and polyphenols. Industrial scale production of this compound from yeast however remains challenging. Using metabolic engineering and a systems biology approach, we developed a Saccharomyces cerevisiae platform strain able to produce 242 mg/L of p-coumaric acid from xylose. The same strain produced only 5.35 mg/L when cultivated with glucose as carbon source. To characterise this platform strain further, transcriptomic analysis was performed, comparing this strain’s growth on xylose and glucose, revealing a strong up-regulation of the glyoxylate pathway alongside increased cell wall biosynthesis and unexpectedly a decrease in aromatic amino acid gene expression when xylose was used as carbon source. The resulting S. cerevisiae strain represents a promising platform host for future production of p-coumaric using xylose as a carbon source.

    更新日期:2019-11-06
  • Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-05
    Daniel Christoph Volke; Johann Rohwer; Rainer Fischer; Stefan Jennewein

    Terpenoids are of high interest as chemical building blocks and pharmaceuticals. In microbes, terpenoids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. Although the MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood. We applied metabolic control analysis to the MEP pathway in Escherichia coli expressing a heterologous isoprene synthase gene (ispS). The expression of ispS led to the accumulation of isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) and severely impaired bacterial growth, but the coexpression of ispS and isopentenyl diphosphate isomerase (idi) restored normal growth and wild-type IPP/DMAPP levels. Targeted proteomics and metabolomics analysis provided a quantitative description of the pathway, which was perturbed by randomizing the ribosome binding site in the gene encoding 1-deoxyxylulose 5-phosphate synthase (Dxs). Dxs has a flux control coefficient of 0.35 (i.e., a 1% increase in Dxs activity resulted in a 0.35% increase in pathway flux) in the isoprene-producing strain and therefore exerted significant control over the flux though the MEP pathway. At higher dxs expression levels, the intracellular concentration of 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP) increased substantially in contrast to the other MEP pathway intermediates, which were linearly dependent on the abundance of Dxs. This indicates that 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), which consumes MEcPP, became saturated and therefore limited the flux towards isoprene. The higher intracellular concentrations of MEcPP led to the efflux of this intermediate into the growth medium. These findings show the importance of Dxs, Idi and IspG and metabolite export for metabolic engineering of the MEP pathway and will facilitate further approaches for the microbial production of valuable isoprenoids.

    更新日期:2019-11-06
  • Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage
    Microb. Cell Fact. (IF 4.402) Pub Date : 2019-11-01
    Javier Garrigós-Martínez; Miguel Angel Nieto-Taype; Arnau Gasset-Franch; José Luis Montesinos-Seguí; Xavier Garcia-Ortega; Francisco Valero

    The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression—heterologous genes included—, thus directly influencing the production kinetics of recombinant protein.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2003-06-05
    Jari Rautio,Kim Bundvig Barken,Juhani Lahdenperä,Antje Breitenstein,Søren Molin,Peter Neubauer

    BACKGROUND: A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules. RESULTS: The sensitivity of the assay was approximately 1.2 x 109 (2 fmol) molecules of target RNA. The developed method was feasible with crude cell lysates of S. cerevisiae carlsbergensis and was evaluated by measuring the levels of 18S rRNA during cell growth and SUC2 mRNA under repressive and inductive conditions. The 18S rRNA expression level followed the changes in the specific growth rate. SUC2 mRNA levels were in good correlation with the measured invertase enzyme activities. CONCLUSIONS: The here presented sandwich hybridisation method was succefully applied for monitoring the amounts of ribosomal RNA and mRNA with high expression level in shake flask cultivation conditions. Sandwich hybridisation method offers a fast and convenient tool for following single key RNA species of interest in the production conditions.

    更新日期:2019-11-01
  • Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2003-05-13
    Mikko I Viitanen,Antti Vasala,Peter Neubauer,Tapani Alatossava

    BACKGROUND: Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. RESULTS: Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-beta-D-thiogalactopyranoside) or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction. CONCLUSION: Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.

