The use of kinetic models of metabolism in design-build-learn-test cycles is limited despite their potential to guide and accelerate the optimization of cell factories. This is primarily due to difficulties in constructing kinetic models capable of capturing the complexities of the fermentation conditions. Building on recent advances in kinetic-model-based strain design, we present the rational metabolic

Pathway engineering offers a promising avenue for sustainable chemical production. The design of efficient production systems requires understanding complex host-pathway interactions that shape the metabolic phenotype. While genome-scale metabolic models are widespread tools for studying static host-pathway interactions, it remains a challenge to predict dynamic effects such as metabolite accumulation

Plant polyphenols, a class of natural plant products with nutritional and medicinal value, can be alternatively produced by microbial cell factories. However, metabolic cross talk and enzyme incompatibility within the microbial host limits their synthesis. Therefore, we developed a sustainability-driven biotechnological process using cytoplasm periplasm combinatorial engineering to producing salvianic

Genetic regulation tools have been examined for their ability to enable sophisticated dynamic control of biosynthesis in microbial cell factories, enhancing the production performance of valuable compounds. However, most genetic tools are pathway- or intermediate-specific, hindering their broad applicability in synthetic biology. Moreover, their potential to balance metabolic fluxes in central metabolism

Bacillus methanolicus is the next workhorse in biotechnology using methanol, an alternative and economical one-carbon feedstock that can be obtained directly from carbon dioxide, as both carbon and energy source for the production of value-added chemicals. The wild-type strain B. methanolicus MGA3 naturally overproduces l-glutamate in methanol-based fed-batch fermentations. Here we generated a B. methanolicus

A genome-scale metabolic model was developed to explore metabolic engineering strategies for thermoacidophilic archaea, with a focus on the genetically tractable Sulfolobus acidocaldarius (Topt 75 °C, pHopt 2.5). S. acidocaldarius is natively neither an autotroph nor a sulfur oxidizer, although its genome suggests that this might have been the case at some evolutionary point. Comparative genomics provided

The increasing demand for biopolymers has positioned poly-γ-glutamic acid (γ-PGA) as a promising alternative to fossil-based polymers due to its biodegradability and biocompatibility. γ-PGA biosynthesis in Bacillus subtilis is closely linked to intracellular glutamate availability, which is typically maintained via the supply of an exogenous glutamate source, a cost-intensive factor for industrial

β-caryophyllene, a plant-derived sesquiterpene, serves as a food flavoring, anti-inflammatory agent, antioxidant, and high-energy fuel source. Extraction of β-caryophyllene from plants is a costly and inefficient process. Therefore, microbial cell factories have been employed for the production of β-caryophyllene. However, the limited yield is insufficient for its industrial application. In this study

The protein-glutaminase (PG, EC 3.5.1.44) specifically targets glutamine residues in proteins and peptides, and has significant potential for enhancing the functional characteristics and processing efficiency of plant proteins. However, natural PG production faces challenges such as low enzymatic yield and difficult genetic manipulation. To address these challenges, a novel self-activating PG expression

Metabolic target and strategy design play a critical role in enhancing the DBTL (Design-Build-Test-Learn) cycle in metabolic engineering. Classical stoichiometric algorithms such as OptForceMust and FSEOF narrow the experimental search space but fail to account for thermodynamic feasibility and enzyme-usage costs, leaving a space for their predictive performance. In this study, we introduce ET-OptME

Isoprenol (3-methyl-3-buten-1-ol) is a precursor to aviation fuels and other commodity chemicals and can be microbially synthesized from renewable carbon streams. Its production has been demonstrated in Pseudomonas putida KT2440 but its titers, rates, and yields have yet to reach commercially viable levels, potentially due to toxicity to the bacterial chassis. We hypothesized that utilization of Tolerization

Gallic acid (GA), a natural phenolic acid antioxidant, has significant therapeutic and industrial applications. However, its traditional manufacturing approach, based on plant extraction, has been associated with risks of environmental pollution as well as a limited range of applications. Consequently, microbial-based production of GA, being more environmental-friendly, is viewed as a potential alternative

