Dual-channel glycolysis balances cofactor supply for l-homoserine biosynthesis in Corynebacterium glutamicum

Li, Ning; Li, Lihong; Yu, Shiqin; Zhou, Jingwen

doi:10.1016/j.biortech.2022.128473

Cited by 20 publications

(5 citation statements)

References 37 publications

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“…To date, only the G378E mutant of Cg HSD (retained activity of 52.7% at 50 mM l -threonine, Figure 4A ) was generated by traditional strain mutagenesis ( Sahm et al, 1996 ), which has been widely employed to develop the producers of downstream products, such as l -threonine and l -isoleucine ( Vogt et al, 2015 ; Wei et al, 2018 ). Metabolic engineering was also made to enhance the catalytic reaction by Cg HSD for l -homoserine production in C. glutamicum by overexpression of a bifunctional analog enzyme from E. coli ( Li et al, 2023 ). In the present study, we demonstrated a promising strategy to engineer the allosteric inhibition in amino acid biosynthesis by direct coupling feedback inhibition with the analog resistance and establishing a subsequent HTS method to screen the semi-rational designed libraries efficiently.…”

Section: Discussionmentioning

confidence: 99%

Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening

Liu,

Liu

et al. 2024

Front. Bioeng. Biotechnol.

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Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum (CgHSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of CgHSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of CgHSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway.

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Section: Discussionmentioning

confidence: 99%

Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening

Liu,

Liu

et al. 2024

Front. Bioeng. Biotechnol.

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“…Therefore, to increase intracellular NADPH and NADH supply, it is necessary to reduce the carbon flux through the EMP pathway and enhance it through the PP pathway. − As reported in numerous studies, the knockout of the pfkA gene can diminish the flux through the EMP pathway and activate the PP pathway, a finding derived from 13 C-labeling experiments . CRISPR/Cas9 gene editing technology was used to knock out the pfkA gene, which encodes 6-phosphofructokinase 1.…”

Section: Resultsmentioning

confidence: 99%

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli

Yang,

Wu,

et al. 2024

J. Agric. Food Chem.

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5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 μg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 μg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 μg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

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“…Increasing the availability of NADPH is commonly used to improve amino acid production by C. glutamicu (Hao et al 2022 ; Li et al 2023 ; Wu et al 2019 ). It is reported that, the affinity of glucose-6-phosphate 1-dehydrogenase (Zwf) toward NADP + can be increased by mutating alanine at position 243th to threonine in C. glutamicum (Becker J 2007).…”

Section: Resultsmentioning

confidence: 99%

Rapid screening of point mutations by mismatch amplification mutation assay PCR

Zhang,

Liu,

Liu

et al. 2024

Appl Microbiol Biotechnol

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Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique to detect point mutation and screen saturation mutation library. In this study, mismatch amplification mutation assay (MAMA) PCR was used to detect point mutation and screen saturation mutation library. In order to fine-tune the expression of odhA encoding 2-oxoglutarate dehydrogenase E1 component, a saturating mutant library of the RBS of odhA was created in Corynebacterium glutamicum P12 based on the CRISPR-Cas2a genome editing system, which increased the l-proline production by 81.3%. MAMA PCR was used to filter out 42% of the non-mutant transformants in the mutant library, which effectively reduced the workload of the subsequent fermentation test and the number of sequenced samples. The rapid and sensitive MAMA-PCR method established in this study provides a general strategy for detecting point mutations and improving the efficiency of mutation library screening. Key points • MAMA PCR was optimized and developed to detect point mutation. • MAMA PCR greatly improves the screening efficiency of point mutation. • Attenuation of odhA expression in P12 effectively improves proline production.

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Dual-channel glycolysis balances cofactor supply for l-homoserine biosynthesis in Corynebacterium glutamicum

Cited by 20 publications

References 37 publications

Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening

Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli

Rapid screening of point mutations by mismatch amplification mutation assay PCR

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