Use Of The CRISPR Base Editing Technology To Optimize ?-carotene Metabolic Pathways

?-carotene is the most stable natural pigment which can be commonly found in nature.It is a non-toxic,safe and nutritious food additive recognized by the Food andAgriculture Joint Committee of the Food andAgriculture Organization of the United Nations,It is also widely used in food industry,feed industry,medicine and cosmetics industry.Currently,?-carotene can be industrially produced using chemical synthesis,biological fermentation,and genetic engineering methods.This study uses CRISPR technology to optimize the metabolic pathway of ?-carotene in E.coli to improve the ability of E.coli to produce ?-carotene.Several genes or the whole metabolic pathways often need to be introduced to microbial cells when microbial cell factories construction using synthetic biology technique,the balance of those genes expression is very important,too high or too low are not conducive to cell growth and yield increase.It needs to optimize each gene expression level.This work,a transcription regulation technique will be constructed based on the CRISPR system using dCas9-RpoZ;then,the technique of multi-gRNA plasmid libraries construction will developed,which are used for multi-gene transcription regulation;finally,the transcription regulation technique combined with the multigRNA plasmid libraries will be constructed for transcriptional regulation of multi-gene in Escherichia coli,and this system will also be used for further verification and application in the regulation of beta-carotene metabolic pathways.In this work,we determined the feasibility of dCas9-RpoZ fusion mediated CRISPR activation(CRISPRa)and CRISPR repression(CRISPRi)system via testing the transcription initiation and elongation of the reporter protein of RFP.Subsequently,we constructed the assembly technique of multiple sgRNAs libraries to perform the application of multiple genes modulation in the MEP and central metabolic pathways,involving eight genes,namely isp D,ip E,isp G,idi,gapA,pgk,gpmA and pyk F.Finally,through our strategy of multiple gene regulation for combinatorial modulating the ?-carotene synthetic pathway genes,.After strain fermentation,a total of 47 strains were obtained.Among them,3 strains showed inhibitory ability to produce ?-carotene,and44 strains showed activation.The titer of ?-carotene varied from 1.1 to 2.1 times that of CAR001(dCas9-RpoZ),the specific production value of ?-carotene displayed 1.2-to2.5-fold increase compared to the original strain CAR001(dCas9-RpoZ),furthermore,we also successfully achieved high production of ?-carotene strain,especially the strain lib 47 produced 125.46 mg/L ?-carotene with a specific production value of 23.91 mg/g,representing a 2.1-fold and 1.8-fold increase over the starting strain CAR001(dCas9-RpoZ).Finally,the "multi-gRNA plasmid" library of 47 strains was grouped and sequenced according to the ability to produce ?-carotene,and the sequence of the recognition sites of multiple gRNA plasmids of each strain was obtained.The results demonstrated that the dCas9-RpoZ based CRISPRa and CRISPRi system indeed had beneficial effects on the multi-gene modulation via multiple sgRNAs libraries.This is the first regulatory approach and metabolic engineering strategy for modulating eight genes simultaneously.