Isolation And Heterologous Expression Of One Key Enzyme For Carbon Fixation From Filamentous Photosynthetic Bacterium Chloroflexus Aurantiacus

A novel carbon dioxide fixation pathway,3-hydroxypropionate cycle, has been discovered in thermophilic filamentous photosynthetic bacteria such as Chloroflexus aurantiacus, a kind of unique bacteria that are distantly related to all other five groups of photosynthetic prokaryotes. The distinct feature of this pathway is3-hydroxypropionate as metabolic intermediate and be secreted into the growth media.3-hygroxypropinate can be used for synthesis many valuable products such as biodegradable polyester PHA in industry. However, either traditional chemical synthesis processes or other bioengineering methods are limited by low efficiency and high cost of starting material and pollution to environment. The purpose of this project is investigation of its reaction mechanism, especially the involvement of some multifunction key enzymes.In this study, we obtained the following results:(1) The optimal growth conditions for3-hydroxypropionate pathway utility are searched. Addition of sodium propionate (0.5g/L) into the growth media reduce the generation time of CFX, thus shorten the mature time from normal14days to less than10days.(2) By using several methods of protein purification, we isolated three samples: the whole protein, soluble protein, and membrane protein from the harvested cell. Based on the knowledge of the binding of biotin to our target protein, after SDS-PAGE and Western blotting analysis, we found one protein in soluble part is about20kDa and probably is one subunit of propionyl-CoA carboxylase.(3) Based on the genome of CFX, the gene coding the subunit of propionyl-CoA carboxylase (PCC-BCCP) is amplified by PCR from genomic DNA, and cloned into Bio-Rad’s vector pPAL-7, transformed into E. coli. The fusion protein is overexpressed after IPTG induction, which shows30kDa (including the8kDa tag) in SDS-PAGE and active in the Western blotting analysis.