Biosynthesis Of Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) From Escherichia Coli Using Glucose As Sole Carbon Source

Polyhydroxyalkanoates (PHAs) are polyesters synthesized by a variety of bacterial strains as intracellular carbon and energy storage compounds grown usually under stress conditions. They are biodegradable, thermoplastic, biocompatible, optically active, piezoelectric polyesters for the application as renewable plastics alternative to petrochemical plastics that received considerable attention. Particular physical and material properties of PHAs enable them for applications in various areas, including industry, agriculture, food industry, chemical engineering, medicine and pharmacy.Poly (3-hydroxybutyrate) [P(3HB)], as the most common type of PHAs, was studied in detail with respective to its production, property and application. However, due to its brittleness and thermal instability, the application of PHB is limited for the replacement of normal plastics. Copolymers consisting various contents of 3-hydroxybutyrate and 4-hydroxybutyrate, namely, P(3HB-co-4HB), attracted many attentions due to the flexible properties ranging from brittle to elastic materials. P(3HB-co-4HB) can be hydrolyzed by both PHA depolymerases and lipases, and its degradation rate is relatively rapid compared with other PHAs.The formation of P(3HB-co-4HB) was first reported by Doi et al in1988. They found that Alcaligenes eutrophus was able to accumulate P(3HB-co-4HB) copolymer when suitable substrate precursor such as 4-hydroxybutyric,1,4-butanediol or y-butyrolactone was added in the medium. However,4HB and related compounds, which were used as the precursor for P(3HB-co-4HB) production, are generally costly and difficult to obtain. This results in the high cost of PHAs containing 4HB as a monomer.Valentin and Dennis introduced genes sucD,4hbD, cat2 together with genes phbCAB from Ralstonia eutropha into E.coli strains, which was confirmed to accumulate P(3HB-co-4HB) with very low 4HB fraction. Inactivation of sad and gabD gene may improve the 4HB content in the copolymer up to 11% when cultivated using glucose as carbon source. Because of both sucD and 4hbD are oxygen-sensitive enzyme, the yield of 4HB was not high. In our study, the succinate degradation pathway in Clostridium kluyveri was also cloned in E.coli. Also gene cat1 was introduced to make the pathway proceed reversal TCA circle. Fermentation result of the recombinant E.coli was under anaerobic condition.P(3HB-co-4HB) production under anaerobic condition was studied in recombinant E.coli WCCS4, which contains mutations in pyruvate formate lyase (pflB), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzymeâ…¡(ptsG). Introduction genes cat1, sucD,4hbD, cat2 together with genes phbCAB can accumulate P(3HB-co-4HB) with above 70% 4HB fraction.In this study, we firstly engineered the anaerobic pathway to synthesis P (3HB-co-4HB) from the sole carbon source glucose by the engineered E. coli WCCS4. The results obtained here proved a theoretical pathway under anaerobic conditions, and also explored new ways to production 4HB in aerobic condition.