High-level and -yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering

Ding, Xiaohu; Yang, Wei; Du, Xiang; Chen, Ning; Xu, Qingyang; Wei, Minhua; Zhang, C. H.

doi:10.1016/j.ymben.2023.06.003

Cited by 17 publications

(4 citation statements)

References 42 publications

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“…In particular, environmentally friendly microbial manufacturing has experienced rapid growth across various fields of food, medicine [ 3 ], and energy, which has brought significant economic benefits and generated substantial social value worldwide [ 4 ]. Recent advances have been made in many commercial cases of microbial manufacturing using high-performance strains in trans-aconitic acid [ 5 ], lipoic acid [ 6 ], cinnamaldehyde [ 7 ], L-leucine [ 8 ] and other products [ 9 ]. Vitamin B 6 encompasses a group of vitamins, namely, pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), along with their corresponding phosphate esters, 5’-pyridoxal phosphate (PLP), 5’-pyridoxine phosphate (PNP), and 5’-pyridoxamine phosphate (PMP) [ 9 – 11 ].…”

Section: Introductionmentioning

confidence: 99%

Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization

Tian,

Liu,

et al. 2024

Microb Cell Fact

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Background Microbial engineering aims to enhance the ability of bacteria to produce valuable products, including vitamin B6 for various applications. Numerous microorganisms naturally produce vitamin B6, yet the metabolic pathways involved are rigorously controlled. This regulation by the accumulation of vitamin B6 poses a challenge in constructing an efficient cell factory. Results In this study, we conducted transcriptome and metabolome analyses to investigate the effects of the accumulation of pyridoxine, which is the major commercial form of vitamin B6, on cellular processes in Escherichia coli. Our omics analysis revealed associations between pyridoxine and amino acids, as well as the tricarboxylic acid (TCA) cycle. Based on these findings, we identified potential targets for fermentation optimization, including succinate, amino acids, and the carbon-to-nitrogen (C/N) ratio. Through targeted modifications, we achieved pyridoxine titers of approximately 514 mg/L in shake flasks and 1.95 g/L in fed-batch fermentation. Conclusion Our results provide insights into pyridoxine biosynthesis within the cellular metabolic network for the first time. Our comprehensive analysis revealed that the fermentation process resulted in a remarkable final yield of 1.95 g/L pyridoxine, the highest reported yield to date. This work lays a foundation for the green industrial production of vitamin B6 in the future.

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Section: Introductionmentioning

confidence: 99%

Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization

Tian,

Liu,

et al. 2024

Microb Cell Fact

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“…Dry cell weight (DCW) was calculated based on the measured OD 600 , as follows: 1 DCW [g/L] = 0.32 × OD 600 + 0.02. 41 High-performance liquid chromatography (HPLC) was used to determine the concentrations of LNnT, LNT II, lactose, glycerol, and glucose. Fermented samples were boiled for 5 min and centrifuged at 17,800 × g for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…The OD 600 of cultured cells was measured using a UV-5100 Spectrophotometer (Metash, China). Dry cell weight (DCW) was calculated based on the measured OD 600 , as follows: 1 DCW [g/L] = 0.32 × OD 600 + 0.02 . High-performance liquid chromatography (HPLC) was used to determine the concentrations of LNnT, LNT II, lactose, glycerol, and glucose.…”

Section: Methodsmentioning

confidence: 99%

High-Yield Synthesis of Lacto-N-Neotetraose from Glycerol and Glucose in Engineered Escherichia coli

Liao,

Lao,

et al. 2024

J. Agric. Food Chem.

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Lacto-N-neotetraose (LNnT) is a neutral human milk oligosaccharide with important biological functions. However, the low LNnT productivity and the incomplete conversion of the intermediate lacto-N-tetraose II (LNT II) currently limited the sustainable biosynthesis of LNnT. First, the LNnT biosynthetic module was integrated in Escherichia coli. Next, the LNnT export system was optimized to alleviate the inhibition of intracellular LNnT synthesis. Furthermore, by utilizing rate-limiting enzyme diagnosis, the expressions of LNnT synthesis pathway genes were finely regulated to further enhance the production yield of LNnT. Subsequently, a strategy of cofermentation using a glucose/glycerol (4:6, g/g) mixed feed was employed to regulate carbon flux distribution. Finally, by overexpressing key transferases, LNnT and LNT II titers reached 112.47 and 7.42 g/L, respectively, in a 5 L fermenter, and 107.4 and 2.08 g/L, respectively, in a 1000 L fermenter. These are the highest reported titers of LNnT to date, indicating its significant potential for industrial production.

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“…Arense et al increased the flux of the TCA cycle by deleting aceK and blocked the byproduct γbutyrobetaine by deleting caiA, eventually, 59.6 mmol L −1 h −1 of L-carnitine was produced in resting cells of the E. coli strain. 53 Our study is the same, using the CRISPR-Cas9 technology for knockout replacement of the gene, but L-HYP cannot be produced spontaneously in microorganisms and the substances to be added in the process are expensive; therefore, we modified the required substrates separately to achieve the generation of L-HYP using microbial fermentation. Finally, as can be seen from Figure 7e, the supply of substrates L-Trp and SAM is not the limiting factor for producing L-HYP in strain HYP-12, whereas the Trp-specific methyltransferase has once again emerged as the limiting factor in strain HYP-12.…”

Section: ■ Associated Contentmentioning

confidence: 99%

Combining Protein Engineering and Pathway Engineering to Achieve Green Biosynthesis of l-Hypaphorine in Escherichia coli from Glucose

Wang,

Sun,

et al. 2024

ACS Sustainable Chem. Eng.

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High-level and -yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering

Cited by 17 publications

References 42 publications

Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization

Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization

High-Yield Synthesis of Lacto-N-Neotetraose from Glycerol and Glucose in Engineered Escherichia coli

Combining Protein Engineering and Pathway Engineering to Achieve Green Biosynthesis of l-Hypaphorine in Escherichia coli from Glucose

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