CRISPR/Cas9 Based Biosynthetic Engineering Of An Artemisinic Acid Producing Strain And A Nystatin Producing Strain

CRISPR/Cas gene editing technology has gradually become the mainstream tool for gene editing,and has been widely used in Saccharomyces cerevisiae and Streptomyces.Among these CRISPR/Cas based tools,CRISPR/Cas9 technology has been widely used for gene insertion,replacement and knock-out due to its simplicity and high efficiency.In this study,CRISPR/Cas9 technology was applied to an engineered high-artemisinic acid-producing yeast S.cerevisiae 1211 and a high-nystain-producing strain Streptomyces sp.ZY01,in order to further increase the industrial application values of the two strains.S.cerevisiae 1211 is an original artemisinic acid-producing strain provided by Zhejiang Hisun Pharmaceutical Co.,Ltd.,but 1%galactose needs to be supplemented to induce the production of artemisinic acid.In order to reduce the fermentation cost of the enterprise(galactose accounts for about 15%),this study intends to knock out the gal80 gene involved in galactose induction.However,it is difficult to find a suitable screening marker for the application of traditional knockout methods,as the strain S.cerevisiae 1211 contains almost all resistance genes known to be available in yeast.Therefore,the first part of this project is to develop a CRISPR/Cas9 technology for gene blocking.Since the CRISPR/Cas9 plasmid pML 104 selected carries the orotin-5’-phosphate decarboxylase gene ura3,which can serve as a selection marker,this study first obtained ura3 mutant strain S.cerevisiae 1211-1 through 5-FOA mutagenesis.A knockout plasmid pML104-gal80 was constructed,and then the negative regulation genes of galactose metabolism(gal80)was knocked out using the CRISPR/Cas9 system mediated by pML104-gal80 to obtain S.cerevisiae 1211-2.The orthogonal flask experiment was used to optimize each parameter within the shake flask fermentation process of S.cerevisiae 1211-2.Under the best fermentation conditions(fermentation medium Ⅲ,1%inoculation amount,fermentation for 84 h),the flask fermentation yield reached 740 mg/L.However,the strain S.cerevisiae 1211-2 was inefficient in the utilization of the carbon source ethanol,which played a decisive role in the accumulation of artemisinic acid in the subsequent 50 L pilot fermentation experiment.At the same time,the growth of the bacteria was relatively slow.We speculated that it was the uracil nutritional deficiency that affected its growth and artemisinic acid production.Thus,we constructed a recombinant plasmid pML104-KanMx4-u based on the CRISPR/Cas9 plasmid pML104-KanMx4(without the ura3 selection marker).The ura3 gene functional region was successfully inserted by CRISPR/Cas9 technology,and finally a modified strain S.cerevisiae 1211-3 was obtained.S.cerevisiae 1211-3 and S.cerevisiae 1211 were subjected to fermentation at two levels(shake flask fermentation and 50 L pilot fermentation,respectively).The HPLC results of the fermentation broth showed that the yield of artemisinic acid of the modified strain S.cerevisiae 1211-3 in the fermentation medium without galactose was equivalent to that of the parental strain S.cerevisiae 1211 under the conditions of galactose addition(1200 mg/L in shake flask fermentation and 20 g/L in pilot fermentation,respectively).The modified strain S.cerevisiae 1211-3 used in industrial production is expected to reduce the cost of artemisinic acid fermentation by 10%-15%,which has high industrial application value.The second part of this project is to cooperate with Zhejiang Zhenyuan Pharmaceutical Co.,Ltd.to carry out a synthetic biological engineering of a high-nystain-producing strain Streptomyces sp.ZY01.In the previous research on its secondary metabolites,an unknown component of nystatin,RT6,has been identified as a cyclic dipeptide compound-Albonoursin,which is an impurity component in the synthesis of nystatin and has a certain cytotoxicity.The purpose of this study is to remove this component by synthetic biology methods.Through genome sequencing and bioinformatics analysis,it is speculated that the RT6 component may be synthesized by 4 genes(albABCD),and the albC gene is the rate-limiting and key enzyme in the synthesis of RT6 components.Therefore,this study intends to block the functional gene albC and the RT6 biosynthetic gene cluster,in order to achieve the goal of removing RT6 invalid components.However,Streptomyces sp.ZY01 is not sensitive to a variety of common antibiotics used for streptomyces(Thiostreptonin,Apramycin,and Kanamycin),and we found that its clonal growth on the Apramycin-containing medium was in a dose-dependent manner.Repeated screening through temperature-sensitive experiments was used to obtain Apramycin-sensitive strains.The fermentation results of the strain showed that the yield of each component of the secondary metabolite was not significantly different from that of the parental strain.Then we selected the plasmid pKCcas9dO carrying the Apramycin resistance gene to construct a series of knockout plasmids carrying different sgRNAs,and transformed the plasmids into Streptomyces sp.ZY01 through protoplast transformation and conjugation transfer for the knockout of albC gene and RT6 biosynthetic gene cluster.