Insights into genetic determinants of volatile fatty acid catabolism in Cupriavidus necator H16.

Abstract

The soil bacterium Cupriavidus necator H16 is a promising host for upgrading waste-derived volatile fatty acids (VFAs) into renewable biochemicals. While bacterial VFA metabolic pathways are well understood, the C. necator genome encodes multiple enzymes for each catabolic step, and the degree of substrate specificity among these homologs is currently unknown. To gain insight into the catabolism of VFA substrates in C. necator, we performed transcriptomics on cells grown with acetate, propionate, butyrate, valerate, or hexanoate as the sole source of carbon and energy. These data revealed that C. necator upregulates multiple sets of genes putatively involved in substrate activation and β-oxidation in response to VFAs. To better understand this redundancy, we performed biochemical and genetic deletion studies of acyl-CoA synthetase enzymes upregulated during growth on VFA substrates. These results demonstrated the functional redundancy of the C. necator VFA catabolism and led to the identification of a gene cluster, H16_B1332-H16_B1337, that contains several genes that are important for the efficient catabolism of hexanoate. Constitutive expression of a second copy of these hexanoate catabolism genes did not improve growth of C. necator on hexanoate, suggesting that other factors (e.g., redox, transport, or toxicity) may be limiting for growth. Collectively, this work provides new insight into how C. necator uses metabolic regulation to effectively utilize VFA substrates and uncovers the important role of the gene cluster H16_B1332-H16_B1337 in the catabolism of hexanoate.

Importance: The development of efficient bioprocesses that utilize waste-derived carbon will be important for ensuring the circularity of carbon flows and the sustainability of new biotechnologies. Unfortunately, carbon substrates that can be reliably sourced from waste are often toxic or inefficient growth substrates for industrially relevant bacteria. A more complete understanding of the regulatory and biochemical mechanisms that bacteria use to respond to and catabolize waste-derived carbon resources will enable metabolic engineering strategies to improve bioconversion of these same resources. In this study, we provide new insight into these mechanisms for an emerging and promising host-feedstock pairing: Cupriavidus necator H16 and volatile fatty acids (VFAs). We anticipate that these insights can be leveraged in future work to engineer C. necator to more efficiently convert VFAs into sustainable protein and bioproducts.