    更新日期:2019-11-01
  • Metabolic networks of microbial systems.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2003-05-13
    Sumana Bhattacharya,Subhra Chakrabarti,Amiya Nayak,Sanjoy K Bhattacharya

    In contrast to bioreactors the metabolites within the microbial cells are converted in an impure atmosphere, yet the productivity seems to be well regulated and not affected by changes in operation variables. These features are attributed to integral metabolic network within the microorganism. With the advent of neo-integrative proteomic approaches the understanding of integration of metabolic and protein-protein interaction networks have began. In this article we review the methods employed to determine the protein-protein interaction and their integration to define metabolite networks. We further present a review of current understanding of network properties, and benefit of studying the networks. The predictions using network structure, for example, in silico experiments help illustrate the importance of studying the network properties. The cells are regarded as complex system but their elements unlike complex systems interact selectively and nonlinearly to produce coherent rather than complex behaviors.

    更新日期:2019-11-01
  • The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2003-02-28
    Vivi Joosten,Christien Lokman,Cees AMJJ Van Den Hondel,Peter J Punt

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted.

    更新日期:2019-11-01
  • Bacterial autoinduction: looking outside the cell for new metabolic engineering targets.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2003-01-23
    Matthew P DeLisa,William E Bentley

    Recent evidence has demonstrated that cell-to-cell signaling is a fundamental activity carried out by numerous microorganisms. A number of specialized processes are reported to be regulated by density-dependent signaling molecules including antibiotic production, bioluminescence, biofilm formation, genetic competence, sporulation, swarming motility and virulence. However, a more centralized role for quorum sensing is emerging where quorum signaling pathways overlap with stress and starvation circuits to regulate cellular adaptation to changing environmental conditions. The interplay of these phenomena is especially critical in the expression of recombinant proteins where elicitation of stress responses can dramatically impact cellular productivity.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2002-10-24
    Adriana M Rosso,Susana A Ferrarotti,Norberto Krymkiewicz,B Clara Nudel

    BACKGROUND: The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related alpha-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans. RESULTS: CGTase activity produced from Bacillus circulans DF 9R was optimised in shake flasks using a combination of conventional sequential techniques and statistical experimental design. Effects of nutrients, including several carbon, nitrogen and mineral sources, were assayed. The selected minimal medium consisted of 1.5 % cassava starch, 0.4 % ammonium sulphate, 0.1 M phosphate buffer, 0.002 % MgSO4 and 0.002 % FeSO4. The optimal concentrations of carbon and nitrogen sources were determined using a central composite design. Maximum CGTase activity obtained in supernatants was 5.8 U/mL in 48 h of incubation. Optimal conditions for enzyme production also included an initial pH of 8.3 and 37 degrees C as the incubation temperature.Cell growth and CGTase production profile were not linked to each other, suggesting that enzyme production/secretion is not growth-associated but mainly a late-log phase event. CONCLUSION: We have screened conditions for optimal CGTase production. The selected minimal medium contained starch, ammonium, Mg2+ and Fe2+ as essential nutrients. As an additional advantage, this medium does not require complex nitrogen sources with varying and unknown composition.

    更新日期:2019-11-01
  • Purification and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2002-06-22
    Luca Martirani,Mario Varcamonti,Gino Naclerio,Maurilio De Felice

    BACKGROUND: Applications of bacteriocins as food preservatives have been so far limited, principally because of their low antimicrobial activity in foods. Nisin is the only bacteriocin of significant use, but applications are restricted principally because of its very low activity at neutral or alkaline pH. Thus the isolation of new bacteriocins active in foods is desirable. RESULTS: We isolated a Bacillus licheniformis thermophilic strain producing a bacteriocin with some novel features, named here bacillocin 490. This bacteriocin was inactivated by pronase E and proteinase K and was active against closely related Bacillus spp. both in aerobic and in anaerobic conditions. Bactericidal activity was kept during storage at 4 degrees C and was remarkably stable in a wide pH range. The bacteriocin was partially purified by elution after adhesion to cells of the food-isolated strain Bacillus smithii and had a rather low mass (2 KDa). Antimicrobial activity against B. smithii was observed also when this organism was grown in water buffalo milk. CONCLUSIONS: Bacillocin 490 is a novel candidate as a food anti-microbial agent since it displays its activity in milk, is stable to heat treatment and during storage, is active in a wide pH range and has bactericidal activity also at high temperature. These features may allow the use of bacillocin 490 during processes performed at high temperature and as a complementary antimicrobial agent of nisin against some Bacillus spp. in non-acidic foods. The small size suggests its use on solid foods.

