Enhancing microbial production of biofuels by expanding microbial metabolic pathways.

Fatty acid, isoprenoid, and alcohol pathways have been successfully engineered to produce biofuels. By introducing three genes, atfA, adhE, and pdc, into Escherichia coli to expand fatty acid pathway, up to 1.28 g/L of fatty acid ethyl esters can be achieved. The isoprenoid pathway can be expanded to produce bisabolene with a high titer of 900 mg/L in Saccharomyces cerevisiae. Short- and long-chain alcohols can also be effectively biosynthesized by extending the carbon chain of ketoacids with an engineered "+1" alcohol pathway. Thus, it can be concluded that expanding microbial metabolic pathways has enormous potential for enhancing microbial production of biofuels for future industrial applications. However, some major challenges for microbial production of biofuels should be overcome to compete with traditional fossil fuels: lowering production costs, reducing the time required to construct genetic elements and to increase their predictability and reliability, and creating reusable parts with useful and predictable behavior. To address these challenges, several aspects should be further considered in future: mining and transformation of genetic elements related to metabolic pathways, assembling biofuel elements and coordinating their functions, enhancing the tolerance of host cells to biofuels, and creating modular subpathways that can be easily interconnected.

Combinatorial biosynthesis of Synechocystis PCC6803 phycocyanin holo-α-subunit (CpcA) in Escherichia coli and its activities.

In order to investigate the feasibility for the biosynthetic pathway of CpcA conjugated protein to be reconstituted in Escherichia coli and its antioxidant ability and protective effect on the growth of E. coli, the minimal biosynthetic pathway in cyanobacteria leading from heme to the formation of the cysteinyl residue of phycocyanobilin with deprosthetic CpcA was reconstituted in E. coli using a relatively simple and effective method. When the constructed plasmid pETDuet-6 bearing five genes involved in the biosynthesis of CpcA was transformed into E. coli, the screened transformant acquired a pronounced blue color. Visualization of proteins on SDS-PAGE gel showed a 29 kDa distinct band, corresponding to the theoretically calculated molecular weight of CpcA. Upon exposure to Zn(2+) and UV illumination, the CpcA band was fluorescent. Western blot analysis using His-tag monoclonal antibody confirmed the expression of CpcA in the recombinant E. coli. After the optimization of critical medium components by response surface methodology, the recombinant cells produced 22.29 mg/l of CpcA. The recombinant CpcA displayed a strong ability to scavenge three free radicals ·OH, ·DPPH, and O2 (-) to protect against the oxidation of linoleic acid and to restore the growth of E. coli cells injured by DPPH and H2O2 at a relatively low concentration. These results lay a good foundation for the production and future use of CpcA.