Climate change is reducing crop yields and increasing price volatility for commodities like cocoa and palm oil, thereby driving the need for sustainable alternatives such as microbial lipid production. The oleaginous yeast Yarrowia lipolytica is a promising platform for lipid synthesis. However, its lipid accumulation has traditionally relied on nitrogen limitation, posing challenges for achieving

Isopropanol (IPA), a versatile chemical with applications in various fields of industries, yet its petroleum-based production raises environmental concerns. In this study, Corynebacterium glutamicum was engineered to enhance IPA production while mitigating CO2 emissions. First, the rational design of secondary alcohol dehydrogenase (SADH) variants with shifted cofactor specificity from NADPH to NADH

Bacterial exopolysaccharide (EPS), as a high-value probiotic product, is known to be biosynthesized by a secondary metabolic pathway to mediate acid stress in lactic acid bacteria. However, a quantitative understanding of cellular resource coordination underlying acid stress-induced EPS production remains lacking. Systematically investigating Lactiplantibacillus plantarum HMX2, a well acknowledged

Dynamic sensing of gas substrates like toxic carbon monoxide (CO) in living microbial cells is often limited due to the lack of suitable biosensors. Here, we integrated the CO-binding transcription activators, CooA and RcoM1, with an O2-independent fluorescent reporter system, Halo-tag, to develop CO-sensing modules (ON/OFF) capable of detecting CO concentrations in the strictly anaerobic acetogenic

Due to limited reserves and excessive carbon emission of fossil fuels, there has been an increasing interest in developing advanced biofuels with high energy density such as alkanes and alkenes. Here we report the design and construction of three heterologous biosynthetic pathways of alkanes and alkenes in oleaginous yeast Rhodotorula toruloides, including the AAR/ADO, UndA/UndB and FAP pathways. The

Genetically engineered microbes are increasingly utilized to produce a broad range of high-value compounds. However, most studies start with only a very narrow group of genetically tractable type strains that have not been selected for maximum titers or industrial robustness. In this study, we used high-throughput screening and parallel metabolic engineering to identify and optimize Saccharomyces cerevisiae

Cyanobacteria represent promising biocatalysts for producing carbohydrates, including sorbitol, a naturally-occurring, fermentable sugar alcohol with conventional uses as a sweetener, pharmaceutical additive, and biodegradable plasticizer. Previously, Synechocystis sp. PCC 6803 was engineered to produce sorbitol, reaching a final titer of 2.3 g/L after 18 days. To improve upon this performance, sorbitol

Ethylene glycol (EG), a major product of the enzymatic degradation of polyethylene terephthalate (PET), provides a promising feedstock for sustainable biomanufacturing. Herein, we developed a novel metabolic pathway using Escherichia coli(E. coli) as a host for the biosynthesis of four-carbon compounds such as 1,4-butanediol (1,4-BDO), 1,2,4-butanetriol (1,2,4-BTO), and succinate, from two-carbon substrates

Palm oil is an attractive feedstock for bioproduction due to its high carbon content and low cost. However, its metabolism generates excess reducing power, leading to redox imbalances and reduced metabolic efficiency in industrial fermentations. Through a model-driven approach integrating flux balance analysis, we activated the Calvin-Benson-Bassham (CBB) cycle in Cupriavidus necator to recycle surplus

The production of recombinant proteins requires the precise coordination of various biological processes, including protein synthesis, folding, trafficking, and secretion. The overproduction of a heterologous protein can impose various bottlenecks on these networks. Identifying and alleviating these bottlenecks can guide strain engineering efforts to enhance protein production. The methylotrophic yeast

Phenazine derivatives, a class of nitrogen-containing heterocyclic compounds, exhibit broad-spectrum antifungal, anticancer, and antimalarial activities. Pseudomonas and Streptomyces are the primary microbial strains responsible for the synthesis of phenazine derivatives. In general, Pseudomonas strains use phenazine-1-carboxylic acid (PCA) as a precursor for enzymatic modification, while Streptomyces

