Abstract
Objectives
To deregulate the purine operon of the purine biosynthetic pathway and optimize energy generation of the respiratory chain to improve the yield of guanosine in Bacillus amyloliquefaciens XH7.
Results
The 5′-untranslated region of the purine operon, which contains the guanine-sensing riboswitch, was disrupted. The native promoter Pw in B. amyloliquefaciens XH7 was replaced by different strong promoters. Among the promoter replacement mutants, XH7purE::P41 gave the highest guanosine yield (16.3 g/l), with an increase of 23% compared with B. amyloliquefaciens XH7. The relative expression levels of the purine operon genes (purE, purF, and purD) in the XH7purE::P41 mutant were upregulated. The concentration of inosine monophosphate (IMP), the primary intermediate in the purine pathway, was also significantly increased in the XH7purE::P41 mutant. Combined modification of the low-coupling branched respiratory chains (cytochrome bd oxidase) improved guanosine production synergistically. The final guanosine yield in the XH7purE::P41△cyd mutant increased by 41% to 19 g/l compared with B. amyloliquefaciens XH7.
Conclusion
The combined modification strategy used in this study is a novel approach to improve the production of guanosine in industrial bacterial strains.
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Acknowledgements
This work was supported by the Science and Technology Planning Project of Guangdong Province (Grant Numbers 2016A050503016, 2016A010105004 and 2013B010404007), the Science and Technology Planning Project of Guangzhou City (Grant Number 201510010191), the National High-tech R&D Program (863 Program) (Grant Number 2014AA021304) and the China Scholarship Council Fund (201606155032).
Supporting information
Supplementary Table 1—Bacterial strains and plasmids used.
Supplementary Table 2—Primers used.
Supplementary Fig. 1—Construction of the expression plasmid pBE-Pw-bgaB.
Supplementary Fig. 2—Construction of the integrative plasmids pKS2-T1 and pKS2-T2.
Supplementary Fig. 3—Construction of the integrative plasmids pKS2-P41, pKS2-P r2 , and pKS2-P43.
Supplementary Fig. 4—Construction of the knockout plasmid pKS2-cyd.
Supplementary Fig. 5—Genetic modification of the purine operon in Bacillus amyloliquefaciens XH7.
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Liao, Y., Ye, Y., Wang, B. et al. Optimization of the purine operon and energy generation in Bacillus amyloliquefaciens for guanosine production. Biotechnol Lett 39, 1675–1682 (2017). https://doi.org/10.1007/s10529-017-2412-4
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DOI: https://doi.org/10.1007/s10529-017-2412-4