Metabolic Engineering Of Pichia Pastoris To Enhance The Biological Utilization Of Acetic Acid

With the rapid development of biotechnology,great concern and application are aroused on microorganism to produce fine and bulk pharmaceutical chemical products.Glucose and glycerol are common carbon sources used in yeast culture,and however,uncommon carbon sources such as methanol,ethanol and acetic acid are increasingly employed.Acetic acid is a kind of extensive carbon source which is economical and environmentally friendly.And it receives wide attention as the precursor of acetyl-CoA,in the synthesis of amounts of fine and bulk pharmaceutical chemical products.In our previous work,P.pastoris has been defined as an excellent host in biosynthesis of pharmaceutical and chemical products of small molecules.In this study,we try to enhance the utilization of acetic acid and evaluate its potential as the carbon source and precursor by metabolic engineering on tolerance,transport and metabolism of acetic acid.At first,the dissociation constant?pKa?of acetic acid is 4.76 and acetic acid shows strong antimicrobial action at low-pH,including a series of signal transduction related to kinase.Therefore,it is the inevitable problem to utilize acetic acid as carbon source.In this study,we screened acetic acid tolerance related genes from the P.pastoris kinase deletion library constructed in our previous study.Then,the ability of tolerance to acetic acid in yeast was enhanced through deletion or overexpression of these genes.The deletion strain ?PAS_chr3₁091?hrkl?showed high sensitivity to acetic acid,thus this gene showed positive effect on acetic acid tolerance.Then we identified the function of Hrkl in enhancing the utilization of acetic acid,with 6-methylsalicylic acid?6-MSA?,which uses acetyl-CoA as precursor.The productivity of the strain overexpressed hrkl was up to 80 mg/g cell,1.55 times of the control.Thus overexpression of Hrk1 improved the biosynthesis of 6-methylsalicylic acid and the utilization of acetic acid.There are two forms of acetic acid in solution,undissociated acetic acid and acetate anion,and they are transported by different plasma membrane proteins,respectively.Since the optimal environment of yeast is acidic,it is of importance to reinforce the transport ability of undissociated acetic acid in yeast.The aquaglyceroporin ScFps1 in S.cerevisiae was overexpressed in P.pastoris to analyze the utilization of acetic acid and the production of 6-MSA.However,neither the utilization of acetic acid or the production of 6-MSA were improved,and the transport of acetic acid might be adequate in P.pastoris in our experimental conditions.The accumulation of acetic acid in cytoplasm usually causes the programmed cell death,so it is crucial to improve the effective convertion of acetic acid.In microorganism,acetic acid is converted to acetyl-CoA by acetyl-CoA synthetase?Acs?or acetate kinase?AckA?/phosphotrans acetylase?Pta?.These acetic acid metabolism related genes were overexpressed and 6-MSA was used as the precursor.The results showed that acetyl-CoA synthetase PpAcs1 from P.pastoris and ScAcs1 from S.cerevisiae played a great role in improving the utilization of acetic acid,and their 6-MSA productivity reached 47.0 mg/g cell and 43.7 mg/g cell,1.39 times and 1.30 times of the control,respectively.However,the AckA/Pta had no remarkable effect.Finally,when both the acetic acid tolerance and metabolism related genes were overexpressed,the utiliazation of acetic acid improved to a high degree.This study effectively improved the biological utilization of acetic acid in yeast through metabolic engineering of acetic acid tolerance,transport and metabolism,providing great foundation to the production of small molecule pharmaceuticals and chemicals with acetic acid and acetyl-CoA as precursors.However,considering the natural toxicity of acetic acid to Pichia pastoris,efficient growth and product synthesis with acetic acid as precursors in P.pastoris still need further research.