Improved production of sublancin via introduction of three characteristic promoters into operon clusters responsible for this novel distinct glycopeptide biosynthesis.

Shengyue Ji,Meng Wang,Abdul Rasheed Baloch,Weili Li,Binyun Cao

doi:10.1186/s12934-015-0201-0

Shengyue Ji, Meng Wang + Show 3 more

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https://doi.org/10.1186/s12934-015-0201-0

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Abstract

BackgroundSublancin is a novel and distinct antimicrobial glycopeptide that can be used as an alternative to conventional antibiotics. The reported production of sublancin by Bacillus subtilis 168 is poor because transcriptional regulatory circuit of sunA, a gene that encodes presublancin, is complex and difficult to control.ResultsA strong inducible and easy to control vegetative σA promoter of Pglv was introduced to replace that of sunA in situ in B. subtilis 1A747 [SPβc, prototroph, the derivative of B. subtilis 168 (trpC2)]. Meanwhile, other two strong promoters of P43 and PluxS were respectively placed before sunI and sunT–bdbA–sunS–bdbB, encoding five functional proteins that involved in the biosynthesis of mature sublancin. 642 mg sublancin was obtained from 1 L culture supernatant of recombinant B. subtilis 1A747 strains. Analysises of electrospray ionization mass spectrometry and circular dichroism spectrum showed that mature sublancin had a molecular weight of 3877.642 Da and displayed a α–helical conformation that are consistent with reported results. In addition, the mature sublancin was proved to be a potent antimicrobial glycopeptide with broad activity spectrum, moderate cytotoxicity and good conditional stability under high temperature, extreme pH and protease–rich environments, thus showing its potential for clinical applications.ConclusionsOur present findings suggest that recombinant B. subtilis 1A747 strains can effectively and efficiently biosynthesize mature sublancin. The replacement of native promoters provides an extra method for production improvement of some other complicated peptides such as nisin and subtilin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0201-0) contains supplementary material, which is available to authorized users.

Highlights

Sublancin is a novel and distinct antimicrobial glycopeptide that can be used as an alternative to conventional antibiotics
Construction of recombinant B. subtilis 1A747 strain Three strong characteristic promoters including vegetative–and–stationary double functional promoter P43 [22], the maltose–inducible promoter Pglv [21] and vegetative promoter PluxS [23], were in situ chromosome–integrated into B. subtilis 1A747 and respectively placed before two genes of sunI and sunA and one gene cluster of sunT–bdbA–sunS–bdbB that are responsible for mature sublancin biosynthesis
Recombinant B. subtilis 1A747 strains were constructed for the efficient biosynthesis of sublancin for commercial applications (Figure 2)

Summary

Introduction

Sublancin is a novel and distinct antimicrobial glycopeptide that can be used as an alternative to conventional antibiotics. The reported production of sublancin by Bacillus subtilis 168 is poor because transcriptional regulatory circuit of sunA, a gene that encodes presublancin, is complex and difficult to control. Other two strong promoters of P43 and PluxS were respectively placed before sunI and sunT–bdbA–sunS–bdbB, encoding five functional proteins that involved in the biosynthesis of mature sublancin. Sublancin (Genbank accession number P68577.1) is a novel distinct peptide that is synthesized by Bacillus subtilis 168. SunA is immediately located downstream of sunI and encodes the presublancin [2], and SunT is an ABC–type transporter with a proteolytic domain that removes the leader peptide from sublancin during its translocation across the membrane [9,10]. SunS is a S–glycosyltransferase that has a CxxS motif; and is involved in biosynthesis of mature sublancin by glucosylating Cys

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