Studies On Macrolactins Biosynthetic Genes In Bacillus Marinus B-9987

Bacillus marinus B-9987 was isolated from the rhizosphere of halophyte Suaeda salsa collected in the intertidal zone of Bohai Bay of Eastern China. B. marinus B-9987 is able to produce macrolactins, which are 24-membered macrolides with antibacterial, antiviral and antitumor activities. But studies on post-translational modification and transcriptional control of this kind of compounds are still unknown. The main skeleton of macrolactins is assembled by trans-acyltransferase (AT) polyketide synthase (PKS). However, some macrolactin derivatives are only obtained after certain post-translational modification steps, such as acylation and glycosylation. Thus, these modification steps are considered to play important roles to get novel bioactive macrolactin derivatives. This article aims to study on the regulatory gene(s) involved in macrolactin biosynthesis and key enzymes in B. marinus B-9987 by the combination methods of bioinformatics, biochemistry, molecular biology as well as natural product chemistry. The main works were as follows:Firstly, we identified the regulatory gene related to macrolactin biosyntheticsis in B. marinus B-9987 and did a pilot study. According to the bioinformatics analysis, bm0677 gene was predicted to code a transcriptional regulator which belongs to the GntR family. Then we determined the function of gene bm0677 by homologous recombination and gene overexpression strategy. The fermentation broth of wild, mutant and overexpression strains were subject to HPLC analysis. According to the results, the production of glycosylated macrolactins and bacillaenes was decreased, while acylated compounds were accumulated in the mutant strain compared with those of the wild type strain at the same fermentation condition. Therefore, it is speculated that gene bm0677 might have a positive regulation on the glycosylation but a nagative regulation on the acylation in B. marinus B-9987. Then we compared the colony morphology and biofilm formation of the wild type and mutant strains. The result showed that the colony surface of the wild type was dry but the mutant strain was wet. The biofilm was observed for wild type but not for the mutant strain. The results above indicated that gene bm0677 may regulate biofilm formation of B. marinus B-9987 as well.Secondly, we tested different reaction conditions, including reaction time, enzyme concentration and substrate concentration,to study the BmmGT1-catalyzed reactions and obtain new glycosylated compounds. We found that BmmGT1 can transfer multiple glucose from UDP-Glucose to different hydroxyl position (7-or 13-or 15-) of macrolactin A under the reaction condition of 0.2 mM of macrolactin A,8 μM of BmmGT1 and 4 mM of UDP-Glucose. Finally, we obtained four new glycosyl macrolactin derivatives and elucidated their structures on the basis of MS and NMR data analysis.The results above are helpful to decifer the regulatory mechanism of macrolactins biosynthesis; and four new macrolactin derivatives we obtained by exploring different enzyme reaction conditions may provide material base for new drug discovery.