A Preliminary Study On The Adaptive Evolution Of Aureobasidium Pullulans And The Regulation Of Calcineurin Signal On Its Tolerance

Aureobasidium pullulans（A.pullulans）is a kind of yeast-like fungus,and it is a potential chassis cell factory,which has a wide range of substrate utilization and strong environmental adaptability.Poly（2-hdyrobutanedioic acid）（P2HBD）,also known as polymalic acid,is a new type of water-soluble aliphatic polyester compound produced by its metabolism.Currently,P2HBD biosynthesis mainly has the following problems:the high fermentation cost of starch carbohydrate raw materials;the fermentation process requires the addition of calcium carbonate（Ca CO₃）as a neutralizer to control the fermentation p H,resulting in a large amount of calcium slag in downstream extraction,causing environmental pollution.Xylose mother liquor is the mother liquor of xylose crystallization in the industrial process of producing xylitol from corncob raw materials.It is a cheap and unused biological processing waste.It mainly contains three monosaccharides:xylose,arabinose and glucose,which can be used as sustainable biomass resources.The previous research of the research group found that A.pullulans can directly utilize all the carbohydrate components in the mother liquor.This subject uses the xylose mother liquor as the raw material and the laboratory-preserved A.pullulans g135 as the starting strain to carry out research on the evolution of high-sugar substrates,low-p H fermentation adaptability and stress resistance engineering,and explore the establishment of a high-concentration xylose mother liquor substrate and a direct fermentation system without p H control（Free-controlled p H）to realize the efficient production of a new generation of P2HBD.The main research conclusions of this paper are as follows:1.Use A.pullulans g135 as the starting strain to perform atmospheric and room temperature plasma mutagenesis（ARTP）,and detect the growth of mutant strains and P2HBD production by bromocresol green screening medium.The experimental results showed that the best mutation time of A.pullulans ARTP was 130 s.Under this mutation time,the positive mutation rate of A.pullulans was higher.After two rounds of ARTP mutagenesis,a mutant strain YB-9 was screened.When the concentration of xylose mother liquor was 120 g/L,the growth of the mutant YB-9 is significantly faster than that of the starting strain.2.Use xylose mother liquor as a substrate to carry out adaptive laboratory evolution（ALE）for strain YB-9 tolerant to high-concentration xylose mother liquor.By gradually increasing the concentration of xylose mother liquor,after about 200consecutive passages,the evolutionary strain JH1-3 that can grow in 300 g/L xylose mother liquor is finally obtained.The fermentation results showed that with the concentration of xylose mother liquor of 210 g/L,the P2HBD yield of the evolved strain JH1-3 reached 59.94 g/L,and the sugar-acid conversion rate was 0.28 g/g,which was10.31%and 3.77%higher than that of the starting strain,respectively.The starting strain g135 and evolutionary strain JH1-3 were tested on a 5 L fermentor.When the mother liquor of xylose was 300 g/L,the fermentation results showed that the yield of P2HBD of the evolutionary strain JH1-3 was 53.34 g/L,compared with the control,the yield of the starting strain g135 P2HBD increased by 13.88%.3.The evolutionary strain JH1-3 was subjected to high-throughput（Microbial microdroplet culture system,MMC）acid resistance evolution.After two rounds of MMC acid resistance evolution experiments（about 800 h）,the strain JH3-5 with obvious growth advantage was screened;under the condition of not controlling the p H value,the yield of P2HBD obtained by fermentation of this strain was 12.21 g/L,compared with the control,the yield of strain JH1-3 increased by 15.73%.4.Through the overexpression of exogenous calcineurin signal regulation,the A.pullulans was engineered to resist stress,and the calcineurin A subunit gene（cna）,B subunit gene（cnb）and the AB subunit gene（cnab）homologous to cna of Beauveria bassiana were expressed in the acid-tolerant evolutionary strain JH3-5.Comparative tests found that overexpression of the cnb gene can effectively improve the stress resistance of A.pullulans.Under the conditions of the concentration of the xylose mother liquor at 300 g/L and the p H value of 2.6,after 26 hours of MMC culture,the OD₆₀₀ of the overexpressing cnb strain reached 2.14,which was much higher than that of other strains.The fermentation results of the 5 L fermentor showed that the P2HBD yield of the acid-tolerant evolutionary strain（b-13）overexpressing cnb reached 32.63g/L,which was an increase of 31.26%compared with the starting strain g135;The results of this paper show that the strategy of combining adaptive evolution and stress-resistant engineering can effectively improve the P2HBD fermentation performance of A.pullulans strains that use xylose mother liquor as non-grain raw materials and do not control the p H for P2HBD fermentation.And these results lay the foundation for exploring and establishing a new generation of organic acid polyester polymer fermentation system.