Studies On The Construction Of Yeast Chassis For Efficient Expression Of Cytochrome P450S Via Genome-Scale Engineering

Plant natural products(PNPs)play important roles in many fields,and Saccharomyces cerevisiae is one of the most commonly used chassis for the synthesis of PNPs due to its unique advantages.The biosynthesis of PNPs often involves the catalysis of one or more cytochrome P450 enzymes(P450s).However,most plant P450s are membrane proteins located in the endoplasmic reticulum,leading to low expression levels and activities in S.cerevisiae,which is one of the main bottlenecks for efficient biosynthesis of PNPs.Although several strategies have been applied to improve the functional expression of P450s in yeast,there is no generally applicable engineering strategy that can improve the functional expression of most P450s.Therefore,this study aims to improve the microenvironment of S.cerevisiae for expressing and folding P450s via genome-scale evolution technology,thereby constructing yeast chassis cells that efficiently expressing P450s.The establishment of a high-throughput screening method coupled with the activity of P450s is the prerequisite for genome-scale evolution of yeast cells.Thus,the betaxanthin(yellow pigment with fluorescence)producing strain catalyzed by the P450 enzyme(CYP76AD1)was selected as the biosensor strain for high-throughput screening of genetic targets at the genome-scale level that can increase the functional expression of P450s in S.cerevisiae.First,the S.cerevisiae cDNA overexpression library was introduced into the biosensor strain to explore the effect of yeast gene overexpression on the functional expression of CYP76AD1 and accordingly the biosynthesis of betaxanthin.The results showed that singlecopy overexpression of yeast genes DAP1,PH023,RFU1,and PIB1 increased the production of betaxanthin to 1.20-,1.12-,1.19-,and 1.13-fold,respectively,while the high-copy overexpression of DAP1 and RFU1 increased the production of betaxanthin to 1.55-and 1.32fold,respectively.Considering a high possibility of the presence of key factors in plants that can promote the functional expression of plant P450s,a genome-scale cDNA overexpression library from Arabidopsis thaliana was introduced into the biosensor strain to explore the effects of plantderived genes on the expression of plant P450s in S.cerevisiae.The overexpression of the A.thaliana genes AtGRP7,AtMSBP1,and AtCOL4 increased the accumulation of betaxanthin to 1.32-,1.86-,and 1.10-fold,respectively.Moreover,when these three genes were overexpressed simultaneously,the production of betaxanthin was increased to 2.36-fold.Transcriptome analysis results showed that the overexpression of these plant genes caused significant changes in the expression levels of multiple genes in yeast,including those related to endoplasmic reticulum expansion,maintenance of membrane protein stability,and response to stress conditions,indicating the complexity of the regulatory network of P450 enzyme expression in yeast.On this basis,the screened A.thaliana genes were overexpressed in the Z-α-santalol producing strain to explore their effects on the functional expression of CYP736A167 and the synthesis of Z-α-santalol.The results showed that simultaneous introduction of the three genes,AtGRP7,AtMSBP1,and AtCOL4,increased the conversion rate of the hydroxylation of αsantalene to Z-α-santalol by up to 2.97-fold.In addition to simple gene overexpression libraries,a CRISPR/Cas(Clustered regularly interspaced short palindromic repeats/associated proteins)technology based multifunctional MAGIC(Multi-functional genome-wide CRISPR system)library,which could simultaneously achieve gene deletion,transcription repression,and transcription activation,was also introduced into the biosensor strain to enable a more comprehensive and diversified genomescale evolution.After the first round of MAGIC evolution,a total of 37 targets that could increase the production of betaxanthin to varying degrees were screened,including targets 1162(SpSg-DOA4),10-32(SpSg-MAC1),10-62(SpSg-DOA1),and 12-75(SpSg-BRO1),which increased the production of betaxanthin to 5.89-,3.02-,2.61-,and 2.59-fold,respectively.The targets screened after the first round of MAGIC genome-scale evolution were introduced into the Z-α-santalol-producing strain,and the results showed that multiple targets could also increase the functional expression of CYP736A167 and the production of Z-α-santalol.The most effective target 10-44(SpSg-CYC8)increased the production of Z-α-santalol to 2.05-fold.Therefore,the second round of MAGIC genome-scale iterative evolution was carried out based on target 10-44,and 33 gene targets that could effectively increase the production of betaxanthin were further screened.Many of the selected targets could also increase the production of Z-αsantalol,and the most effective target 31-47(SpSg-GET2)further increased the production of Z-α-santalol to 1.42-fold.Finally,the screened targets were tested in a vindoline-producing strain,which harbored multiple P450s.The simultaneous expression of targets 10-44 and 3147 increased the production of vindoline to 1.64-fold,indicating that the screening gene targets were likely to have general applicability to improve the functional expression of P450s in yeast.In conclusion,this study performs genome-scale evolution in S.cerevisiae using yeast cDNA overexpression library,A.thaliana cDNA library,and CRISPR/Cas-based multifunctional MAGIC library,with an aim to improve the microenvironment for functional expression of P450s in S.cerevisiae.Many genetic targets were screened to improve the expression levels and activities of several P450s,and accordingly the production of betaxanthin,Z-α-Santalol.and vindoline.Combined with synthetic biology and metabolic engineering strategies,this study is of great significance for the construction of S.cerevisiae cell factories for efficient biosynthesis of plant natural products.