The non-natural amino acid 4-nitrophenylalanine is a crucial pharmaceutical ingredient and has extensive utility in protein engineering. Here, we demonstrated the production of 4-nitrophenylalanine by Escherichia coli with AurF, 4-aminobenzoate N-oxygenase from Streptomyces thioluteus. Firstly, eight distinct gene combinations, encompassing four variants of papA and two of papBC, were evaluated to

Chinese Hamster Ovary (CHO) cells are widely used in the pharmaceutical industry to produce therapeutic proteins. Increasing the productivity of CHO cells through media development and genetic engineering is a significant industry objective. Past research demonstrated the benefits of modulating fatty acid composition of CHO cells through genetic engineering. In this study, we describe an alternative

Psilocybin, the prodrug to the psychoactive compound in ‘magic’ mushrooms, is currently being studied in clinical trials as a treatment for severe mental health conditions, such as depression and anxiety. Previous reports of psilocybin biosynthesis as reconstituted in E. coli reported maximum titers of 1.16 g/L, exclusively using genes from the most common recreationally used mushroom, Psilocybe cubensis

Given sustainability and scalability concerns of using sugar feedstocks for microbial bioproduction of bulk chemicals, widening the feedstock range for microbial cell factories is of high interest. Methanol is a one-carbon alcohol that stands out as an alternative feedstock for the bioproduction of chemicals, as it is electron-rich, water-miscible and can be produced from several renewable resources

HEK293 cells are a versatile cell line extensively used in the production of recombinant proteins and viral vectors, notably Adeno-associated virus (AAV) (Bulcha et al., 2021). Despite their high transfection efficiency and adaptability to various culture conditions, challenges remain in achieving sufficient yields of active viral particles. This study presents a comprehensive multi-omics analysis

Formate, a biologically accessible form of CO2, has attracted interest as a renewable feedstock for bioproduction. However, approaches are needed to investigate efficient routes for biological formate assimilation due to its toxicity and limited utilization by microorganisms. Cell-free systems hold promise due to their potential for efficient use of carbon and energy sources and compatibility with

Polyhydroxyalkanoates (PHAs) are biodegradable and environmentally sustainable alternatives to conventional plastics, yet their adoption has been hindered by the high production costs and scalability challenges. This study employed unbiased genomics approaches to engineer Cupriavidus necator H16, an industrial strain with intrinsic capabilities for PHA biosynthesis, for enhanced utilization of oil-based

A continual goal in metabolic engineering is directing pathway flux to desired products and avoiding loss of pathway intermediates to competing pathways. Encapsulation of the pathway is a possible solution, as it creates a diffusion barrier between pathway intermediates and competing enzymes. It is hypothesized that bacteria use organelles known as bacterial microcompartments - proteinaceous shells

The recent clinical success of genetically modified T-cell therapies underscores the urgent need to accelerate fundamental studies and functional screening methods in T lymphocytes. However, a facile and cost-effective method for efficient genetic engineering of T-cells remains elusive. Current approaches often rely on viral transduction, which is labor-intensive and requires stringent biosafety measures

Biosynthetic gene clusters (BGCs) in microbial genomes play a crucial role in the biosynthesis of diverse secondary metabolites (SMs) with pharmaceutical potential. However, most BGCs remain silent under conventional conditions, resulting in the frequently repeated discovery of known SMs. Fortunately, in the past two decades, the heterologous expression of BGCs in genetically tractable hosts has emerged

Methanol is a promising sustainable alternative feedstock for green biomanufacturing. The yeast Yarrowia lipolytica offers a versatile platform for producing a wide range of products but it cannot use methanol efficiently. In this study, we engineered Y. lipolytica to utilize methanol by overexpressing a methanol dehydrogenase, followed by the incorporation of methanol assimilation pathways from methylotrophic

Progesterone and its steroidal derivatives, including androgens, estrogens, glucocorticoids and mineralocorticoids are extensively utilized in pharmacotherapy, serving as predominant agents in anti-inflammatory, contraceptive, and anticancer treatments. From the 1990s to the present, scientists attempted to biosynthesize steroids such as progesterone and hydrocortisone from sugars in engineered microbial

