The Molecular Breeding Of Guanosine Industrial Producer Based On Whole Genome Sequencing And Systems Biology

Aguanosine industrial producer Bacillus amyloliquefaciens XH7was used as starting strain inthis work. In order to improve the efficiency of breeding and create high yielding stablegenetic engineering bacteria of guanosine, it was studied to improve guanosine productionwith whole genome sequencing and systems biology analysis. The main results were achievedand shown as follows:(1) Whole-genome sequencing of Bacillus amyloliquefaciens XH7Whole-genome sequencing of XH7was carried out by Solexa sequencing to produce629Mb filtered sequences, representing a160-fold coverage of the genome. The sequences wereassembled into76contigs and23scaffolds using the SOAPdenovo package. Scaffolds'relative position was identified by blasting with the published Bacillus amyloliquefaciensFZB42genome sequence. Then, special primers were designed to amplify the sequences ofinner gaps and outer gaps of the scaffolds. Finally, the identified PCR products and thescaffold make up of the whole genome sequence. The genome sequence of Bacillusamyloliquefaciens XH7was deposited in GenBank under the accession number CP002927.The complete genomic information of the Bacillus amyloliquefaciens XH7is contained on asingle circular chromosome of3,939,203bp with an average GC content of45.82%. Atotal of4,204protein coding genes,75tRNA genes, and7rRNA operons were identified.Comparative genomics analysis revealed that an approximately1.26Mb DNA fragment wasinverted, and multiple inactive gene realated with guanosine high-yield were identified.(2) Systems biology analysis of guanosine biosynthesisBased on Bacillus subtilis metabolic model iYO844, systems biology methods were usedto analyze the theoretical conversion yields of guanosine and in silico strain modification.Besides, two strategies including the flux distribution comparison analysis (FDCA) methodand product stress-flux distribution comparison analysis (PS-FDCA) method were employedto predict potential gene targets in order to improve the guanosine production. The resultswere reliable and could provide guidance for the follow-up strain modification.(3) Genetic transformation system of Bacillus amyloliquefaciens and molecular breedingA protocol for electroporation-competent cells preparation and transformation of XH7 strain was created. Based on the results of in silico strain improment for guanosine production,four genes (prs, purF, guaB, vgb) were transformed into XH7strain. The results of shakingflask fermentation showed that overexpression of prs and purF genes enhanced the guanosineproduction yield by10.8%and20.9%, while overexpression of guaB genes did not enhancethe guanosine production yield, but caused a slight decrease of guanosine production and asignificant decrease of growth rate. The expression of vgb caused no significant increase ofproduction yield, but vgb expression promoted cell growth, shortened fermentation period andreduced energy consumption.(4) Gene-knockout technology of Bacillus amyloliquefaciens and molecular breedingA gene-knockout technology based on temperature-sensitive plasmid pKS1wasestablished, and pur operon duplication mutants, phosphotransferase system and deoDdeficient mutants were constructed based on the results of in silico strain improment forguanosine production. As the total length of pur operon is12kb, it is very difficult to clone itdirectly. A chloramphenicol resistance gene and a pur operon promoter about1kbhomologous sequence were inserted into3' end of pur operon terminator. By increasing theconcentration of chloramphenicol in the medium, the pur operon was induced doubling in thegenome. The max copy number of the pur operon was about11by qPCR. Meanwhile, theproduction yield was increased by25.5%. In guanosine fermentation experiments, theguanosine production yields of ptsG genetic defect strains was increased by23.9%, while thatof ptsHI genetic defect strain was decreased by81.8%. These results showed that ptsGdeletion was benefit to increase the carbon flux into the pentose phosphate pathway, whileptsHI deletion completely blocked the glucose use. The deoD genetic defect strain did notincrease the guanosine production yield, which suggested that the protein DeoD encoded bydeoD did not degradate the guanosine.In this study, the molecular breeding of guanosine industrial producer XH7was carriedout based on systems biology. The results show that the method is reliable and effective, andcan provide new ideas and research directions for further follow-up molecular breeding. Theplatform constructed by this work is also suitable for other kinds of products research. Infuture, the platform will get well developed step-by-step with the development of systemsbiology.