Optimizing Hexose Utilization Pathways of Cupriavidus necator for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions

Abstract

<i>Cupriavidus necator</i> is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the <i>phaC1AB1</i> operon) can effectively enhance the production of <i>C. necator</i>, but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of <i>C. necator</i> in this study by implementing strategies such as blocking the Entner-Doudoroff pathway, completing the phosphopentose pathway by expressing the <i>gnd</i> gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden-Meyerhof-Parnas pathway by expressing the <i>pfkA</i> gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD₆₀₀ of the <i>phaC1AB1</i>-knockout strain increased by 44.8% with <i>pfkA</i> gene expression alone, and by 93.1% with <i>gnd</i> and <i>pfkA</i> genes expressing simultaneously. During autotrophic fermentation, <i>gnd</i> and <i>pfkA</i> genes raised the OD₆₀₀ of <i>phaC1AB1</i>-knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the <i>pfkA</i> gene on the production of <i>C. necator</i>, an alanine-producing <i>C. necator</i> was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the <i>phaC1AB1</i> operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the <i>pfkA</i> gene. The results indicate that completing the Embden-Meyerhof-Parnas pathway by expressing the <i>pfkA</i> gene is an effective method to improve the growth and production of <i>C. necator</i>.