Effect Of Interfering The Biosynthetic Genes Expression By CRISPRi On The Ectoine Production

Ectoine is a natural compatible solute,which exists in many different kinds of microorganisms.It can not only regulate the osmotic pressure,but also stabilize enzyme activity and protect cells against extreme environment(freeze-thaw,drying,ionizing radiation,high temperature,etc.).It also used as an oxidant to prevent the membrane function of cells from dameging,and it used to treat neurodegenerative diseases.Because of its excellent protective properties,ectoine has broad application prospects in the fields of biomedicine,biological manufacturing and chemical industry.An engineered strain ECT01(Escherichia coli W3110 ybeM::PT7-ectABC,tyrR::PT7-ectABC)was constructed in this laboratory.The strain has some problems,such as low conversion and inadequate metabolic network.In order to solve these problems,we had interfered the expression of biosynthetic genes by CRISPRi and further improved the conversion and the yield of ectoine.This study had completed the following works:In order to improve the conversion and the yield of ectoine,we had interfered the expression of biosynthetic genes by CRISPRi.Firstly,gene dcas9 which can express dCas9 protein and interfere gene expression)was integrated into the yncl site.Because the promoter xylF had been used in strain ECT01,gene dcas9 was controlled by the promoter xylF.The strain ECT02(ECTO1,yncI::PxyIF-dcas9)was constructed.The targeting genes are aceE and zwf,and the interfering sites are the-35 box region,the ATG region and the end of gene,respectively.On the basis of ECT02,strains ECT03-1/2/3 and ECT04-1/2/3 were constructed.The results of shake flask fermentation showed that the yields of ECT03-1/2/3 and ECT04-1/2/3 were 37.7%,8.4%,4.2%and 59.3%,27.9%,2.9%lower than that of strain ECT02-K(ECT02,plasmid pGRB without sgRNA)respectively.In addition,the results of real-time PCR showed that it has little effect on transcription of gene aceE whether xylose added as inducer.So the promoter xylF was not tight.This leads to translating dCas9 protein too early,which affects the growth and metabolism of strains.In order to interfere the expression of the biosynthetic genes accurately,we had screened more tighter promoter among three promoters(Pte,Para,Prha).In strain ECT01,the biosynthetic genes of ectoine controlled by promoter T7.The transcription of promoter T7 requires T7 RNA polymerase,and the gene T7RNAP controlled by PxyIF.In this study,the promoter of gene T7RNAP replaced by three promoters.The tightness of each promoter could be determined by detecting the yield of ectoine.Strains ECT05-07 were constructed on the basis of ECT01.The results of shake flask fermentation showed that the yied of strains ECT06 was 0.27±0.03 g/L without inducers,and that of strain ECT06 was 11.88 ±0.89 g/L when inducers were added.The experimental results showed that Para has good tightness and the highest yield of ectoine,so Para is the most suitable for interfered experiments.Gene dcas9 that controlled by Para was integrated into the yncl site.Strain ECT08(ECT06,yncI::Pua-dcas9)was constructed.The genes zwf,aceE,gltA,pfkA,pyk,sucA and sucCD were interfered on the basis of ECT08.The interfering sites were the ATG region and the middle region of gene.The strains ECT09-15 were constructed.The results of shake flask fermentation showed that the yields of interfering genes aceE and sucA were 14.12±0.15 g/L and 12.83±0.24 g/L respectively,which were 21.1%and 10.0%higher than that of ECT08-K(11.66±0.16 g/L).The yield of interfering gene sucCD was not significantly different from that of ECT08-K.The yields of interfering genes zwf,gltA,pfkA and pykF were 38.2%,26.8%,25.8%and 5.8%lower than that of ECT08-K respectively.Finally,the strain of interfering the middle region of gene aceE has the most significant positive effect.The yield of this strain reached 14.12 g/L,which was 21.1%higher than that of the original strain.The conversion reached 31.4%,which was 21.2%higher than that of the original strain(25.9%).The plasmid system is unstable and it makes heavy burden on bacterias.The sgRNA used to interfere the middle region of gene aceE was:integrated into the xylA site on the basis of ECT08.Four promoters with different intensity(Pt7,Ptrc,Para,PJ23119)were selected.The strains ECT16-19 were constructed.The results of shake flask fermentation showed that the yields of ectoine were 12.44 g/L,11.17 g/L,4.38 g/L and 11.21 g/L,respectively.The yields of ECT16 were 5.8%higher than that of ECT08(11.75±0.18 g/L),while ECT17-19 were 4.9%,62.7%and 4.5%lower than ECT08,respectively.