Gradient Constitutive Promoters Screening In Pdseudomonas Denitrificans And Its Application In Vitamin B₁₂Production

Vitamin B₁₂(VB₁₂),also referred to as cobalamin,is extensively used.Due to complex technology and low efficiency of chemical synthesis,it is solely produced via microbial fermentation.Therefore,obtaining high VB₁₂ producing strains is significant.Pseudomonas denitrifians is one of the main strains for industrial production of VB₁₂ due to its high growth rate and VB₁₂ yield.There are lots of research on improving VB₁₂ yield via metabolic engineering.But research on promoter screening of Pseudomonas denitrifians is scare.Promoter screening is an important metabolic engineering tool to balance metabolic flux and increase product yield.Traditional promoter screening and cloning methods of prokaryotes include screening based on promoter probe plasmid and cloning based on PCR.These strategies are efficient under certain conditions,but also have limitations.Recently,RNA-Seq technology increases range and accuracy of promoter screening,becoming a good tool for promoter screening on genome scale.In the thesis,an-industrial vitamin B₁₂producing strain PD320 was used for fermentation in 250 mL and 500 mL shake flask.The RPKM value for describing gene expression on genome scale,was obtained on RNA-Seq of 4 samples at 72 h and 96 h.Strength of promoters PE.coli-tutfA,PE.coli-tufB and P? were measured by fluorescence of the reporter gene gfp.The result showed that fluorescence of the strain Pd320/pBBR122-P?-gfp was distinctly higher than PD320/pBBR122-PE.coli-tufA-gfp and PD320/pBBR122-PE.coli-tufB-gfp,indicating promoter P? has the highest strength.With tuf in P.denilrificans as the reference gene,three gradient promoters were selected based on RPKM value and affirmed based on gfp fluorescence.Then these promoters were used to drive RCcobA2 in recombinant strains.Results on fermentation showed that VB₁₂yield of PD320/pBBR122-P?RCcobA2?124.09 mg/L?increased by 12.6%?29%and 35%compared with PD320/pBBR122-P?-RCcobA2?110.16 mg/L?,PD320/pBBR122-P?-RCcobA2?96.2 mg/L?and the control PD320/pBBR122?91.72 mg/L?.These results indicate that RNA-Seq has wide range and high accuracy for promoter screening,lay the foundation for promoter evaluation and application.The gradient promoters obtained facilitated promoter recognition in P.denitrificans and improving product yield via precise control of particular genes in synthetic biology.