The Expression Of Multiple Copies Of Thmg And Ggs1 Improves The Synthesis Of ?-carotene By The Engineered Strain Yarrowia Lipolytica YL-C1

Yarrowia lipolytica is an ideal strain for biosynthesis of ?-carotene.Our laboratory had successfully constructed the recombinant strain synthesizing P-carotene,but the following problems remain unresolved.For one thing,the recombinant strain is unstable and the ?-carotene content is low due to the fact that all the genes in the recombinant strain are freely expressed,and the plasmid and the bacteria are not simultaneously replicated.For another,the genetic engineering technology currently used in Y.lipolytica is less efficient.Therefore,this study intended to construct the stable recombinant strain of ?-carotene synthesis in Yarrowia lipolytica.On this basis,the content of ?-carotene was increased by regulating metabolic balance.Finally,the Crispr-cas9 technology was initially explored.The main contents and results were described below:(1)Construction of the stable recombinant strains YL-C1 producing ?-carotene.The optimal host strain was selected as polf by biosynthesis of ?-carotene in polf and the oil-producing strain polf-mfe--pex10-,and the Ku70 gene was knocked out in polf to improve the efficiency of homologous recombination.Based on this,the stable recombinant strain producing ?-carotene was constructed by integrating the gene Thmg,Ggs1,CarRA,CarB into the Y.lipolytica Polf(?Ku70)genome using snf site.And after 60 generations of the recombinant strain,the stability of the recombinant strain decreased slowly and the content of ?-carotene reached 2.34 mg/g DCW.(2)Enhancing ?-carotene production by regulating the genes copy numbers of Thmg and Ggs1.The intermediate product HMG-CoA and FPP were measured involved in the(3-carotene synthesis pathway in Y.lipolytica polf(?Ku70).When the strain contained 2Thmg,the content of ?-carotene reached the highest,14.12 mg/g DCW.When the strain contained 1Ggs1,the content of P-carotene was the highest,11.86 mg/g DCW.(3)The Crispr-cas9 system was explored in Y.lipolytica.The key factors affecting the Crispr-cas9 system were studied,and determined that the efficiency of homologous recombination increased 25%when the gene Ku70 was deleted;the optimal Donor DNA length was 100 bp;the higher the Donor DNA concentration,the higher the efficiency of the Crispr-cas9 system;In the scoring system of the target site,the efficiency of the Crispr-cas9 system was the highest when the score of the specificity,ecommended for guides expressed in cells,recommended for guides expressed in cells,out-of frame and out-targets is about 100,60,40,40 and 0.This study constructed the stable recombinant strain YL-C1 producing ?-carotene,which had certain guiding significance for the biosynthesis of carotenoids.At the same time,the Crispr-cas9 system was initially explored in the Yarrowia lipolytica,which laid the foundation for the use of Crispr-cas9 system.