Construction And Application Of A Robust Escherichia Coli Strain With A Dual Protection System

Escherichia coli?E.coli?is one of the most widely used expression host in the field of gene engineering.However,there have been some problems of microbial contamination and phage infection during the fermentation process,which have not been solved at present.This study uses E.coli as the research object,aiming to construct a robust engineered E.coli which can resist the contamination by undesired microbes and the infection by phages.Two new metabolic pathways of rare nitrogen and phosphorous sources?an anti-hybrid system?were introduced in E.coli,which can express formamidase and phosphite dehydrogenase to specifically catalyze formamide and phosphite to become ammonium ions and phosphate as nitrogen source and phosphorus source respectively for E.coli growth to resist the contamination by undesired microbes.On this basis,the anti-phage system?CRISPR/Cas9?was introduced to endue the engineered E.coli the ability to resist the infection by phages.The robust E.coli constructed with the dual protection system will have very great potential application in fermentation engineering.By analyzing the transcriptome of Paenibacillus pasadenensis CS0611 strain with independent intellectual property right,our research group found that the genome of P.pasadenensis CS0611 contained a formamidase gene,whose full-length sequence was cloned by PCR.In addition,Klebsiella pneumoniae strain OU07,which could utilize phosphite,was screened and the full-length sequence of phosphite dehydrogenase gene was successfully cloned.The sequence information and catalytic structure of the two enzyme genes and corresponding proteins were analyzed in detail,and it was found that the complete open reading frames of formamidase gene and phosphite dehydrogenase gene were composed of 1014 bp and 1011 bp,and encoded 337 and 336 amino acids,respectively.Moreover,formamidase derived from P.pasadenensis CS0611 belonged to the nitrilase superfamily,was a hexamer,and included 3 AB dimers,each of which was composed of A and B units.The secondary structure of formamidase was composed of 24.6%alpha helix,27.0%beta fold and 48.4%turn.The amino acid residues of Glu60,Lys129 and Cys162 constituted the catalytic triad of formamidase to combine formamide.Phosphite dehydrogenase derived from K.pneumoniae strain OU07belonged to the D-2-hydroxy dehydrogenase family.The secondary structure of phosphite dehydrogenase was composed of 35.4%alpha helix,11.9%beta fold,and 52.7%of turn,and this phosphite dehydrogenase had 99.4%sequence identity with phosphite dehydrogenase from Pseudomonas stutzeri WM88.The formamidase gene and phosphite dehydrogenase gene were inserted into expression vectors respectively,and the recombinant plasmids were efficiently expressed in E.coli.After the total protein was separated and purified by GST-tagged column,pure recombinant FmdA and PtxD were obtained,and then the enzymatic properties of the two recombinant enzymes were systematically studied.Studies found that the optimal reaction temperatures for FmdA and PtxD were 30 ? and 35 ?,meanwhile,the optimal reaction pHs were 6.5 and 7.0,respectively.Under the optimal reaction conditions,the specific activities of recombinant FmdA and recombinant PtxD were 10.0 U/mg and 10.8 U/mg,respectively.Afterwords,stability studies showed that recombinant FmdA was stable at temperatures below 40 ? and pH range of 6-8,and recombinant PtxD was stable at temperatures below 30 ? and pH range of 5-8.Furthermore,metal ions Cu²⁺,Fe²⁺,Zn²⁺and surfactant SDS significantly inhibited the recombinant FmdA to catalyze formamide.Metal ions Li⁺,Zn²⁺,Mo²⁺,Fe³⁺had serious inhibitory effect on recombinant PtxD to catalyze phosphite.Cu²⁺?Pb²⁺and surfactant SDS could completely or almost completely inhibit the catalytic activity of recombinant PtxD.In addition,EDTA had no obvious inhibitory effect on the two recombinant enzymes,indicating that both of them might belong to non-metallic enzymes.Enzymatic properties studies have shown that the optimal temperature and pH of recombinant FmdA and PtxD were close to the optimal growth conditions of E.coli.In this paper,these two enzyme genes were constructed into the same recombinant plasmid?as an anti-hybrid system?by fusion expression and co-expression,and successfully expressed in E.coli.The resulting recombinant E.coli could use formamide and phosphite as nitrogen and phosphorus sources respectively in auxotrophic MOPS medium for growth and reproduction.The results showed that the recombinant E.coli obtained by the construction method of fusion expression had the fastest growth rate and the highest fluorescence intensity of green fluorescent protein.This recombinant E.coli could effectively resist the microbial contamination in auxotrophic MOPS medium,and was always the dominant bacteria in the cultivation process.Subsequently,the plasmids of CRISPR/Cas9 system?anti-phage system?were introduced into the recombinant E.coli that already had the antimicrobial system,the resulting robust E.coli strain in LB liquid medium could effectively resist the infection of T7phage,but in auxotrophic MOPS medium the growth of the robust E.coli grew slowly and reached the stationary on the 6th day,moreover,the efficiency of resistance to phage was reduced.To accelerate the growth rate of the robust E.coli,the promoter that regulated the fusion gene was optimized in this study.It was found that moderate-strength constitutive promoters?BBa?J23101 and BBa?J23106?were the most conducive to the growth of the engineered E.coli and could increase the growth rate significantly.In addition,by integrating plasmids with the same replicator,the number of plasmid types in the dual system was reduced to two which were mutually compatible.The results showed that the growth of the optimized robust E.coli in auxotrophic MOPS medium reached the stationary after about 8 h cultivation time,which was 7.5 times higher than that before optimization,and could resist the infection of T7 phage with a concentration of up to 2×10⁷ PFU/m L,and maintain high-efficiency protein synthesis ability.Using the robust E.coli as the host bacteria,three types of enzyme genes?epoxide hydrolase gene,carbonyl reductase gene,and lipase gene?were efficiently expressed,and a robust E.coli Rob3 expressing epoxide hydrolase was used as whole cell catalyst to enantioselectively resolve racemic glycidyl o-methylphenyl ether?rac-o-GMPE?for preparing?R?-o-GMPE.Studies showed that under the conditions of a reaction temperature of 25 ? and pH 7.0,?R?-o-GMPE could be prepared from 20 m M rac-o-GMPE by E.coli Rob3 cells with the maximum yield of 23.0%and ee of 99.2%within 12 min reaction,and the specific activity of wet cells was 6416.7 U/g.In order to reduce the inhibitory effect of the substrate on catalysis and the toxic effect of the products on the cells,a biphasic system?aqueous buffer:butyl acetate=0.5:1?was developed to catalyze 3 M rac-o-GMPE.The initial reaction rate was increased to549.7 m M/h,the final ee was 91.7%,the yield was 17.1%and the space-time yield was 3.66g/L/h.