Metabolic Engineering Of Saccharomyces Cerevisiae To Synthesize β-glucan Efficiently

β-glucan is an important component of yeast cell wall and has various biological activities,such as antioxidant and anti-radiation.It is widely used in biomedicine,functional food and skincare products.This study reconstructed the synthesis pathway ofβ-glucan in Saccharomyces cerevisiae and further expanded the available carbon sources through metabolic engineering to improve the solubility ofβ-glucan.We ultimately realizing the green and sustainable production ofβ-glucan.The main findings of the study are as follows.（1）The key pathways ofβ-glucan synthesis were regulated by using the"push-pull-inhibit"strategy commonly used in metabolic engineering to achieve efficient synthesis ofβ-glucan.Firstly,the genes encodingβ-glucan synthase and its regulatory subunit were overexpressed in the starting strain of Saccharomyces cerevisiae CEN.PK2-1C,which"pulled"the flow of the key precursor UDP-glucose to theβ-glucan synthesis pathway,and the yield ofβ-glucan reached 38.16 mg·g^-1.Then,the metabolic flux was"pushed"towards the UDP-glucose synthesis pathway by increasing the copy number of hxk1,pgm2 and ugp1genes to enhance the precursor UDP-glucose synthesis pathway,increasing the yield to 43.17mg·g^-1;after that,the competing pathways related toβ-glucan synthesis were further knocked out.And the key gene alg5 of the glycosylation pathway was determined to be more conducive toβ-glucan synthesis and increased its yield to 49.07 mg·g^-1.（2）By introducing the xylose metabolic pathway,the range of available carbon sources was expanded.Firstly,the xylose redox pathway was introduced to equip the recombinant strain S_xyl with the ability to metabolise xylose and improve the utilisation of xylose by expressing the HXT7（F79S）transporter protein;then,by knocking out the protein kinase encoding gene snf1,the glucose blocking effect in the cell was lifted and the co-utilisation of xylose and glucose was achieved,using a mixed sugar containing xylose and glucose.Finally,the fermentation conditions were optimized and the optimal fermentation conditions were determined as follows:50 g·L^-1 KCl was added to the fermentation medium,the inoculum was 4%and the fermentation time was 96 h.At this time,the yield ofβ-glucan could reach98.54 mg·g^-1.（3）Modulation of the expression of titin transglycosylase and endo-β-glucanase reduced the molecular weight ofβ-glucan and improved the solubility ofβ-glucan.Down-regulation of transglycosylase expression by replacing the promoters of transglycosylase-encoding genes crh1 and crh2 with weak promoters showed that the above strategy did not have a significant effect onβ-glucan solubility.Therefore,the choice of using a galactose-inducible promoter to regulate the expression of endo-β-glucanase,which hydrolysesβ-glucan in the cell wall during the late stage of fermentation,successfully reduced the molecular weight ofβ-glucan from 1×10⁶ g·mol^-1 to 2×10⁶ g·mol^-1 to 1.26×10⁵ g·mol^-1 and increased the solubility to6.589 g·L^-1.