Production Of Itaconic Acid From Waste Cooking Oil Through Subcellular Compartmentalization In Yarrowia Lipolytica

Itaconic acid(IA)is an unsaturated dicarboxylic acid,which is widely used in the production of chemical industry,cosmetics,medicine and other industries.Chemical synthesis is generally used in industrial production or microbial Aspergillus terreus of IA,but these methods will have the problems of low utilization of raw materials,difficult separation and purification of products and complex fermentation and culture process,resulting in the risk of bacterial contamination.Therefore,seeking suitable microorganisms to produce IA is a research upsurge in recent years.Yarrowia lipolytica is a kind of unconventional yeast that has been widely studied.It has a variety of unique physiological and metabolic advantages.One of the remarkable advantages is that it can grow on a variety of substrates,especially cheap renewable substrates such as waste cooking oil(WCO).In addition,Y.lipolytica can also secrete a large number of organic acids.The above shows that yeasts are ideal microbial cells for producing IA.In this study,the Y.lipolytica was successfully engineered to produce IA by introducing the heterologous cis-aconitic acid decarboxylase gene(CAD)from A.terreus.Next,by adding the enhanced peroxisome targeting signal(ePTS1)sequence after the CAD gene,the CAD gene was successfully located in the peroxisome of Y.lipolytica,and the compartmental production of IA in the peroxisome was realized.In order to improve IA production by the engineered yeast Y.lipolytica strain,eight genes involved in theβ-oxidation pathway were overexpressed individually and another two genes,lipases and a protein required for peroxisome assembly.Subsequently,the isocitric acid lyase and carnitine acetyltransferase genes in the above strains were knocked out separately,and the results showed that this strategy led to further improvement of IA production.Finally,the high-yield strain was fermented in the bioreactor to detect the ability to synthesize IA.The main research contents are as follows:(1)The CAD gene fragment from A.terreus HAT418 genome was amplified,and the recombinant plasmid pYLEX1-CAD containing CAD gene was constructed with pYLEX1 as vector.And after linearization,it was recombined onto the Y.lipolytica Po1 g ku70Δgenome to obtain the recombinant strain Po1g-CAD.The original strain Po1 g ku70Δ and the recombinant strain Po1g-CAD were analyzed and detected after fermentation culture.The results showed that the recombinant strain Po1g-CAD could successfully heterologous synthesis IA with a yield of 33.13 mg/L,which proved that the genes of A.terreus selected in this paper achieved functional expression in the host of Y.lipolytica.(2)To implement the compartmental localization strategy,we found the sequence ePTS1 that can be localized to the peroxisome of Saccharomyces cerevisiae.To verify whether it works in Y.lipolytica,we chose to use green fluorescent protein(hrGFPO)in conjunction with the Nile red dye.ePTS1 is added after the hrGFPO gene sequence(before terminators),and Nile red is added to the fermentation broth for observation under a laser scanning confocal microscope.The results showed that the green fluorescence overlapped almost exactly with the red fluorescence,indicating that the ePTS1 sequence could successfully localize the gene in the peroxisome in the Y.lipolytica.(3)To explore the ability of Y.lipolytica to convert WCO into IA in the peroxisome compartment.After the successful ePTS1 sequence was added to the CAD gene sequence,the yeast was transformed for fermentation culture,and the fermentation products were analyzed and detected by gas chromatography-mass spectrometry.The results showed that the yield of recombinant strain Po1g-CAD-ePTS1 synthesized IA in the peroxisome was1.58 g/L.(4)In order to increase the yield of IA,10 recombinant strains were constructed by overexpressing 10 functional genes of the IA production pathway of Y.lipolytica.Detection after fermentation culture showed that peroxisome thiolase(POT1)is a rate-limiting enzyme in the β-oxidation pathway to increase IA yield.The corresponding Y.lipolytica engineering strain Po1g-CAD-ePTS1-POT1(Po1g-2G)yielded 2.42 g/L of IA.(5)To further increase the yield of IA,we chose to knock out the relevant gene.The isocitric acid lyase(ICL)and carnitine acetyltransferase(CAT)genes were knocked out on the basis of the above-mentioned high yield engineered strains,and after fermentation culture,it was learned that the ICL knockout strain synthesized IA 3.33 g/L.(6)In order to achieve optimal IA yields,the above-mentioned highest yield engineered strains are cultured in bioreactor.The resulting optimal medium formulation with 59 g/L WCO,16 g/L yeast extract and 8 g/L tryptone was fermented in a 5 L bioreactor at 30 ℃,1 vvm without pH control,and the final itaconic acid yield was 54.55 g/L.In conclusion,the ability of heterologous synthesis of IA from Y.lipolytica was studied in this study by using the metabolic engineering and subcellular compartmentalization strategies and the conversion of WCO into IA was also successfully demonstrated in the engineered Y.lipolytica strains.The success of this study also provides reference value for the future transformation of Y.lipolytica to use WCO to synthesize more valuable industrial products.