    更新日期:2019-11-01
  • Stress responses and replication of plasmids in bacterial cells.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2002-06-22
    Grzegorz Wegrzyn,Alicja Wegrzyn

    Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage lambda that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

    更新日期:2019-11-01
  • Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2018-01-14
    Scott Bottoms,Quinn Dickinson,Mick McGee,Li Hinchman,Alan Higbee,Alex Hebert,Jose Serate,Dan Xie,Yaoping Zhang,Joshua J Coon,Chad L Myers,Robert Landick,Jeff S Piotrowski

    BACKGROUND Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. RESULTS Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. CONCLUSIONS We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.

    更新日期:2019-11-01
  • Metabolomics investigation of recombinant mTNFα production in Streptomyces lividans.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2015-10-10
    Howbeer Muhamadali,Yun Xu,David I Ellis,Drupad K Trivedi,Nicholas J W Rattray,Kristel Bernaerts,Royston Goodacre

    BACKGROUND Whilst undergoing differentiation, Streptomyces produce a large quantity of hydrolytic enzymes and secondary metabolites, and it is this very ability that has focussed increasing interest on the use of these bacteria as hosts for the production of various heterologous proteins. However, within this genus, the exploration and understanding of the metabolic burden associated with such bio-products has only just begun. In this study our overall aim was to apply metabolomics approaches as tools to get a glimpse of the metabolic alterations within S. lividans TK24 when this industrially relevant microbe is producing recombinant murine tumour necrosis factor alpha (mTNFα), in comparison to wild type and empty (non-recombinant protein containing) plasmid-carrying strains as controls. RESULTS Whilst growth profiles of all strains demonstrated comparable trends, principal component-discriminant function analysis of Fourier transform infrared (FT-IR) spectral data, showed clear separation of wild type from empty plasmid and mTNFα-producing strains, throughout the time course of incubation. Analysis of intra- and extra-cellular metabolic profiles using gas chromatography-mass spectrometry (GC-MS) displayed similar trends to the FT-IR data. Although the strain carrying the empty plasmid demonstrated metabolic changes due to the maintenance of the plasmid, the metabolic behaviour of the recombinant mTNFα-producing strain appeared to be the most significantly affected. GC-MS results also demonstrated a significant overflow of several organic acids (pyruvate, 2-ketoglutarate and propanoate) and sugars (xylitol, mannose and fructose) in the mTNFα-producing strain. CONCLUSION The results obtained in this study have clearly demonstrated the metabolic impacts of producing mTNFα in S. lividans TK24, while displaying profound metabolic effects of harbouring the empty PIJ486 plasmid. In addition, the level of mTNFα produced in this study, further highlights the key role of media composition towards the efficiency of a bioprocess and metabolic behaviour of the host cells, which directly influences the yield of the recombinant product.

    更新日期:2019-11-01
  • Expression of RCK2 MAPKAP (MAPK-activated protein kinase) rescues yeast cells sensitivity to osmotic stress.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2015-06-13
    V Kumar,A J Hart,T T Wimalasena,G A Tucker,D Greetham

    BACKGROUND Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars during beverage or bioethanol fermentations. These fermentations are characterised by high osmotic stress on a yeast cell, with selected brewing fermentations beginning at 20-25% fermentable sugars and bioethanol fermentations at 13% fermentable sugars. RESULTS RCK2 encodes for a MAPKAP (MAPK-activated protein kinase) enzyme and was identified on a locus by QTL analysis in yeast cells under osmotic stress, RCK2 expression was placed under a tetracycline regulatable vector and rescued glucose, sorbitol or glycerol induced osmotic stress in an rck2 null strain. A strain overexpressing RCK2 had significantly faster fermentation rates when compared with the empty vector control strain. CONCLUSIONS Presence of RCK2 increased rates of glucose utilisation (~40 g glucose in first 8 h) during a 15% glucose fermentation and concurrent production of ethanol when compared with empty vector controls. Tolerance to osmotic stress using the tetracycline regulatable vectors could be turned off with the addition of tetracycline returning a rck2 null strain back to osmotic sensitivity.

