Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis

Microbial Cell Factories

Research

Dingyu Liu¹  Zhiwen Wang¹  Ting Shi¹  Yongcheng Wang¹  Guanglu Wang¹  Jing Fu¹  Tao Chen¹  Xueming Zhao¹ 

[1] Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China;Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, China;Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, China;Edinburgh-Tianjin Joint Research Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin, China;

关键词: Bacillus subtilis;    Purine pathway;    Transcription repression;    Feedback inhibition;    Deregulation;    Riboflavin;   

DOI  :  10.1186/s12934-014-0101-8

 received in 2014-04-14, accepted in 2014-07-06,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundPurine nucleotides are essential metabolites for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and biosynthesis of several amino acids and riboflavin. Owing to the pivotal roles of purines in cell physiology, the pool of intracellular purine nucleotides must be maintained under strict control, and hence the de novo purine biosynthetic pathway is tightly regulated by transcription repression and inhibition mechanism. Deregulation of purine pathway is essential for this pathway engineering in Bacillus subtilis.ResultsDeregulation 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.ConclusionsA sequential optimization strategy was applied to deregulate the rib operon and purine pathway of B. subtilis to create genetic diversities and to improve riboflavin production. Based on the deregulation of purine pathway at transcription and metabolic levels, an extended application is recommended for the yield of products, like inosine, guanosine, adenosine and folate which are directly stemming from purine pathway in B. subtilis.

【 授权许可】

Unknown   
© Shi et al.; licensee BioMed Central 2014. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.