Analysis And Construction Of Knock-out Vectors For RocG Gene And Ure Gene In Bacillus Subtilis Natto

Natto is a Japanese traditional soybean product with a high nutritionalvalue and various healthy functions. It is fermented by strains of Bacillussubtilis natto. However, the high content of ammonia contributes a negativeinfluence to its flavour. CGMCC No.2801, a B. subtilis natto strain withhigh nattokinase activity was isolated from commercial natto. Theammonia content in natto product, which fermented by this strain, canreach347.7±24.31mg/kg.To improve natto’s quality and flavor, modification of metabolicpathways in B. subtilis natto is a good choice. The major metabolicpathways of ammonia production in B. subtilis natto are transdeaminationand transamination catalyzed by glutamate dehydrogenase(GDH), andurease is an enzyme that catalyzes the hydrolysis of urea into carbondioxide and ammonia. It is reported that gene rocG and gene ure code forGDH and urease respectively in B. subtilis natto. Thus site-directedmutagenesis and gene knock-out of rocG and ure may be efficient ways toobtain low ammonia production strains.In this study, gene rocG and gene ure were cloned by PCR usinggenomic DNA of CGMCC No.2801as the template. The primers weredesigned according to the sequence of B.subtilis168. Comparison of GCcontent about coding areas of rocG and ure, amino acid sequences of GDHand urease between CGMCC No.2801and other9reference Bacillusstrains were made. The results showed that both genes and amino acid sequences of GDH and urease in different Bacillus strains have highhomology and conservatism. High stability of genes and coding productsindicates that the enzyme activities can only be changed by modification ingenes.Therefore, site-directed mutagenesis and gene konck-out may be oneof the reasonable and effective solutions to reduce ammonia production. Inaddition, analysis of evolutionary tree based on rocG and ure showed thatGDH and urease in CGMCC No.2801was more closed with B. subtilisthan B. amyloliquefaciens, especially with B. subtilis168. So B. subtilis168can be taken as a reference strain to clone untranslated regions of rocGand ure and construct their knock-out vectors.Gene structures wereanalyzed by NCBI and software Mega5.05. Promoter of rocG andpromoter of ure were similar with-10sequence and-35sequence inprocaryote microbiology, and σApromoter in B. subtilis, respectively. Bothpromoters have high transcription efficiency so as to product abundantGDH and urease to catalyze the reactions of ammonia production.Plasmid pMUTin4, the integrative vector for gram-positives genomics waschosen to construct the knock-out vectors. After analysed its multiplecloning site (MCS) and the restriction sites in1,000bp flanking sequencesof rocG and ure,4restriction sites Hind Ⅲ(AAGCTT), Xho Ⅰ(CTCGAG),Not Ⅰ(GCGGCCGC)and BamH Ⅰ (GGATCC) were chosen forconstruction of knock-out vectors. Resistance gene nisI for screening andflanking sequences of two targeting gene for homologous recombinationwere introduced into pMUTin4, in the order of upstream, nisI anddownstream. The results of restriction endonuclease analysis and DNAsequencing showed that the recombinant knock-out vectors pMUTin4-rocG::nisI and pMUTin4-ure::nisI were constructed successfully. Thesevectors can be used to construct a rocG or ure knock-out B. subtilis nattomutant by homologous recombination, which may produce natto with low ammonia content.