    更新日期:2019-11-01
  • Production of recombinant protein by a novel oxygen-induced system in Escherichia coli.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2014-04-09
    Antonino Baez,Nadim Majdalani,Joseph Shiloach

    BACKGROUND The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes. RESULTS The soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence. By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW). CONCLUSIONS Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery.

    更新日期:2019-11-01
  • A generalised module for the selective extracellular accumulation of recombinant proteins.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2012-05-30
    Yanina R Sevastsyanovich,Denisse L Leyton,Timothy J Wells,Catherine A Wardius,Karina Tveen-Jensen,Faye C Morris,Timothy J Knowles,Adam F Cunningham,Jeffrey A Cole,Ian R Henderson

    BACKGROUND It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. RESULTS Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. CONCLUSIONS The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.

    更新日期:2019-11-01
  • Triauxic growth of an oleaginous red yeast Rhodosporidium toruloides on waste 'extract' for enhanced and concomitant lipid and β-carotene production.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2018-11-21
    Gunjan Singh,Sweta Sinha,K K Bandyopadhyay,Mark Lawrence,Ram Prasad,Debarati Paul

    BACKGROUND Vegetable 'mandi' (road-side vegetable market) waste was converted to a suitable fermentation medium for cultivation of oleaginous yeast Rhodosporidium toruloides by steaming under pressure. This cultivation medium derived from waste was found to be a comparatively better source of nutrients than standard culture media because it provided more than one type of usable carbon source(s) to yeast. RESULTS HPLC results showed that the extract contained glucose, xylose and glycerol along with other carbon sources, allowing triauxic growth pattern with preferably usage of glucose, xylose and glycerol resulting in enhanced growth, lipid and carotenoid production. Presence of saturated and unsaturated fatty acid methyl esters (FAMEs) (C14-20) in the lipid profile showed that the lipid may be transesterified for biodiesel production. CONCLUSION Upscaling these experiments to fermenter scale for the production of lipids and biodiesel and other industrially useful products would lead to waste management along with the production of value added commodities. The technique is thus environment friendly and gives good return upon investment.

    更新日期:2019-11-01
  • Correction to: Indirect and direct routes to C-glycosylated flavones in Saccharomyces cerevisiae.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2018-07-30
    Katherina Garcia Vanegas,Arésu Bondrup Larsen,Michael Eichenberger,David Fischer,Uffe Hasbro Mortensen,Michael Naesby

    Upon publication of this article [1], it was brought to our attention that revised Fig. 1 supplied by the author during proof correction was unfortunately not presented in the original version of the article. The revised Fig. 1 is given in this erratum.

    更新日期:2019-11-01
  • Correction to: Bioprospection of actinobacteria derived from freshwater sediments for their potential to produce antimicrobial compounds.
    Microb. Cell Fact. (IF 4.402) Pub Date : 2018-06-07
    Zothanpuia,Ajit Kumar Passari,Vincent Vineeth Leo,Preeti Chandra,Brijesh Kumar,Chandra Nayak,Abeer Hashem,Elsayed Fathi Abd Allah,Abdulaziz A Alqarawi,Bhim Pratap Singh

    Upon publication of this article [1], it was brought to our attention that Figs. 3, 4 and 5 are incorrectly presented in the original version of the article. The figures were inadvertently swapped in the original submission and published. Figure 3 should be treated as Fig. 5; Fig. 4 should be 3 and Fig. 5 should be Fig. 4.

    更新日期:2019-11-01
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