Engineering Escherichia coli for enhanced production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in larger cellular space

Polyhydroxyalkanoates (PHA) are intracellularly accumulated as inclusion bodies. Due to the limitation of the cell size, PHA accumulation is also limited. To solve this problem, Escherichia coli was enlarged by over-expression of sulA gene to inhibit the cell division FtsZ ring assembly, leading to the formation of filamentary E. coli that have larger internal space for PHA accumulation compared with rod shape E. coli. As a result, more than 100% increases on poly(3-hydroxybutyrate) (PHB) contents and cell dry weights (CDW) were achieved compared with its control strain under same conditions. The enlarged cell strategy was applied to the production of poly(3-hydroxybutyrate-. co-4-hydroxybutyrate) or P(3HB-. co-4HB) by sad, gabD, essential genes ispH and folK knockout E. coli harboring two addictives and thus stable plasmids consisting of P(3HB-. co-4HB) producing genes, including phaCAB operon, orfZ, 4hbD, sucD, essential genes ispH and folK as well as the sulA. The so constructed E. coli grew in glucose to form filamentary shapes with an improved P(3HB-. co-4HB) accumulation around 10% more than its control strain without addition of 4HB precursor, reaching over 78% P(3HB-. co-4HB) in CDW. Importantly, the shape changing E. coli was able to precipitate after 20. min stillstand. Finally, the filamentary recombinant E. coli was not only able to produce more P(3HB-. co-4HB) from glucose but also allow convenient downstream separation from the fermentation broth.