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An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain


The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMICg::ISm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDHCg) by NADP-GapDHCa from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ significantly between these strains (14.1 ± 1.8 g/L vs 16.2 ± 1.1 g/L). Enhancing the carbon flux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icdCg were introduced into C. glutamicum with NADP-GapDHCa and pEC-ABNCE increased L-leucine yield (54.3 ± 2.9 g/L) and improved cell growth (OD562 = 83.4 ± 7.5) in fed-batch fermentation because the resultant strain C. glutamicum XL-1 ΔMICg::ISm GCg::GCa Pzwf-D1 Picd-D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the first report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.

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Glucose-6-phosphate dehydrogenase


Malic enzyme


Isocitrate dehydrogenase


Glyceraldehyde 3-phosphate dehydrogenase


Acetohydroxyacid isomeroreductase


Branched-chain amino acid transaminase


6-Phosphogluconate dehydrogenase


Chloramphenicol acetyltransferase


Reactive oxygen species


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The authors would like to acknowledge Prof. Xiaoyuan Wang for plasmids pDXW-11 from State Key Laboratory of Food Science and Technology in Jiangnan University.


This work was financially supported by the National Key Research and Development Program of China (2021YFC2100900), the Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University (KLIB-KF 202004), and Fundamental Research Funds for the Central Universities [No. JUSRP115A19].

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Jian-Zhong Xu and Sheng-Ling Chen designed the research. Jian-Zhong Xu, Sheng-Ling Chen, and Ting-Shan Liu performed the experiments, analyzed the data, and prepared the figures. Sheng-Ling Chen, Ting-Shan Liu, and Jian-Zhong Xu wrote the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jian-Zhong Xu.

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Chen, SL., Liu, TS., Zhang, WG. et al. An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain. Int Microbiol (2022).

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  • Corynebacterium glutamicum
  • NADPH auxotroph
  • Promoter engineering
  • L-leucine production