Polyhydroxyalkanoate synthesis in recombinant and environmental microbes.

摘要

Polyhydroxyalkanoates (PHAs) represent a class of natural polyesters that are biodegradable and made from renewable resources. These biopolymers offer a sustainable alternative to petrochemical-derived plastics. New expression systems and metabolic engineering strategies have been developed, and novel biosynthetic genes have been discovered to facilitate the use of recombinant microbes for PHA bioplastics production.; The main focus has been on the copolymer poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV). It has commercially desirable physical and mechanical properties that are determined by its 3-hydroxyvalerate (HV) content.; PHBV synthesis was reconstituted in recombinant E. coli, and the effects of varying factors such as minimal media, carbon source, vector backbone, and PHA synthesis operon source were surveyed. Results indicated that all these variables were important in determining copolymer content and composition. Furthermore, the discovery that expression of the PHA synthesis operon from Acinetobacter sp. RA3849 led to a higher HV fraction in the copolymer than the expression of the Ralstonia eutropha operon provided a tool for the metabolic engineering of PHBV composition.; To control copolymer composition, a strain of Salmonella enterica serovar Typhimurium mutant in the ability to convert propionate to propionyl-CoA (the key precursor in the synthesis of HV) was metabolically engineered with the pathways for propionyl-CoA and polymer synthesis. A system has been developed in which the inducible expression of the propionyl-CoA synthetase gene (prpE) is varied while the Acinetobacter PHA synthesis operon (phaBCA) is coexpressed under the control of an independent promoter at a fixed substrate (glycerol and propionate) concentration. Using this expression system, copolymer composition can be controlled between 2 and 25 mol % HV by varying the IPTG level in the medium. This may have application in making biodegradable plastics with specific properties for processes where the propionate composition of the feed steam is not adjustable.; Finally, PCR-based molecular techniques were used to identify unique PHA synthase genes in the environmental microorganisms in a lab-scale activated sludge reactor performing enhanced biological phosphorus removal. Nine novel PHA synthase fragments were discovered. This may eventually lead to the metabolic engineering of new PHA copolymers with interesting properties for sustainable plastics production in recombinant microbes.