The β-Poly(L-Malic Acid)Fermentation Optimization Of Aureobasidium Melanogenum And The Study Of Its Biosynthesis Mechanism

β-Poly（malate）（PMLA）is a water-soluble polyester formed by the polymerization of L-malic acid,which possessed good applications in the biomedical industry,drug carriers,and biomaterials.In this paper,the production of PMLA by Aureobasidium melanogenum was taken as the research object.Firstly,the fermentation process of PMLA was optimized through correlation analysis,and the PMLA production was predicted by machine learning algorithms by collecting fermentation samples,and then the key enzymes affecting the biosynthesis of PMLA were studied through transcriptomics and genomics.Finally,the transformation method suitable for A.melanogenum was established,and the key genes were knocked out to investigate the effect of different genes on the biosynthesis of PMLA in A.melanogenum,the specific research contents were as follows:（1）The effects of different substrates on PMLA production were investigated by adding corn steep liquor（CSL）,magnesium sulfate,manganese sulfate,vitamin B₁,vitamin B₂,and nicotinamide at different concentrations to the fermentation medium.The PMLA production and four secondary indexes,p H,biomass,viscosity,and osmotic pressure,were measured to investigate the relationship between different substrate additions,PMLA production,and four secondary indexes.By optimizing the fermentation technic of CSL addition,the PMLA production increased by 40.2%.Then,different machine learning algorithms were used to build a model to predict PMLA production.The predicted results showed that the BP neural network obtain a better prediction on PMLA production with secondary indexes,and the Mean absolute error（MAE）of the test set is 4.556 g/L.This result indicated that the production of PMLA can be detected in real-time by forecasting the production of PMLA through secondary indexes to improve the fermentation efficiency.At the same time,the pictures of the fermentation medium after 144 h fermentation were collected for the convolution neural network modeling and prediction.The results showed that the model could accurately predict the PMLA production of fermentation medium pictures and the prediction accuracy of the test set was about 90%.（2）Through the transcriptomics sequencing of the fermentation medium of high PMLA and the low PMLA production group,8 treatments,a total of 24 transcriptome samples were collected.Then,the transcriptome was assembled,annotated,and analyzed for differentially expressed genes（DEGs）.Finally,10 genes were selected that may relate to the PMLA biosynthesis by clustering analysis.These genes were located in mitochondria,glyoxysome,and cytoplasm through bioinformatics analysis.Among them,an NRPS（Non-ribosomal peptide synthetase）was found to encode an NRPS-like protein,which may be the key protein for polymerizing L-malic acid to form PMLA.The protein may locate in the cytoplasm.Therefore,the cytosolic L-malic acid should be polymerized to form the PMLA.At the same time,it was found that some genes related to the TCA cycle,glyoxylate,and gluconeogenesis pathway were up-regulated in the high-production group.（3）The second and third-generation sequencing technologies were employed to sequence the genome of A.melanogenum,which was then assembled to obtain the assembly results.The genome was also structurally annotated with transcriptome data,which was then annotated with the different functional databases.The structure of PCKA（Phosphoenolpyruvate carboxykinase（ATP））and MASY（Malate synthetase）related to the synthesis of PMLA were predicted and compared with the existing crystal results on the Protein data bank（PDB）.It was found that the crystal structure of these two proteins in A.melanogenum was consistent with that of PDB,and the functions of these two proteins may relate to malate metabolism.（4）The CRISPR and homologous recombination gene editing methods suitable for A.melanogenum were established,Then,we select the CRISPR method to knock out PCKA,MASY,ACOT9,and NRPS genes.In the transcriptomics analysis,the expression of PCKA was increased by about 5 times in the high-production group compared with the low-production group,but after the knock-out of PCKA,we found that the PMLA production has not been affected,which indicated that the biosynthesis of PMLA was not related to the gluconeogenesis.After the deletion of MDHC,MASY ACOT9,it was found that the production and yield of PMLA decreased,while after the deletion of the NRPS gene,the strain did not produce any of PMLA.Therefore,we concluded that PMLA is polymerized by NRPS in A.melanogenum,which polymerized the cytosolic L-malic acid to form PMLA,of which 30%of L-malic acid comes from the glyoxylate pathway,20%of L-malic comes from the pyruvate carboxylase pathway,and 50%comes from the TCA cycle.Then,we added potassium acetate to the fermentation medium,which may improve the glyoxylate pathway.The fermentation result showed that adding 4 g/L potassium acetate in the fermentation medium can increase the PMLA production by about 17%compared with adding 3 g/L CSL.At the same time,the enzyme activity of MASY was increased in the fermentation medium by adding CSL solely and adding CSL and potassium acetate simultaneously.We finally found that the PMLA production could reach 77.8 g/L by adding CSL and potassium acetate in a 5-L fermentor that employed the batch-fermentation method,which increased by 59.72%compared to the orginal control group of 48.71g/L.