Construction,Characterization And Application Of Constitutive Gradient Intensity PUTR Library In Pediococcus Acidilactici

Pediococcus acidilactici was generally recognized as safe（GRAS）that was common to lactic acid bacteria,in addition to the advantages of the simple metabolic background,and excellent resistance to high temperatures and salt.These characteristics made it a high-potential chassis microorganism that can be used for food biosynthesis.In the process of constructing more efficient microbial cell factories of P.acidilactici,improving strain performance through metabolic engineering or synthetic biology method was the most effective strategy.However,there was currently a lack of gene regulatory elements of P.acidilactici,which restricted the construction and metabolic regulation of microbial cell factories.To improve the gene expression system of P.acidilactici,90 promoter-5’-UTR complexes（PUTRs）with gradient protein expression intensity were screened from the genome of P.acidilactici DY15 based on RNA-seq.Firstly,we characterized the transcription and protein expression levels of the PUTR library.Then,by analyzing the structure of PUTR,we obtained the conserved sequences of promoter regions and 5’-UTR sequences.On this basis,a set of combinatorial PUTRs with higher and stable protein expression levels was constructed by promoter engineering.Next,the stability of the PUTR library in the overexpression of different genes and the application of different strains was verified.Finally,the PUTR library was applied to improve the yield of phenyllactic acid（PLA）.The highest yield of PLA reached 8.12 g·L^-1 in 5-L fermentor,demonstrating the potential of the PUTR library in the metabolic engineering modification of P.acidilactici.The specific research content was as follows:（1）Construction and characterization of constitutive PUTR library of P.acidilactici.To mine endogenous gradient intensity PUTRs,the gene transcription levels within the genome were analyzed by RNA-seq.Total 90 PUTRs of with transcriptional intensity（FPKM）spans from 3.46 to 2,432,822 were screened,and the PUTR plasmid library was constructed.Subsequently,the PUTR library was characterized at the transcription level and the fluorescence level,respectively.RT-qPCR results showed that the transcription level of the PUTR library was 0.059%to 2010%of the promoter P₃₂.And the fluorescence intensities of the PUTR library in the early-log,mid-log,post-log,and stationary phases were 0.75%to 236%,0.81%to 228%,0.77%to 245%,and 0.56%to 230%of P₃₂,respectively.Finally,the first 15strong PUTRs were selected to verify their expression stability under special environmental pressure,among which 8 PUTRs showed excellent stability.（2）Engineering modifications based on conservative structural analysis to construct high-intensity combinatorial PUTRs.The sequence prediction of the-35 and-10 regions of 90PUTRs was performed using the BPROM model,and it was found that the-35 and-10 regions showed the conserved sequence features of“TTGANN”and“TATAAT”,respectively.Furthermore,the spacer sequence lengths were mostly distributed between 16～18 bp,which were consistent with the basic features of theσ⁷⁰ promoter.Three high transcriptional intensity PUTRs and four high translational intensity PUTRs were selected by comparing the transcriptional and translational intensities of different PUTRs,and the 5’-UTR regions of these PUTRs were determined by rapid-amplification of cDNA ends（RACE）.Finally,by combining promoter region with 5’-UTR,12 combinatorial PUTRs were constructed.And all 11combinatorial PUTRs except P_rpsU-UTR_rplM had significantly higher fluorescence intensity than the original PUTR,in which P_rpsU-UTR_ldhL showed the highest fluorescence expression intensity,853%of P₃₂.（3）Pervasive application of constitutive PUTR library gradients for the regulation of different genes.In order to verify the universality of the PUTR library,the combinatorial PUTR was selected to express red fluorescent protein（RFP）.The protein expression level of RFP was close to that of GFP expression,indicating that the PUTR library was capable of gradient-regulated and stable expression of different genes.In addition,overexpression of the transaminase AT in different strains using PUTR all led to the increase of PLA production,indicating the suitability of the PUTR library for strains with different genetic backgrounds.Finally,the transaminase AT was overexpressed in P.acidilactici DY15 using gradient-intensity PUTR.The highest yield of 950.6 mg·L^-1 was achieved by strain DY15-P_hpp1-UTR_rplM-AT,which was 12.4%and 79.2%higher than that of DY15-P₃₂-AT and the wild-type strain,respectively.Finally,strain DY15-P_hpp1-UTR_rplM-AT was fermented in a 5-L bioreactor,and the highest PLA production was 8.12 g·L^-1.The conversion rate of phenylalanine to phenyllactic acid was 92%.In addition,it was confirmed that phenyllactic acid can insert into DNA by spectroscopic analysis and molecular docking simulation,which fleshed out the antibacterial mechanism of phenyllactic acid.And it also showed that the phenyllactic acid had high application potential in food preservation industry.