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Construction Of High Riboflavin Producing Bacillus Subtilis By Metabolic Engineering

Posted on:2015-01-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:T ShiFull Text:PDF
GTID:1220330485491667Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
In this work, we have developed an efficient, precise and scarless method for rapid multiple genetic modifications without altering the chromosome of B. subtilis. Subsequently, a Bacillus subtilis cells factory containing minimal beneficial mutations was reconstructed by reverse metabolic engineering. Furthermore, the riboflavin biosynthetic pathway and purine biosynthetic pathway were deregulated and overexpressed to enhance the riboflavin production.The markerless mutation delivery system employs upp gene as a counter-selectable marker, double-strand break (DSB) repair caused by exogenous endonuclease Ⅰ-Scel and comK overexpression for fast preparation of competent cell. The DSB repair is a potent inducement for stimulating the second intramolecular homologous recombination, which not only enhances the frequency of resolution by one to two orders of magnitude, but also selects for the resolved product. This method has been successfully and reiteratively used in B. subtilis to deliver point mutations, to generate in-frame deletions, and to construct large-scale deletions.Based on the beneficial mutation analysis, we attempt to reconstruct the riboflavin overproducer by using iterative markerless mutation delivery system starting from scratch. It produced a series of engineered strains with clean genetic background from BSW4 to BSW54. BSW54 represented the highest riboflavin production (298.90±8.20 mg/L) with most beneficial mutations, including RibC (G199D), ribG+(G+39A), CcpN (A44S), YhcF (R90*), YvrH (R222Q), YwaA (Q68*), PurA (P242L).Genetic manipulations of rib operon were previously applied on the reconstructed B. subtilis strain to direct the necessary high levels of carbon flow through the riboflavin biosynthetic pathway. We tried three different ways to deregulate the rib operon by overexpression of the key gene (ribA), substitution the native promoter ribP1 and the RFN regulatory region ribO with strong promoter P43 and disruption of ribO. It was surprising that such high relative transcription levels of rib genes (ribG and ribA) were achieved by only overexpression of the key gene ribA. And overexpression of ribA in mutant BS89 led to approximately 1.4 times increase of riboflavin production from 210.25±3.73 mg/L to 506.45±3.50 mg/L, coupling with significant enhancement of the specific riboflavin production rate from 32.85±2.66 μmol/g CDW/h to 41.65±1.52 μmol/g CDW/h, indicating ribA overexpression could raise the metabolic flux of rate-limiting step, reflecting its direct contribution on riboflavin overproducing phenotype.Deregulation of purine pathway was attempted to improve purine nucleotides supply, based on a riboflavin producer B. subtilis strain with modification of its rib operon. To eliminate transcription repression, the pur operon repressor PurR and the 5’-UTR of pur operon containing a guanine-sensing Riboswitch were disrupted. Quantitative RT-PCR analysis revealed that the relative transcription levels of purine genes were up-regulated about 380 times. Furthermore, site-directed mutagenesis was successfully introduced into PRPP amidotransferase (encoded by purF) to remove feedback inhibition by homologous alignment and analysis. Overexpression of the novel mutant PurF (D293V, K316Q and S400W) significantly increased PRPP amidotransferase activity and triggered a strong refractory effect on purine nucleotides mediated inhibition. Intracellular metabolite target analysis indicated that the purine nucleotides supply in engineered strains was facilitated by a stepwise gene-targeted deregulation. With these genetic manipulations, we managed to enhance the metabolic flow through purine pathway and consequently increased riboflavin production 3-fold (826.52 mg/L) in the purF-VQW mutant strain.
Keywords/Search Tags:Bacillus subtilis, riboflavin, markerless mutation delivery system, inverse metabolic engineering, reconstruction, purine biosynthetic pathway
PDF Full Text Request
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