Metabolic engineering for the production of aliphatic and aromatic containing PHAs

Abstract

Escherichia coli strain was metabolically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate [P(3HB-co-3HV)] using citramalate pathway derived 2-ketobutyrate, as a precursor for propionyl-CoA. Two different metabolic pathways for the production of propionyl-CoA from 2-ketobutyrate were constructed. The first one is composed of Dickeya dadantii 3937 2-ketobutyrate oxidase (2KO) or E. coli pyruvate oxidase mutant (PoxB V380A L253F) for the conversion of 2-ketobutyrate into propionate and Ralstonia eutropha propionyl-CoA synthetase (PrpE) for further activation of propionate into propionyl-CoA. Second one is consisted of E. coli 2-ketobutyate synthases (TdcE) or Clostridium difficile (PflB) for direct conversion of 2-ketobutyrate into propionyl-CoA The recombinant E. coli XL1-Blue strain equipped with citramalate pathway harboring E. coli poxB V380A L253F and R. eutropha prpE genes together with R. eutropha PHA biosynthesis genes successfully produced P(3HB-co-2.3mol%3HV) up to 61.7 wt% of polymer content . Also, further increased 3HV fraction up to 5.5 mol% was achieved by deletion of the prpC and scpC genes.

E. coli XL1-Blue strain was engineered to produce aromatic monomer, phenyllactic acid in E. coli XL1-Blue strain. First, the negative feedback inhibition of key enzyme such as aroF and pheA was released by site directed mutagenesis. Second, tolerance test to know how much concentration of 5-phenyllactic, E. coli can tolerate, tolerance test was conducted using 0, 1, 5, 7 g/L of phenyllactic acid. The cell growth was dramatically inhibited by addition of phenyllactic acid in a dose dependent manner and cell was lysed more than 7 g/L of phenyllactic acid. Based on previous result and report, essential genes for amplification of shikimate and phenyllactic acid production was introduced in the E. coli XL1-Blue strain, resulting in 17 mg/L of phenyllactic acid production in MR medium. This study demonstrates the possibility for the biosynthesis of aliphatic and aromatic PHA from biomass.