Activity Analysis Of Promoters From Chloroviruses And Their Application In Threonine Biosynthesis

The transformation of metabolic pathways of platform organisms such as E.coli and accumulation of useful metabolites are the core content of modern fermentation engineering and synthetic biology.Promoters are important regulatory elements for gene regulation and metabolic pathway modification.By changing the promoter’s activity,the transcription level of the target gene can be regulated to accumulate metabolites.In the previous work of our laboratory,a group of strong chlorella virus promoters N63,N37,and N40 were screened from random fragments of the chlorella virus genome by shotgun method;the transcription of the reporter genes under the control of these promoters was detected by RT-qPCR.Abundance,the transcription initiation site was determined by RACE-PCR,the core structure of-10 region and-35 region was speculated,and these promoters were truncated and other mutations.Each mutant promoter was linked to the promoter detection plasmid pKK232-8 by molecular techniques such as PCR,so that it could express the chloramphenicol acetyltransferase gene cat.Thereafter,the transformants carrying the chlorella virus-derived strong promoter-recombinant plasmid-transformed strains were cultured in LB medium containing 450 μ g / mL and the growth curve was measured to indirectly analyze the transcriptional regulation activity of the promoters.In this study,the lacZ gene was amplified from the E.coli genome and fused with the codon-optimized egfp gene by overlap PCR.The RBS sequence of the chloramphenicol acetyltransferase gene cat was inserted between the two to construct a strong promoter tac from a known bacterium.Or the artificial operon controlled by the strong promoter of chlorella virus selected from the growth curve experiment,replacing the reporter gene cat of the promoter detection plasmid pKK232-8,and obtaining a novel dual-reporter promoter detection plasmid for chlorella Detection of virus-derived promoter activity.Qualitative and quantitative analysis of the reporter gene egfp using laser confocal microscopy and flow cytometry,respectively;using2-nitrophenyl-β-D-galactopyranoside(ONPG)as the reaction substrate,andIVmeasuring its products The specific activity of lacZ gene was measured by2-nitrophenol(ONP).The plasmid-transformed E.coli DH5α strain showed green fluorescence under a laser confocal microscope,and the fluorescence intensity was quantitatively determined using a flow cytometer;the transformed strain showed blue cells on a plate containing X-β-Gal;cells The lysate can react with colorless ONPG to form yellow ONP.This reaction can be used for enzyme activity analysis.The experimental results show that both reporter genes can be correctly expressed in E.coli.Through quantitative analysis and transcription level analysis of the lacZ and egfp dual reporter genes,it was confirmed that the promoter activity of the strong promoter of chlorella virus is stronger than that of the tac promoter.The original cat gene on lacZ-egfp and pKK232-8 plasmid was amplified from the double-reporter plasmid by overlap PCR,and the RBS sequence of the chloramphenicol acetyltransferase gene cat was inserted between them to construct a strong promoter from known bacteria.The tac or artificial operon controlled by the strong promoter of chlorella virus selected from the growth curve experiment was used to obtain a three-reporter promoter detection plasmid,which was applied to the screening of strong promoters of chlorella virus or environmental virus group samples.The strong promoter screened by this plasmid can directly use the reporter genes lacZ and egfp to perform in situ detection of transcriptional regulation activity,thereby improving the efficiency of promoter selection and analysis.Recombinant plasmids constructed by promoters N37,N40,N63 and their mutants controlling the single reporter gene / selective marker gene cat and the three reporter gene lacZ-egfp-cat were transformed into E.coli DH5α,and they contained 450μg / mL.The growth curve of chloramphenicol in LB medium shows that cat located at the distal end of the three reporter gene operon can be expressed normally in most recombinant plasmids,which proves the feasibility of this strategy;this system can also be used to study promoters Primer activity for relatively distant genes in tandem expression systems.This study further applied a strong promoter derived from chlorella virus to the replacement of the key gene / operon thrABC promoter in the threonine biosynthesispathway of E.coli in order to increase the accumulation of threonine.Using CRISPR /Cas9 technology,the threonine promoter and its leader in the threonine biosynthetic pathway were knocked out,and the strong chlorella virus promoter and the RBS sequence of the chloramphenicol acetyltransferase gene cat were knocked in.Construction of engineering bacteria E.coli K-12 MG1655 ΔilvA ΔmetA ΔlysA ΔtdhΔtdcC : rhtc ΔthrLΔthrLp/ptac,E.coli K-12 MG1655 ΔilvA ΔmetA ΔlysA ΔtdhΔtdcC : rhtc ΔthrLΔthrLp/pN63,E.coli K-12 MG1655 ΔilvA ΔmetA ΔlysA ΔtdhΔtdcC : rhtc ΔthrLΔthrLp/pN37.Among them,the production of threonine in shake flask fermentation of the strain in which the N37 promoter was knocked in increased from 12.88 g / L of the engineering strain to 32.32 g / L.