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Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum d-lactate productivity under oxygen deprivation

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Abstract

We previously demonstrated the simplicity of oxygen-deprived Corynebacterium glutamicum to produce d-lactate, a primary building block of next-generation biodegradable plastics, at very high optical purity by introducing heterologous D-ldhA gene from Lactobacillus delbrueckii. Here, we independently evaluated the effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production in C. glutamicum. We consequently show that while the reactions catalyzed by glucokinase (GLK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase had positive effects on d-lactate productivity by increasing 98, 39, 15, 13, and 10 %, respectively, in 10 h reactions in minimal salts medium, the reaction catalyzed by pyruvate kinase had large negative effect by decreasing 70 %. The other glycolytic enzymes did not affect d-lactate productivity when each of encoding genes was overexpressed. It is noteworthy that all reactions associated with positive effects are located upstream of glycerate-1,3-bisphosphate in the glycolytic pathway. The d-lactate yield also increased by especially overexpressing TPI encoding gene up to 94.5 %. Interestingly, overexpression of PFK encoding gene reduced the yield of succinate, one of the main by-products of d-lactate production, by 52 %, whereas overexpression of GAPDH encoding gene increased succinate yield by 26 %. Overexpression of GLK encoding gene markedly increased the yield of dihydroxyacetone and glycerol by 10- and 5.8-fold in exchange with decreasing the d-lactate yield. The effect of overexpressing glycolytic genes was also evaluated in 80 h long-term reactions. The variety of effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production is discussed.

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Acknowledgment

We thank Crispinus A. Omumasaba for critical reading of the manuscript.

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Authors and Affiliations

  1. Research Institute of Innovative Technology for the Earth (RITE), 9-2, Kizugawadai, Kizugawa-shi, Kyoto, 619-0292, Japan

    Yota Tsuge, Shougo Yamamoto, Masako Suda, Masayuki Inui & Hideaki Yukawa

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  1. Yota Tsuge
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  2. Shougo Yamamoto
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  3. Masako Suda
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  4. Masayuki Inui
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  5. Hideaki Yukawa
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Correspondence to Hideaki Yukawa.

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Tsuge, Y., Yamamoto, S., Suda, M. et al. Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum d-lactate productivity under oxygen deprivation. Appl Microbiol Biotechnol 97, 6693–6703 (2013). https://doi.org/10.1007/s00253-013-4986-7

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