Construction Of Genetically Engineered Strain Of Yarrowia Lipolytica Producing Itaconic Acid

Itaconic acid is a platform chemical with broad application prospects,and an important chemical raw material in the fields of polymer materials,coatings,and drug synthesis.At present,the production of itaconic acid is dominated by the fermentation of Aspergillus terreus.However,Aspergillus terreus as a fermentation production strain has many shortcomings,such as high nutritional requirements and long fermentation cycles,which increase the cost of itaconic acid fermentation production.The development of genetic engineering and metabolic engineering has laid the foundation for the construction of genetically engineered bacteria to solve the above-mentioned defects.The selection of chassis strains has a greater impact on the construction of genetically engineered bacteria.Yarrowia lipolytica has the innate ability to tolerate low pH,fast growth,strong substrate adaptability,and strong synthesis of citric acid in the metabolic process.It provides a good foundation for the construction of itaconic acid synthesis pathway,and is an itaconic acid production chassis strain with great development potential.In this study,Yarrowia lipolytica was used as the chassis strain,and by expressing the Aspergillus terreus cis-aconitic acid decarboxylase gene CADA,the metabolic pathway of itaconic acid was constructed in Yarrowia lipolytica.Glucose was used as the substrate,the production of itaconic acid reached 50 mg/L.In response to the problem of low itaconic acid production,the aconitase gene A CO and the mitochondrial carboxylic acid transporter gene MTT were further expressed to strengthen the transmembrane supply of cis-aconitic acid.The production of itaconic acid reached 330 mg/L,an increase of about 6.7 times.In order to avoid feedback inhibition of citric acid in the glycolysis process,acetic acid was used to replace glucose as the carbon source,and the acetyl-CoA synthase gene ACS,the citrate synthase gene CAS,and the mitochondrial carboxylic acid transporter gene MTT were overexpressed.The production of itaconic acid precursor-citric acid in the engineering bacteria reached 11.77 g/L,realizing the understanding of Yarrowia lipolytica using acetic acid metabolism to produce citric acid.On this basis,combined with the work described above,through the introduction of the cis-aconitic acid decarboxylase gene CADA,the final itaconic acid production reached 1.2 g/L,which was 2.6 times higher than that of the engineered bacteria using glucose as the carbon source.In this study,by constructing acetic acid-utilizing strains,it was proved that Yarrowia lipolytica has the great potential of using acetic acid to produce itaconic acid,which laid the foundation for the subsequent construction of itaconic acid genetically engineered bacteria.