Rhodotorula sp. is a red yeast that has emerged as a promising host for microbial cell factories. Under specific conditions, Rhodotorula sp. can accumulate lipids that constitute over 70% of its dry cell weight, underscoring its potential in lipid compound production. Additionally, it can utilize a variety of carbon sources, including glucose, xylose, and volatile fatty acids, and exhibits high tolerance

This study aimed to develop a metabolically engineered Escherichia coli strain capable of producing oviedomycin, a type II angucyclinone polyketide compound with anticancer activity. We first addressed the challenges of in vivo reassembly of the type II polyketide synthase machinery in E. coli. These included co-expressing molecular chaperones, rare tRNAs, and a fusion tag to enhance the solubility

Lycopene, a potent carotenoid with high antioxidant capacity and extensive applications, holds significant potential for sustainable production via microbial engineering, particularly with the rising interest in methanol as an ideal non-grain feedstock for a carbon-negative economy. In this study, Komagataella phaffii was systematically engineered to enhance lycopene production using glucose and renewable

Genome-scale metabolic models (GEMs) have been widely utilized to understand cellular metabolism. The application of GEMs has been advanced by computational methods that enable the prediction and analysis of intracellular metabolic states. However, the accuracy and biological relevance of these predictions often suffer from the many degrees of freedom and scarcity of available data to constrain the

Malonyl-CoA is the major precursor for the biosynthesis of diverse industrially valuable products such as fatty acids/alcohols, flavonoids, and polyketides. However, its intracellular availability is limited in most microbial hosts, hampering the industrial production of such chemicals. To address this limitation, we present a multilevel optimization workflow using modern metabolic engineering technologies

Aldehydes are ubiquitous metabolites in living cells. As reactive electrophiles, they have the capacity to form adducts with cellular protein thiols and amines, leading to potential toxicity. Dynamic regulation has proven to be an effective strategy for addressing the accumulation of toxic metabolites. However, there are limited reports on applying dynamic control specifically to mitigate aldehyde

Advances in genomics technologies have generated large data sets that provide tremendous insights into the genetic diversity of taxonomic groups. However, it remains challenging to pinpoint the effect of genetic diversity on different traits without performing resource-intensive phenotyping experiments. Pan-genome-scale metabolic models (panGEMs) extend traditional genome-scale metabolic models by

Microbial poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate), abbreviated as P(3HB-4HB-3HV) or P34HBHV, is a flexible polyhydroxyalkanoate (PHA) material ranging from softness to elasticity depending on the ratios of various monomers. Halomonas bluephagenesis, as the chassis of the next generation industrial biotechnology (NGIB) able to grow contamination free under open unsterile conditions

Correct folding of endoplasmic reticulum (ER)-localized membrane proteins, such as cytochrome P450, endows a synthetic biology host with crucial catalytic functions, which is of vital importance in the field of metabolic engineering and synthetic biology. However, due to complexed interaction with cellular membrane environment and other proteins (e.g., molecular chaperone) regulation, a substantial

Microbial fatty acids (FAs) hold significant potential as alternatives for the oleochemical industry. However, expanding the functional and structural diversity of microbial FA-derived products is essential to fully leverage this potential. Methyl-branched-chain FAs (MBFAs) are of particular interest as high-performance industrial compounds. This study examines the ability of the Escherichia coli FA

Converting polyethylene (PE) into valuable materials, particularly ones that are better for the environment than the incumbent plastics, not only helps mitigate environmental issues caused by plastic waste but also alleviates the long-standing problem of microbial fermentation competing with food supplies. However, the inherent robustness of PE due to its strong carbon-carbon bonds and high molecular

With increasing concern over environmental problems and energy crises, interest in the biological conversion of CO2 into bioproducts is growing. Although microalgae efficiently utilize CO2, their metabolic engineering remains challenging. In contrast, while synthetic biology tools are advanced for many heterotrophic bacteria, these organisms cannot directly utilize CO2. As such, a modular co-culture

