Improved production of D-pantothenic acid in Escherichia coli by integrated strain engineering and fermentation strategies
Introduction
D-pantothenic acid (D-PA), as a crucial water-soluble B-complex vitamin, is the precursor of coenzyme A (CoA) and an acyl carrier protein (ACP) (Eggersdorfer et al., 2012; Leonardi and Jackowski, 2007). With its unique physiological characteristics, D-PA has a series of potential health benefits (Chohnan et al., 2014; Ismail et al., 2020; Patassini et al., 2019). Besides, D-PA is also widely used in the food and cosmetic industries, especially in the feed industry (Postaru et al., 2015). The commercial production of D-PA has currently always relied on developed chemical synthesis routes (Eggersdorfer et al., 2012; Tigu et al., 2018). The classical chemical process involves highly toxic raw materials, cumbersome optical resolution, and cyanide-containing wastewater pollution (Smith and Song, 1996; Rocha et al., 2019). The increase in environmental pollution and energy shortages has made the production of D-PA by eco-friendly methods receive widespread attention from researchers.
Microbial fermentation of natural renewable resource to produce D-PA is considered an attractive alternative method due to its environmentally friendly and sustainable characteristics (Leonardi and Jackowski, 2007). With the development of metabolic engineering, microbial pathway modification has opened the possibility of D-PA microbial production by engineered Corynebacterium glutamicum and Escherichia coli (Huser et al., 2005; Zhang et al., 2019). When glucose is the carbon source, the biosynthesis pathway of D-PA consists of two parts (Fig. 1). Among them, ketopantoate hydroxymethyl transferase (KPHMT) encoded by panB and pantothenate synthetase (PS) encoded by panC are the key rate-limiting steps in the D-PA biosynthetic pathway (Chassagnole et al., 2003; Webb et al., 2004). Overexpression of the panBC operon in C. glutamicum and replacement of the original promoter of panBC by trc in E. coli resulted in increased D-PA accumulation (Huser et al., 2005; Zhang et al., 2019). The key precursors (pyruvate and ketoisovalerate) of D-PA biosynthesis can also be used to synthesize l-isoleucine and l-valine, respectively (Wang et al., 2018; Wang, 2019). Therefore, the reduction of D-PA competition pathway (l-isoleucine and l-valine biosynthesis) and the co-expression of four genes ilvBNCD have been successfully used to reduce the consumption of key precursors and increase the utilization of ketoisovalerate to obtain D-PA production (Sahm and Eggeling, 1999). Besides, a mutation in the coaA gene encoding pantothenate kinase reduced the conversion of D-PA to coenzyme A (CoA), which is also an effective metabolic method to improve the biosynthesis of D-PA, and in the fed-batch fermentation, the result of 28.45 g/L D-PA was obtained (Zhang et al., 2019). Although various efforts have been made to increase the metabolic flux of the microorganisms-based D-PA biosynthetic pathway, the low yield and productivity of D-PA are still the defects of the D-PA microbial fermentation production, which directly reduces the market competitiveness of microbial fermentation due to the complexity and strict regulation of the D-PA biosynthetic pathway (Webb et al., 2004).
In addition, the interaction between the fermentation behavior of the strain and the metabolic network was generally ignored, which may result in a limited increase in D-PA production (Liu et al., 2012). Therefore, the metabolic engineering of D-PA producers should focus on the optimization of the cycle between strain improvement and fermentation performance (Lee et al., 2009; Liu et al., 2012). In general, only the excellent strains combined with the most suitable fermentation process can exert their best production performance. Furthermore, many factors can affect the production of D-PA during fermentation, such as feeding strategy, which may affect cell growth viability and product synthesis (Biglari et al., 2020; Trondle et al., 2018; Zou et al., 2020). Therefore, further consideration should be given to the optimization of the biological process.
