Construction Of Pseudomonas Putida-Escherichia Coli Artificial Consortium To Synthesize Mcl-PHA From Glucose And Xylose

Polyhydroxyalkanoate(PHA)is a substance that some microorganisms under restricted conditions store energy in cells.The physical properties of PHA are similar to traditional petroleum-based plastics.In order to reduce production costs,established the P.putida KT2440-E.coli MG1655 artificial consortium uses xylose and glucose to produce mcl-PHA,which solves the problem of high substrate cost.Two strains were genetically engineered respectively.On the basis of the previously modified E.coli,knocked out the fad D gene improved the secretion ability of long-chain fatty acids;heterologous expression the acyl carrier protein thiolipase gene from castor that promotes fatty acid production,which improved the fatty acid production of the bacteria.After fermentation optimization,using 20g/L xylose the engineered strain E.coli produces 6.44 g/L of acetic acid and 2.51 g/L of FFA.Compared with the previous research,the output of acetic acid has increased by 0.94g/L,and the output of FFA is 2.7 times that of the original research.Optimized P.putida fatty acid β-oxidation pathway,including knocking out the fad A fad B genes,overexpression of the pha J gene that promotes the production of PHA precursors,improving the utilization of fatty acid substances of the strain.Overexpression of the acs gene in the acetic acid utilization pathway improves the acetic acid utilization capacity of the bacteria.The engineered P.putida uses glucose,acetic acid and caprylic acid can produce 0.75 g/L mcl-PHA,and the yield is about twice that of the wild strain.Co-culture engineering E.coli and P.putida with mixed sugars,two bacteria prefer different carbon sources,which avoids substrate competition between the two bacteria.E.coli uses xylose to produce acetic acid and FFA.P.putida consumes glucose,acetic acid and FFA to accumulate mcl-PHA,which solves the problem of high acetic acid concentration inhibits the growth of E.coli.The mutually beneficial symbiosis relationship between "nutrition supply" and "detoxification" has been established.After fermentation optimization,the artificial consortium uses 20 g/L mixed sugar can produce mcl-PHA 1.32 g/L,which is compared with the previously constructed artificial consortium improved 2 times.