Regulation Of Glyoxylate Pathway To Strengthen The Biosynthesis Of Polymalic Acid In Aureobasidium Pullulans

Aureobasidium pullulans is a yeast-like fungus that grew widely in plants,ocean and glacier,which could produce polymalic acid(PMA),pullulans,enzyme preparation,antibacterial peptides and other metabolites.PMA is a novel polyester used in drug carrier,biomedicine and chemical industry due to its good advantages of water solubility,biodegradability,biocompatibility and modification.In this study,A.pullulans CCTCC M2012223 was used as the original strain,combined with metabolomics,environmental stresses and metabolic engineering to investigate carbon metabolism characteristics and the role of key pathway in the biosynthesis of PMA.It was found that sucrose as a dominant carbon source can promote the synthesis of PMA from glyoxylate cycle.Environmental stress found that ethanol can specifically increase the expression and enzyme activity of malate synthase in the glyoxylate shunt.Further,overexpression of endogenous genes and transcription regulator can strengthen the carbon metabolic flux of glyoxylate cycle,as results that significantly increased the production and yield of PMA.These key research results were described as follows:The effect of different carbon sources on the synthesis of PMA showed that sucrose was the best carbon source for PMA production in A.pullulans,in which PMA production increased by 38.9% compared with that from glucose(20.38 ± 2.32 g/L)in shake flask.Analyzing the intracellular metabolites of sucrose and glucose at different time points,a totally of 81 metabolites were detected.Comparative analysis found that sucrose had 27 different metabolites versus glucose fermentation for 48 h and 30 different metabolites at 72 h.Metabolites,of which 26 different metabolites,including valine,leucine were increased.Under sucrose condition,pyruvate decreased,and citric acid and glycolic acid increased,that indicated the accumulation of intracellular carbon was decreased,and the flux of glyoxylate cycle was strengthened.Thus,it was concluded that the glyoxylate shunt was an important pathway for the biosynthesis of PMA.The stress of different solvents on A.pullulans for the synthesis of PMA were investigated.The results showed that 1%(v/v)ethanol can obviously promote PMA production via increasing the expression level of isocitrate lyase(ICL)and malate synthase(MLS)in glyoxylate pathway,which were up-regulated by 4.3 and 25.6-fold,respectively.Meanwhile,the enzyme activity of MLS was 23-fold higher than that of control.These results suggested that ethanol stress can specifically up-regulate the expression level of mls to promote the synthesis of PMA in A.pullulans.The PMA titer increased by 20.8% reached 30.33 ± 1.40 g/L compared with the control in 5 L stirred-tank frmentor.To further explore the key role of glyoxylate shunt in the biosynthesis of PMA,key genes of glyoxylate shunt,isocitrate lyase and malate synthase were overexpressed and subcellular localized.Isocitrate lyase genes(g1842.t1 and g260.t1),and malate synthase gene(mls)fused with enhanced green fluorescent protein(e GFP)were constructed,respectively.Laser confocal microscopy observed that ICL2 and MLS protein were located in peroxisome,and ICL1 widespread in the cytoplasm.Then,in 5 L stirred-tank fermentor,PMA production of mls overexpressed strain reached 35.84 ± 0.94 g/L,and PMA titer was further increased by 33.3% under ethanol stress and reached 39.96 ± 0.38 g/L compared with wild type(29.98 ± 0.74 g/L).As for overexpression of icl1,which has a higher PMA titer of 47.58 ± 0.54 g/L,productivity of 0.57 g/L·h,yield of 0.55 g/g under ethanol stress.In addition,under ethanol stress,PMA production with sucrose as carbon source was increased by 63.9% and reached 49.13 ± 1.57 g/L.Subsequently,the overexpression strain of Cat8,a global transcription regulator of icl and mls,was constructed,in which the expression level of cat8 was 9.86-fold,and icl2(g1842.t1)and mls were 2.66 and 2.33-fold compared with wild-type,respectively.It was indicated that the Cat8 regulator can strengthen the glyoxylate shunt in peroxisome to promote PMA production.In a 5 L fermentor,PMA production arrived 53.05 ± 1.88 g/L and increased by 77.0 % under ethanol stress with a correspondingly productivity of 0.63 g/L·h and yield of 0.63 g/g.In conclusion,glyoxylate shunt was regarded as the poteinal key pathway for PMA biosynthesis in A.pullulans through metabolomics analysis.Under the ethanol stress,the activity of malate synthase in glyoxylate shunt was specifically enhanced,and indicated that the glyoxylate pathway played an important role in the biosynthesis of PMA.Endogenous genes and transcription factor were further engineered to regulate the glyoxylate pathway and achieved the highest yield of PMA.This study integrated substrate regulation,environmental stress,endogenous genetic strengthen and global transcription factor to reveal the metabolic regulation of PMA biosynthesis,which will provide new methods guiding metabolic engineering for PMA production.