Functional Study Of Genes Related To 2-phenylethanlo And Tryptophol Production In Wide-type Saccharomyces Cerevisiae KMLY1-2

The production of aromatic alcohols(such as 2-phenylethanol,tryptophol,etc.)in Saccharomyces cerevisiae is mainly through de novo biosynthesis and Ehrlich pathway.Aromatic alcohols,as a class of higher alcohols,are widely used in medicine,cosmetics,food additives and other fields,and have high research value.At present,it’s mainly obtained by chemical synthesis and extraction from nature.Chemical synthesis of aromatic alcohols has many harmful substances,high environmental pollution and low purity.The low yield and high price of extraction from plants are not suitable for industrial production.However,many microorganisms have the ability to biosynthesize aromatic alcohols,so the biosynthesis of aromatic alcohols by bacteria or fungi has become a research hotspot.In this study,S.cerevisiae KMLY1-2,which was isolated from a steamed bread leavening agent,was used as the research target strain.Based on the previous transcriptomic analyses results,eight key genes were selected and their roles in aromatic alcohol production were verified by homologous recombination knockout technology.Then,the CRISPR/Cas9 gene editing platform of S.cerevisiae was constructed using uli1 gene,which encodes the unfolded protein response inducing protein.Then,combined with the relevant literature and further transcriptomic analyses,six genes were selected from the genome,and their effects on the production of 2-PE and TOL were investigated using the CRISPR/Cas9 gene editing system.In order to reveal the molecular mechanism of ULI1 protein in regulating the biosynthesis of 2-PE,the uli1 gene mutant strain was used for transcriptome sequencing analysis because the mutation of uli1 gene could significantly reduce the production of target alcohols.The main findings of this study are as follows:(1)Eight genes(thi4,syml,ctt1,pdc1,ald4,hsp104,pal and pck1)suspected to be related to the biosynthesis of 2-PE and TOL were identified from the transcriptomic analyses result of KMLY1-2 strain producing different levels of aromatic alcohols.These eight genes were knocked out in Saccharomyces cerevisiae KMLY1-2-a(MTAa)strain by homologous recombination.The 2-PE production of IVΔhsp104 strain increased by 5.05%.The Δpal strain showed a 36.61% decrease in2-PE yield and a 10.14% increase in TOL yield.The TOL yield of Δthi4 strain decreased by 5.34%.The TOL production of Δpck1 strain increased by 10.99%.This study laid a theoretical foundation for further engineering the aromatic alcohol metabolism pathway of Saccharomyces cerevisiae KMLY1-2 by CRISPR/Cas9 system.(2)Using uli1 gene as the target gene,CRISPR/Cas9 editing system was established in KMLY1-2-a strain,and the method of chemical transformation of PEG-lithium acetate,construction of recombinant p Cf B3052 and donor DNA,and the PCR verification of mutant strains were improved.Three different strains of uli1 gene mutation strain(frameshift mutation,base mutation and complete-gene loss mutation)were successfully obtained.By HPLC analysis of the aromatic alcohol production of three uli1 mutant strains,it was found that the mutation of uli1 gene resulted in a significant decrease in the production of 2-PE and TOL,and different mutation types had different effects on the yield.Among them,the frameshift mutant strain Δuli1-(2)had the largest decrease in 2-PE yield,which decreased from 1294.52 mg/L of the wild type to 822.61 mg/L,with a decrease rate of 36.43%.The yield of TOL decreased from 252.35 mg/L of the wild type to 187.35 mg/L,with a decline rate of25.75%.(3)Seven genes(ald3、pal、idh2、pha2、thi4、uli1 and adh5)were selected from the 2-PE and TOL metabolic pathway or other pathways by combining literature and transcriptome analysis.The CRISPR/Cas9 gene editing system was used for functional study.By using the homologous recombination repair donor DNA,the gene was completely deleted or replaced with other genes,or plasmid overexpression was performed in the gene-knockout strain.Finally,11 different types of recombinant strains and 3 gene overexpression strains were constructed(a-Δald3,a-Δald3::uli1,a-Δpal,a-Δpal::uli1,a-Δidh2,a-Δidh2::uli1,a-Δpha2,a-Δpha2::uli1,a-ΔⅩ-3::uli1,a-ΔⅫ-3::uli1,a-ΔⅫ-3::thi4,a-Δald3-p Y26-thi4,a-Δald3-p Y26-uli1 and a-Δald3-p Y26-adh5).In HPLC analysis,pal gene mutation strain were found to significantly increase TOL but decreased 2-PE production.The mutation of idh2 gene could increase the yield of 2-PE and TOL by 8.65% and 12.75%,respectively.Incorporation of uli1 expression cassette into the genome in the form of donor DNA resulted in a small increase in the yield,but the improvement was not as significant as that of the overexpression plasmid.(4)To further study the molecular mechanism of uli1 gene affecting 2-PE and TOL production,KMLY1-2-a and a-Δuli1 were cultured in transformation medium TM-7P,and transcriptome sequencing analysis was performed.GO enrichment analysis showed that the expression of ALD3 was up-regulated,which was mainly responsible for the oxidation of 2-phenylacetaldehyde to 2-phenylacetic acid,and could directly reduce the production of 2-PE,which was consistent with the decrease of 2-PE production in Δuli1 strain.The expression of thi4 gene is down-regulated during 2-PE production in Δuli1 strain,which explains why overexpression of thi4 gene in KMLY1-2-a-Δald3 strain using p Y26 overexpression plasmid increased 2-PE production.KEGG enrichment analysis showed that genes that co-regulated prephenate and phenylpyruvate were down-regulated during phenylalanine metabolism,which led to insufficient upstream products of 2-PE synthesis or promoted the metabolism of 2-PE to other pathways,ultimately resulting in the decreased production of 2-PE in a-Δuli1 strain.