Investigations into Multiple Acyl‐CoA Assimilation Pathways in a Single Organism

Abstract

By 2050 there could be around 12 billion tons of plastic in landfills and the natural environment, which could be avoided by using a more environmentally friendly alternative: biologically produced plastic, or bioplastic. Polyhydroxybutyrate (PHB) is a bioplastic whose precursor is an intermediate in the ethylmalonyl‐CoA pathway (EMCP), a pathway for acetyl‐CoA assimilation in bacteria. The genes that encode the enzymes for two different acetyl‐CoA assimilation pathways, the glyoxylate bypass (GB) and the EMCP, have been observed in two PHB‐producing bacteria: Rhodobacter capsulatus and Paracoccus denitrificans. It was previously observed that P. denitrificans asynchronously uses both pathways when grown with acetate. We produced R. capsulatus fusion strains by fusing the gene for a fluorescent protein, mCherry, to the gene of a key enzyme of the EMCP or GB and observed the fluorescence during growth with acetate. Tracking fluorescence allowed us to track the accumulation of each key enzyme, which indirectly indicates the activity of the pathway. We observed that unlike P. denitrificans, the GB in R. capsulatus is not essential to growth with acetate. Understanding the use/occurrence of the EMCP in bacteria is a step to being able to manipulate the EMCP to produce commercially relevant levels of bioplastic and related products.