Synthesis Of Acetoin?2,3-butanediol And Glycolate From Acetate As Carbon Source

Acetate is a natural organic acid that can be used as a low-cost industrial raw material.Acetoin,2,3-butanediol and glycolate are important chemical products and can be used as medicines,organic synthesis intermediates and biofuels.Using acetate as a low-cost carbon source for microbial fermentation can reduce the consumption of biomass resources such as glucose in the fermentation industry.In this thesis,the synthesis of acetoin,2,3-butanediol and glycolate by microorganisms using acetate as carbon source was studied.First,a newly identified marine microorganism Neptunomonas concharum was cultured and found to be able to effectively use acetate and succinate as carbon sources for growth,while glucose and xylose could not be consumed.Next,the transformation conditions of N.concharum were studied,and the specific conditions of electrotransformation were optimized.Under the optimal conditions,the transformation efficiency reached 3×103 cfu/?g DNA.On this basis,the acetolactate synthase gene alsS,the acetolactate decarboxylase gene alsD of Bacillus subtilis and the butyltransferase gene budC of Klebsiella pneumoniae were expressed in N.concharum,and acetate was constructed as a carbon source.The metabolic pathway for the synthesis of acetoin and 2,3-butanediol.In the TYS medium containing acetate,the yield of acetoin and 2,3-butanediol was 0.11 g/L after 48 h fermentation.Considering that acetate is a carbon source,the intracellular pyruvate precursor may be insufficiently supplied.Overexpressing the malate dehydrogenase gene maeA from Escherichia coli enhances the synthesis of malic acid to pyruvate,which increases the yield of acetoin to 0.56 g/L,2,3-butanediol was increased to 0.39 g/L.In addition,this thesis studies the synthesis of glycolate from Escherichia coli using acetate as a carbon source.In the E.coli K12,the xylulose kinase gene xylB,the hydroxymalonic acid semialdehyde synthase gene gcl,the malate synthase A gene aceB,the malate synthase G gene glcB,and the glycolate oxidase gene glcD were sequentially knocked out.At the same time,the isocitrate lyase gene aceA,isocitrate dehydrogenase kinase/phosphatase gene aceK,glyoxylate/hydroxypyruvate reductase gene ycdW were over-expressed,and the metabolic pathway for synthesizing glycolic acid with acetate as carbon source was constructed.In the shake flask culture with acetate as the carbon source,the yield of glycolate reached 1.78 g/L after 48 h fermentation.On this basis,the overexpression of the citrate synthase gene gltA enhances the tricarboxylic acid cycle/glyoxylate cycle,overexpresses the phosphotransacetylase/acetate kinase gene pta-ackA to enhance acetic acid assimilation,and the yield of glycolic acid is increased to 2.38 g/L.Finally,by adjusting the pH of the medium,the yield of glycolate reached 2.75 g/L.