Optimization And Chromosomal Expression Of Polyhydroxyalkanoates Related Genes In Escherichia Coli

Polyhydroxyalkanoates (PHAs) are microbial polyesters with variable mechanicalproperties and great potential for industrial applications. Current researches onmetabolic engineered Escherichia coli for PHA production mainly rely on unstableplasmid-based systems, and the yield of short-chain-length and medium-chain-lengthcopolymers SCL-MCL PHA in E. coli is relatively low. This study focused on theoptimization of PhaC2Ps, a PHA synthase with wide substrate specificities, to promoteSCL-MCL PHA production in E. coli, and on the stabilized PHA production throughchromosomal expression of PHA-related genes.The PHA synthase PhaC2Psfrom Pseudomonas stutzeri1317along with itsmutants were used for PHA production in E. coli. Codon optimization of the doublemutant PhaC2PsQKST enhanced its protein expression level, and the introduction of ahairpin structure in the5’ untranslated region led to stabilized transcription. Both PHBand SCL-MCL PHA productions were enhanced, along with higher CDW. Compared tothe double mutant, the PHB and SCL-MCL PHA contents using optimized synthasewere increased by2.6-fold and2.2-fold, reaching29.7wt%and3.93wt%, respectively.Investigation on13chromosomal integration sites indicated that the expressionlevels of PHA-related genes were higher when integrated into potential active sites oftranscription, such as yheO, gltA and asnB. The copy number of the genes also affectedtheir expression levels. No PHB accumulation was observed when one copy of phbCABor phaC2AB was integrated, while the presence of11copies of phbCAB or12copies ofphaC2AB led to PHB production of5.18wt%and3.01wt%, respectively.Using Cre-loxP recombination system, a recombinant E. coli strain with about50copies of phbCAB on its chromosome was constructed in asnB site, which benefitedgene expression with little influence on cell growth. The strain could accumulate PHBup to34.1wt%, equivalent to a moderate-copy-number plasmid system. The strain wasproven to be more stable than plasmid-based system in PHB production, indicating thatthis method can be used to engineer E. coli for industrial PHA production.