The biosynthesis of 5-hydroxyvaleric acid (5-HV) from glucose via the l-lysine degradation pathway cocurrently generates by-products, including l-lysine, 5-aminovaleric acid (5-AVA), and glutaric acid (GTA), which are closely interconnected with the 5-HV biosynthesis pathway. This study focuses on developing a highly selective 5-HV production system in Corynebacterium glutamicum. Initial strategies

The utilization of microorganisms to transform biomass into biofuels and biochemicals presents a viable and competitive alternative to conventional petroleum refining processes. Halomonas species are salt-tolerant and alkaliphilic, endowed with various beneficial properties rendering them as contamination resistant platforms for industrial biotechnology, facilitating the commercial-scale production

The oleaginous yeast Rhodosporidiumtoruloides has been exploited for many bioproducts, including several terpenes, owing to its oleaginous nature and biomass inhibitor tolerance. Here, we built upon previous (E)-α-bisabolene work by iteratively stacking the complete mevalonate pathway from Saccharomyces cerevisiae onto a multicopy bisabolene synthase parent strain. Metabolomics and proteomics verified

L-threonine, one of the three major amino acids, plays a vital role in various industries such as food, feed, pharmaceuticals, and cosmetics. Currently, the fermentation-based production of L-threonine has evolved into an efficient, cost-effective, and environmentally friendly industrial process. Escherichia coli and Corynebacterium glutamicum, as the industrial workhorses of amino acids production

Methanol-based biomanufacturing holds great promise for sustainability but is currently limited by the slow growth and low efficiency of natural or synthetic methylotrophic strains. In contrast, the thermophilic methylotroph Bacillus methanolicus exhibits rapid growth, high-temperatures tolerance, and efficient methanol utilization in defined mineral medium, making it a promising candidate for industrial

Biosynthesis of 12-aminododecanoic acid (ADDA) directly from biomass-derived sugars would enable a more sustainable process for manufacturing the engineering polymer Nylon 12. ADDA biosynthesis is currently hindered by the cytotoxicity of dodecanoic acid (DDA) to growing cells, and the accumulation of the overoxidized byproduct dodecanedioic acid (DDDA). In this study, these challenges were addressed

CRISPR interference (CRISPRi) is a versatile tool for high-throughput phenotypic screening. However, rational design and synthesis of the single-guide RNA (sgRNA) library required for each genome-wide CRISPRi application is time-consuming, expensive, and unfeasible if the target organisms lack comprehensive sequencing and characterization. We developed an ultra-dense random sgRNA library generation

Polyhydroxyalkanoates (PHA) with high 3-hydroxyvalerate (3HV) monomer ratios lead to their accelerated biodegradation and improved thermal and mechanical properties. In this study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a broad range of 3HV ratios were produced and characterized using the next generation industrial biotechnology (NGIB) chassis Halomonas bluephagenesis (H. bluephagenesis)

Saccharomyces cerevisiae predominantly ferments sugar to ethanol, irrespective of the presence of oxygen, which is known as the Crabtree-effect. Traditional methods rely on static controls of glycolytic flux to make S. cerevisiae Crabtree-negative, which are not favorable for future biomanufacturing applications. Considering native metabolic pathways typically harness dynamic regulatory networks, we

Palmitoleic acid (POA, C16:1Δ9) is widely recognized for its preventive and therapeutic effects in various chronic and cardiovascular diseases, but the current production practices based on plant extraction are both economically and ecologically unsustainable. Although Yarrowia lipolytica is capable of producing POA, it only accumulates to a small percentage of total fatty acids. The present study

The oxidative formation of AcCoA limits the glycolytic pathway yield (YPGLY) for citric acid due to the NADH overflow and carbon loss as CO2. An interesting approach to enhance product yields is the incorporation of carbon-conserving pathways. This study assesses the potential of a carbon-conserving AcCoA pathway, the glycolysis alternative high carbon yield cycle (GATHCYC), to improve citric acid