Engineered E. coli for Diverse PHA Production

Four PhaC amino acid sequences were chosen based on the BLASTP search results. These phaC genes were chemically synthesized with optimized codon usage in E. coli by Eurofins Genomics Co. Ltd. (Tokyo, Japan) for plasmid construction and evaluation. E. coli LSBJ, a fadB fadJ double-deletion strain of E. coli LS5218 [fadR601, atoC (Con)] (Tappel et al., 2012a (link)), was used as the host strain for PHA biosynthesis. This strain is an ideal host for non-native PHA production because of its ability to take up a wide variety of substrates to be incorporated into PHA homo- and copolymers, and bench-level scale-up methodologies available for overall production (Tappel et al., 2012b (link); Levine et al., 2016 (link); Pinto et al., 2016 (link); Fadzil et al., 2018 (link); Furutate et al., 2021 (link); Scheel et al., 2021 (link)). A broad-host-range plasmid pBBR1MCS-2 (Kovach et al., 1995 (link)) harboring the genes encoding the PhaCs to be evaluated, the lac promoter region, the (R)-specific enoyl-CoA hydratase gene from A. caviae (phaJ_Ac), the 3-ketothiolase gene (phaA) from Ralstonia eutropha H16, and the acetoacetyl-CoA reductase gene (phaB) from R. eutropha H16, termed pBBR1-phaCsAB_ReJ_Ac, was used for the expression of PhaCs (Supplementary Figure S1). For phaAB expression, the R. eutropha pha promoter and terminator regions were located upstream and downstream of their genes, respectively. To enhance the supply of 3HHx, 3H4MV, and 3H2MB monomers, the plasmid pTTQ-PCT (Furutate et al., 2017 (link)) containing the propionyl-CoA transferase (PCT) gene from Megasphaera elsdenii (pct) (Taguchi et al., 2008 (link)) was introduced into the E. coli LSBJ strain (Supplementary Figure S1).