Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering.

Cui-Cui Miao,Yan-Bing Lu,Lin-Li Han,Hong Feng

doi:10.3390/microorganisms8071030

Cui-Cui Miao, Yan-Bing Lu + Show 2 more

Open Access

https://doi.org/10.3390/microorganisms8071030

Copy DOI

Journal: Microorganisms	Publication Date: Jul 12, 2020
Citations: 12	License type: CC BY 4.0

Affiliation: Sichuan University

Abstract

Bacillus subtilis is an ideal host for secretion and expression of foreign proteins. The promoter is one of the most important elements to facilitate the high-level production of recombinant protein. To expand the repertoire of strong promoters for biotechnological applications in Bacillus species, 14 highly transcribed genes based on transcriptome profiling of B. pumilus BA06 were selected and evaluated for their promoter strength in B. subtilis. Consequently, a strong promoter P2069 was obtained, which could drive the genes encoding alkaline protease (aprE) and green fluorescent protein (GFP) to express more efficiency by an increase of 3.65-fold and 18.40-fold in comparison with the control promoter (PaprE), respectively. Further, promoter engineering was applied to P2069, leading to a mutation promoter (P2069M) that could increase GFP expression by 3.67-fold over the wild-type promoter (P2069). Moreover, the IPTG-inducible expression systems were constructed using the lac operon based on the strong promoters of P2069 and P2069M, which could work well both in B. subtilis and B. pumilus. In this study, highly efficient expression system for Bacillus was constructed based on transcriptome data and promoter engineering, which provide not only a new option for recombinant expression in B. subtilis, but also novel genetic tool for B. pumilus.

Highlights

Bacillus subtilis, a rod-shaped Gram-positive soil bacterium, has been used as a model microorganism both in basic research and biotechnological applications for more than a century [1,2,3,4]
Bacillus subtilis WB600 [37] and B. pumilus BA06 were used for recombinant expression
Transcriptome profiling of B. pumilus BA06 was carried out by RNA-seq, which showed more than 96% genes had FPKM values less than 2000

Summary

Introduction

A rod-shaped Gram-positive soil bacterium, has been used as a model microorganism both in basic research and biotechnological applications for more than a century [1,2,3,4]. It can produce and secrete abundant industrial proteins [5]. In order to obtain large amounts of recombinant proteins, it is an important way to use high-copy plasmids and strong promoters. Promoters are important regulatory elements, which facilitate high-level gene expression and recombinant protein production [6,8]. Transcriptomic data are useful information for screening strong promoters in biotechnological applications [24]

Methods

Results

Discussion

Conclusion