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

Abstract

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

Keywords: Cupriavidus necator; alanine; cell density; hexose utilization pathway; metabolic engineering; poly-β-hydroxybutyrate.

Figure 1

Hexose utilization pathways and metabolic engineering of C. necator for alanine production. The endogenous ED pathway was blocked by the knockout of glucose-6-phosphate 1-dehydrogenase (encoded by the zwf gene). The EMP pathway and PP pathway were completed by expressing the pfkA gene (encoding 6-phosphofructokinase) and gnd gene (encoding 6-phosphogluconate dehydrogenase) from E. coli, respectively. The phaC1AB1 operon was knocked out to increase the supply of precursors. The alaD gene (encoding L-alanine dehydrogenase) and alaE gene (encoding an alanine exporter in E. coli) were heterologously expressed for the synthesis and export of L-alanine. nagE, encoding a subunit of a putative GlcNAc-specific phosphotransferase system; nagR, encoding a putative GntR-type transcriptional regulator of nagE; edd, encoding phosphogluconate dehydratase; eda, encoding 2-keto-3-deoxy-6-phosphogluconate aldolase; ldhA1A2, encoding D-lactate dehydrogenase; phaA, encoding acetyl-CoA acetyltransferase; phaB, encoding acetoacetyl-CoA reductase; phaC, encoding poly(3-hydroxybutyrate) polymerase.

Figure 2

Effect of the ED and PHB synthesis pathways on the growth of C. necator. All of the zwf1, zwf2, and zwf3 genes were knocked out in both CnΔ6Δzwf and CnΔ6ΔzwfΔPHB. The phaC1AB1 operon was knocked out in both CnΔ6ΔPHB and CnΔ6ΔzwfΔPHB. (A) The growth curves of C. necator during heterotrophic fermentation with 30 g/L fructose as the sole carbon source. (B) The growth curves of C. necator during autotrophic fermentation with CO₂ as the sole carbon source.

Figure 3

Effect of the pfkA and gnd genes on the growth of different C. necator strains. Both the pfkA and gnd genes were constitutively expressed with promoter P_phaC1 in plasmids. Heterotrophic fermentation using 30 g/L fructose as the sole carbon source and autotrophic fermentation using CO₂ as the sole carbon source. Both the pfkA and gnd genes were cloned from E. coli. (A) The changes in the growth of CnΔ6 during heterotrophic fermentation; (B) the changes in the growth of CnΔ6ΔPHB during heterotrophic fermentation; (C) the changes in the growth of CnΔ6ΔzwfΔPHB during heterotrophic fermentation; (D) the changes in the growth of CnΔ6 during autotrophic fermentation; (E) the changes in the growth of CnΔ6ΔPHB during autotrophic fermentation; (F) the changes in the growth of CnΔ6ΔzwfΔPHB during autotrophic fermentation.

Figure 4

Effect of different alaD genes on the growth and alanine titer of strain CnAla. The highest OD₆₀₀ and alanine titer of the strain were observed with the constitutive (by P_phaC1, marked with P) or inducible (by araCP_BAD, marked with araP) expression of different alaD genes in plasmids during heterotrophic fermentation with 30 g/L glucose as the sole carbon source. The alaD_gs, alaD_ls, and alaD_NADPH genes were codon-optimized from G. stearothermophilus, L. sphaericus, and B. subtilis, respectively.

Figure 5

Effect of the expression of the gnd and pfkA genes on the growth and the alanine production of strain CnAlaD. Both the gnd and pfkA genes were expressed in plasmids. Heterotrophic fermentation using 30 g/L glucose as the sole carbon source. Arrows represent the time point to add inducer. (A) The growth curves of strain CnAlaD with different plasmids. (B) The highest titer and yield of glucose converted to alanine of strain CnAlaD with different plasmids.