Metabolic Engineering Of Escherichia Coli For Poly(3-hydroxybutyrate)

As the best characterized member of the PHA, poly(3-hydroxybutyrate)(PHB) is a polyester of 3-hydroxybutyrate. As biodegradable and biocompatible materials with diverse properties, PHB has drawn industrial attentions such as pharmaceuticals industry for their potential applications in many fields. A series of engineered E.coli JM109 mutants were constructed that could efficiently produce PHB in this study. The respiratory chains of E.coli were firstly analyzed. Then we solved the problem of simultaneous fermentation problem between the glucose and xylose by heterologous expression of xylose isomerase gene(xylA), xylulokinase gene(xylB) and xylose transporter gene(araE) from Bacillus subtilis. Meanwhile, the co-expression of the xylanase system and the PHB system in E. coli resulted in the production of xylan-derived PHB. The main results were shown as follows:Firstly, a comparative study was carried out with E.coli JM109 that was modified in its aerobic respiratory chain. Seven mutants were constructed here. LJ02(?appB), LJ03(?ndh), and LJ23(?appB?ndh)accumulated up to 4.53 g/L, 6.16 g/L, and 4.61 g/L of PHB with 20 g/L glucose. Under the same condition, LJ02, LJ03, and LJ23 produced 2.78 g/L, 3.50 g/L, and 1.82 g/L of PHB 20 g/L with 20 g/L xylose. Among them, LJ03 exhibited 0.32 g(g glucose)-1 which reached 66.67% of the maximum theoretical yield. The PHB production of this strain reached 28.23 g/L in a 5-L fermentor study with a yield of 0.25 g(g glucose)-1. These results indicated that inactivating thecytochrome bd-II oxidase or/and NDH-II dehydrogenase of the aerobic respiratory chain is a simple and effective strategy to improve PHB biosynthesis in E. coli.Further, we solved the problem of simultaneous fermentation problem between the glucose and xylose by heterologous expression of xylose isomerase gene(xylA), xylulokinase gene(xylB) and xylose transporter gene(araE) from Bacillus subtilis. LJ02, LJ03, and LJ23 produced 3.87 g/L, 4.10 g/L and 4.15 g/L PHB with 10 g/L glucose and 5 g/L xylose.At last, the co-expression of the xylanase system and the PHB system in E. coli resulted in the production of xylan-derived PHB. LJ02, LJ03, and LJ23 produced 2.08 g/L, 2.02, and 2.29g/L PHB with 5 g/L xylan and 10 g/L xylose. The result showed that overexpression of talA could improve the production of LJ02 and LJ23 using 20 g/L xylose as the carbon source. However, overexpression of talA decreased PHB accumulation of LJ03 unexpectedly. Further overexpression of the pentose phosphate pathway decreased the production of xylan-derived PHB, suggesting the metabolic burden was generated in engineeried strains by overexpression of the talA gene.The results showed that engineered strain E.coli has potential applications for PHB production.