Metabolic Engineering Of Bacillus Subtilis For Efficient Production Of Riboflavin

Riboflavin is an essential nutrient element for human and animals.It has important industrial value and is widely used in food additives,pharmaceutical industry and feed additives.In recent years,a series of metabolic modifications have been carried out for Bacillus subtilis,which has greatly increased the titer of riboflavin.However,the synthesis of riboflavin needs to consume a lot of oxygen,so the dissolved oxygen of fermentation broth is the key factor limiting the synthesis of riboflavin.Secondly,the precursor of riboflavin is synthesized by pentose phosphate pathway,and the insufficient supply of precursors also seriously limits the synthesis of riboflavin.Moreover,the current metabolic engineering strategy for riboflavin is more traditional,more use of gene overexpression and knockout to optimize the metabolic pathway,and lack of accurate and flexible gene expression regulation.Riboflavin synthesis involves pentose phosphate pathway,purine pathway and riboflavin synthesis pathway,and there are complex regulatory networks.In the past,some pathways or single modules were often modified in metabolic engineering,and there was a lack of overall module adaptation and metabolic flow optimization.This study takes riboflavin as the target product,adopts the strategy of combining comparative transcriptome analysis,traditional metabolic engineering and synthetic biological elements to alleviate the restriction of dissolved oxygen on riboflavin,and maximizes resource utilization by balancing gene expression in modules and metabolic flow between modules,so as to greatly improve the yield of riboflavin.The main results are as follows:(1)Based on transcriptome data analysis,the effect of dissolved oxygen on key genes in riboflavin synthesis was identified,and the restriction of dissolved oxygen on riboflavin synthesis was alleviated through the combination of metabolic transformation and dynamic regulation.There were significant differences in the expression levels of more than 1628 genes in the genome of riboflavin producing strain B.subtilis RF1 induced by low dissolved oxygen,including riboflavin synthesis,purine metabolism,nitrogen metabolism and ribosome metabolism.The genes of metabolic pathways related to riboflavin synthesis,such as purine pathway and riboflavin synthesis pathway,were significantly down regulated,and the expression levels of pur D and pur F were down regulated 69.55 and 23.92 times,respectively.Knockout of purine synthesis inhibitor Pur R relieved the feedback inhibition of purine pathway,and the riboflavin production increased by 25.23%,indicating that low dissolved oxygen inhibited purine pathway through Pur R.At the same time,the expression of gln R and tnr A genes induced by low dissolved oxygen was significantly increased.Using antisense RNA strategy to down regulate the expression of the two genes resulted in the increase of riboflavin production by 12.05%and 23.37%,respectively.These results suggest that dissolved oxygen affects riboflavin synthesis by affecting intracellular nitrogen metabolism.The promoter P_{gln R} of gene gln R was used to control the expression of vgb gene to improve the oxygen utilization rate,and the titer of riboflavin was increased by 29.42%;Finally,the combination of effective genes using metabolic engineering strategy increased the yield of riboflavin by 45.51%to 10.71 g/L.(2)A tunable intergenic regions(TIGRs)library was constructed in B.subtilis to coordinate the expression of multiple genes on the operon.Firstly,an operon library containing reporter gene and TIRG sequence was constructed and characterized in E.coli and B.subtilis.In E.coli,the relative expression ratio of egfp and m Cherry genes regulated by TIRG library changed in the range of more than 160 times,while in B.subtilis,the relative expression ratio of the two genes changed in the range of more than 70 times;Therefore,TIRG library was used to coordinate the expression of key riboflavin synthesis genes zwf,ywlf,and rib BA construct synthetic operon library,then screen the optimal gene expression combination;The riboflavin titer of the selected engineering strain RF1-L3 increased by 64.35%in shake flask fermentation.The analysis of intermediate metabolites showed that the concentration of riboflavin synthesis precursor PRPP in the cells of the engineering strain increased significantly,indicating that sufficient precursor supply ensured a large amount of riboflavin synthesis;Finally,fed-batch fermentation in a 5-L bioreactor increased the yield of riboflavin by 59.27%to 11.7 g/L.(3)The 5?UTR sequence suitable for B.subtilis was screened,and a 5?UTR library was constructed to improve and balance the gene expression on rib operon.Firstly,a natural 5?UTR sequence was screened from B.subtilis 168 genome by flow cytometry.The expression level of EGFP was increased by 2.5 times.It was found that the expression level of EGFP was increased by 5.8 times when the length was 19 bp;Based on the ability of 5?UTR to enhance gene expression,the sequence was used to enhance the expression of each gene on the rib operon.The riboflavin titer increased by 69.15%compared with the control group BSU0(without 5?UTR),indicating that the enzyme encoded by rib BA gene is the rate limiting enzyme for riboflavin synthesis;The random base"N"was introduced into the 5?UTR sequence to construct the 5?UTR library,and the reporter gene was used to characterize the coordination ability of the 5?UTR library for gene expression.In the single reporter gene library,the expression range of EGFP was more than 180 times,while in the double gene reporter library,the fluorescence values of EGFP and MCherry varied in the range of 0-10⁵(a.u.);The 5?UTR library was used to coordinate the gene expression on the rib operon,the synthetic rib operon library was constructed,and the optimal combination was selected.Compared with the original strain,the riboflavin titer was increased by 1.09 times.(4)Using promoters of different intensities to optimize the expression between modules to achieve the adaptation between modules.Firstly,based on transcriptome data analysis,appropriate integration sites were selected in the genome.The deletion of genes cyd and ccp N promoted riboflavin synthesis,so the two genes were selected as integration sites;The P₄₃promoter library was constructed and the expression activity of the library was further characterized.The library changed the expression of EGFP in the range of more than 1000 times,indicating that the library has great regulatory ability;Then,three promoters with different intensities of high activity,medium activity and low activity were selected from the library to control the expression of the module,and their transcription levels were analyzed by RT-PCR;The three promoters were used to control zwlf-TIGR-ywlf-TIGR-rib BA(ZYR)and optimized rib operon,and the optimal module combination was screened.Among them,P43-143-ZYR/P43-Srib increased riboflavin production by 60.9%;Finally,fed-batch fermentation in 5-L bioreactor showed that the riboflavin titer of the engineering strain reached 18.9 g/L,which was 122%higher than that of the original strain,and the riboflavin production intensity reached0.394 g/L/h.