In our previous study, an engineered strain (E. coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA) with overexpressing of panBC operon showed a D-PA production of 1.99 g/L from 20 g/L glucose (Zhang et al., 2019). In this work, we focused on enhancing the production of D-PA by improving the availability of pyruvate and regulating the intracellular redox homeostasis significantly in engineered E. coli. First, derivative strains containing deletions of aceF gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) and mdh gene encoding the malate dehydrogenase were constructed to increase the utilization of pyruvate. Then, the effects of increased NADPH supply by overexpression of four types of genes on D-PA biosynthesis were investigated: glucose-6-phosphate dehydrogenase, transhydrogenase, NAD kinase and glucose-6-phosphate dehydrogenase coupling with NAD kinase. Besides, a novel culture strategy combining dissolved oxygen (DO)-feedback feeding and betaine addition has further increased the production of D-PA.
Section snippets
Strains and plasmids construction
Strains and plasmids used in this study were listed in Table 1. E. coli DH5α (Tsingke, Hangzhou, China) was used as a general host for plasmid construction. The D-PA-producing engineered E. coli strains DPA/pTrc99A-panBC were described from preliminary work (Zhang et al., 2019). All DNA manipulations were performed according to the instructions of standard molecular cloning procedures. Oligonucleotides were purchased from Tsingke Technology (Hangzhou, China). Genomic DNAs were isolated from
Effective D-PA production by enhancing pyruvate pool
Metabolic engineering to increase the supply of precursors is a very effective strategy to improve the synthesis of target products (Li et al., 2017). Pyruvate is one of the important precursors for D-PA production (Fig. 1). The “moderate” strategy to reduce competing metabolic branching processes has been attested to be effective in increasing the pyruvate pool (Li et al., 2017). Several pathways to consume pyruvate were considered, including the conversion of pyruvate to acetyl-CoA catalyzed
Conclusion
In conclusion, this study successfully adopted strain improvement and culture process control strategies to increase the production of extracellular D-PA. The results showed that pyruvate contributes to the biosynthetic pathway of D-PA instead of the consumption of pyruvate to acetyl-CoA by knocking out the aceF gene. Moreover, the knockout of mdh gene to modify TCA further improved the pyruvate pool and resulted in an increase in the production of D-PA. Redox cofactor regulation indicated that
CRediT authorship contribution statement
Shuping Zou: Conceptualization, Investigation, Formal analysis, Data curation, Writing - original draft. Kuo Zhao: Investigation, Formal analysis, Validation, Writing - original draft. Heng Tang: Investigation, Data curation. Zheng Zhang: Investigation, Data curation. Bo Zhang: Investigation, Data curation. Zhiqiang Liu: Conceptualization, Methodology, Writing - review & editing, Funding acquisition. Yuguo Zheng: Writing - review & editing, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This research/project is supported by the National Key Research and Development Project (2019YFA0905000).
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2022, Biochemical Engineering JournalCitation Excerpt :β-Alanine has been widely applied in various industries, such as medicine [2], food [3], and feed [4]. Particularly, β-alanine, along with D-pantoate, is an important substance for the synthesis of D-pantothenic acid (vitamin B5) [5], a key precursor for the coenzyme A and acyl carrier protein biosynthesis [6]. Known as one of the 12 most potential three-carbon chemical products in the world [7], the market demand of β-alanine has been gradually increased.
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2022, Biochemical Engineering JournalCitation Excerpt :There was almost no accumulation of residual sugars and the accumulation of acetate was 24.54 g/L during the feeding fermentation, which was reduced by half compared with the constant rate feeding and intermittent feeding. This may be due to the advantage of DO-feedback feeding, which can alleviate substrate inhibition and DO limitation and improve the yield of the target product [8,21]. The nutritional deficiency caused by the weakening of the isoleucine pathway was compensated by supplementing with 4 mL of 40 g/L isoleucine at 12 h of fermentation to stimulate the growth of the strain, as well as by secondary induction of IPTG at 72 h of fermentation when the bacterium reached a certain level (Fig. 5D).
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2022, Process BiochemistryCitation Excerpt :Too fast or too slow cell growth can lead to slow D-PA biosynthesis. These trends were similar to those in previous reports [9–11]. As shown in Fig. 5A, the production of D-PA had stalled from 33 h to 40 h.