Characterization of genome-reduced Bacillus subtilis strains and their application for the production of guanosine and thymidine.
BACKGROUND
Genome streamlining has emerged as an effective strategy to boost the production efficiency of bio-based products. Many efforts have been made to construct desirable chassis cells by reducing the genome size of microbes. It has been reported that the genome-reduced Bacillus subtilis strain MBG874 showed clear advantages for the production of several heterologous enzymes including alkaline cellulase and protease. In addition to enzymes, B. subtilis is also used for the production of chemicals. To our best knowledge, it is still unknown whether genome reduction could be used to optimize the production of chemicals such as nucleoside products.


RESULTS
In this study, we constructed a series of genome-reduced strains by deleting non-essential regions in the chromosome of B. subtilis 168. These strains with genome reductions ranging in size from 581.9 to 814.4 kb displayed markedly decreased growth rates, sporulation ratios, transformation efficiencies and maintenance coefficients, as well as increased cell yields. We re-engineered the genome-reduced strains to produce guanosine and thymidine, respectively. The strain BSK814G2, in which purA was knocked out, and prs, purF and guaB were co-overexpressed, produced 115.2 mg/L of guanosine, which was 4.4-fold higher compared to the control strain constructed by introducing the same gene modifications into the parental strain. We also constructed a thymidine producer by deleting the tdk gene and overexpressing the prs, ushA, thyA, dut, and ndk genes from Escherichia coli in strain BSK756, and the resulting strain BSK756T3 accumulated 151.2 mg/L thymidine, showing a 5.2-fold increase compared to the corresponding control strain.


CONCLUSIONS
Genome-scale genetic manipulation has a variety of effects on the physiological characteristics and cell metabolism of B. subtilis. By introducing specific gene modifications related to guanosine and thymidine accumulation, respectively, we demonstrated that genome-reduced strains had greatly improved properties compared to the wild-type strain as chassis cells for the production of these two products. These strains also have great potential for the production of other nucleosides and similar derived chemicals.
Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.|College of Life Science, Shihezi University, Shihezi, 832000, China.
Yang
Y
Li
Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Xujun
X
Zhu
Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.|Tianjin Vocational College of Bioengineering, Tianjin, 300462, China.
Xueyu
X
Zhang
Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Jing
J
Fu
Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Zhiwen
Z
Wang
Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China. chentao@tju.edu.cn.|Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan, 430068, China. chentao@tju.edu.cn.
Tao
T
Chen
Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Xueming
X
Zhao
D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial; D006151:Guanosine / Q000378:metabolism*; D060847:Metabolic Engineering; D010727:Phosphoric Diester Hydrolases / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism*
D029968:Escherichia coli Proteins; C406334:ushA protein, E coli; D006151:Guanosine; D010727:Phosphoric Diester Hydrolases; D013936:Thymidine
Bacillus subtilis; Chassis cell; Genome reduction; Guanosine; Nucleosides; Thymidine