Studies On UP Elements Screening And Construction And Application Of Chlorovirus-derived Semi-synthetic Promoters

Enhancing the expression of target genes by increasing the initiation strength of promoters and thereby increasing the yield of metabolites has been a hot topic in recent metabolic engineering research.UP Elements are A-and T-rich DNA fragments that can enhance promoter expression by contacting the α-subunit carboxy-terminal domain(αCTD)of RNA holoenzymes to activate the core RNA polymerase enzyme.In this study,a randomly mutated fragment of the UP Elements was combined with a core promoter from the Chlorella virus genome to form a semi-synthetic promoter,and the randomly mutated UP elements was screened by constructing a dual reporter gene plasmid;the screened UP elements with a promoter-enhancing effect was constructed on other core promoters and validated with dual reporter genes.On this basis,the endogenous promoter thr Lp of thr LABC operon for L-threonine biosynthesis in E.coli genome was replaced with a semi-synthetic promoter in the threonine metabolic pathway by gene editing(CRISPR/Cas9),and the stronger initiation activity of the screened semisynthetic promoter was further verified by comparison of threonine production.The main studies are as follows.The pKK232-8 plasmid was used as a template to construct the chloramphenicol acetyltransferase gene cat,the enhanced green fluorescent protein gene egfp and the core promoter N63 from the Chlorella virus genome obtained in our laboratory by PCR,enzyme digestion and enzyme ligation techniques into the vector p KK232-8 to obtain the p KK232-8-N63-egfp-cat dual reporter plasmid.The medium was designed using Design Expert 12.0 based on the Box-Behnken(BBD)principle and optimized by using the fluorescence intensities of the green fluorescent protein expressed by the dual reporter plasmid transformed to E.coli DH5αas the response indices,and the optimal medium formulation was obtained as follows: yeast extract 2.8%,glucose 2%,tryptone 2.5%,sodium chloride 1%,p H 7,which was ready to use for the subsequent expression verification of the semisynthetic promoter containing the UP elements using the dual reporter gene plasmid.The artificial UP elements were synthesized by random mutations on the non-conserved sequences of the known UP elements,and then the randomly mutated UP elements fragments were fused with our Chlorella virus genome-derived core promoter N63 to form a semisynthetic promoter shotgun library.The randomly mutated UP elements were screened in LB medium supplemented with 450 μg/m L chloramphenicol to obtain a series of UP elements that increased the initiation strength of the core promoter,and the screened UP elements were sequenced in order to validate their effects further.The UP elements with enhanced regulatory activities were fused with another Chlorella virus genome-derived promoter N37,which was previously studied in our laboratory,and inserted into the egfp-cat dual reporter gene plasmid.The growth curves and the fluorescence intensities of the transformed strains were measured,and it was found that the semi-synthetic promoters with the screened UP elements had stronger initiation activities,indicating that these UP elements had similar promoter-enhancing effects on the core promoters other than N63,and demonstrating that they showed wide range of promoter-enhancing activities in some extent.By replacing the promoter of thr ABC,a key gene operon in the E.coli threonine biosynthesis pathway,with the selected semi-synthetic promoters with UP elements,N63UP94 p and N37UP94 p,and constructing bioengineered E.coli K-12 MG1655 Δilv A Δmet AΔlys A Δtdh Δtdc C Δthr Lp::N63UP94p and E.coli K-12 MG1655 Δilv AΔmet A Δlys A ΔtdhΔtdc C Δthr Lp::N37UP94p via the CRISPR/Cas9 gene editing tool,and using the previously bioengineered E.coli K-12 MG1655 Δilv A Δmet A Δlys A Δtdh Δtdc C Δthr Lp::N63p and E.coli K-12 MG1655 Δilv A Δmet AΔlys A Δtdh Δtdc C Δthr Lp::N37p in the laboratory as controls.The knocked-in promoters that replaced the native thr LABC promoter increased the threonine production from 6.8 g/L to 15.38 g/L for N63UP94 and from 15.05 g/L to 20.68 g/L for N37UP94,demonstrating that the randomly mutated UP elements could enhance the production of the target products by enhancing the promoter activities.