Studies On The PKS Gene Cluster Promoter Of A Macrolactins Producing Bacillus Sp.

Macrolactins are a novel class of macrolides which have many biological activities,including antibacterial,immune inhibition,anti-tumor,etc.They have immense application value in medicament.At present the yield of macrolactins by undomesticated bacterial strains fermentation is relatively low,which greatly limited the reasearch and application of macrolactins.Thus it has very important significance to increase the production of macrolactins by related biosynthestic gene cluster modification.A wild macrolactins-producing bacterial named as ZJU-2011,which was found by our laboratory,was researched in this study.It was identified as a new strain of Bacillus amyloliquefaciens based on the results of DNA sequencing.The sequence comparison results of the high conservative AT structure and possible promoter region of the PKS gene cluster with reference gene cluster confirmed the existence of PKS gene cluster and the promoter region,which was 1Kb in front of the first module.We constructed the promoter detection vectors using GFP as reporter gene.Comparison results showed the original promoter of PKS gene cluster was weaker than the P43 promoter.Further research showed that the original promoter could be regulated by glucose and 37? was its optimal temperature.A promoter mutant library was constructed on the basis of P43 promoter by saturated mutation.Optimization results showed that the optimal annealing temperature of the CPEC cloning was 61.6? and the optimal PCR cycle number was 25.LacZ was chosen as the reporter gene to construct the promoter mutant library,?-Galactosidase activity was used to represent the promoter activity.11 positive mutants were found and the activity of mutant labelled "7" was 167.7%of the P43 promoter.The feasibility of gene knockout by homologous recombination in ZJU-2011 was studied.The optimal electro-transformation conditions were as follows:1%glycine,OD600?0.9,the plasmid was demethylated before transformation,electric field intensity 21KV/cm,resistance 200?.We constructed an integration vector pIEFMLN using mazF toxic protein as the counter-selection marker.The positive transformant of homologous single-crossover was successfully obtained.But experiments showed that the probability of homologous double-crossover was lower than 1/1500.Thus,we deduced that gene knock-out by homologous recombination was not feasible in Bacillus amyloliquefaciens ZJU-2011.We tried to establish the CRISPR-Cas9 gene knock-out system of Bacillus amyloliquefaciens.The CRISPR-Cas9 related gene knock-out vector pHYpCasMLN was successfully constructed and confirmed to be transformed into ZJU-2011.