Construction and development of an auto-regulatory gene expression system in Bacillus subtilis

Abstract

BackgroundBacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA).ResultsThe promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the −10 and −35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level.ConclusionThe auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression system that has an expression pattern that is split between cell-growth and over-expression, leading to an increase in cell density and elevating the overall expression levels of heterologously expressed proteins. The broad applicability of this system and inducer-free expression property in B. subtilis facilitate the industrial scale-up and medical applications for the over-production of a variety of desired proteins.

Sugarcane growth and nutrition levels are differentially affected by the application of PGPR and cane waste

“…Several isolates of Bacillus , especially Bacillus subtilis directly affect plant growth promotion, by the mechanisms such as acting as nitrogen fixers, as well as producers of indol butyric acid (IBA) (Araujo, Henning, & Hungria, ), indol acetic acid (IAA), and siderophores (Puente, Bashan, Li, & Lebsky, ), in addition to biocontrol activity (Ali, Charles, & Glick, ; Hanif et al., ; Oslizlo et al., ) and activities of protein expression relevant to the pharmaceutical and industrial sector (Guan et al., ). Bacillus subtilis has such varied functional capacities, which suggests that it has a versatile potential to be used as a plant growth promoter.…”

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

“…To comprehensively compare the expression levels between commonly used strong constitutive promoters and P43′-riboE1, three wild-type promoters P43, P aprE and P srfA , which have been characterized in previous studies [19, 20], were employed to express the GFP reporter. BSG43, BSG04, and BSG03 carrying the three strong promoters respectively, were cultured in LB for 31 h. The BSG11 harbouring pBSG11 was treated with 8 mM theophylline for 24 h. Then, the final expression level was determined by SDS-PAGE analysis.…”

“…Inverse-PCR was used to insert or substitute target genes into the plasmid as previously described [20]. Dpn I (New England BioLabs) was used to digest the template plasmid after the inverse-PCR reactions.…”

“…The fluorescence measurements were conducted in accordance with a previously reported procedure, with some modifications [20]. In brief, the cultured recombinant B. subtilis cells were collected by centrifugation at 4000 rpm for 5 min and then the supernatant was discarded.…”

Safe preparation of beefy meaty peptide with Bacillus subtilis

“…Bacillus subtilis ( B. subtilis ) has been regarded as a ‘generally recognised as safe’ (GRAS) microbe (Guan et al , ; Guan et al , ). Since it does not secret any exotoxin or endotoxin, B. subtilis owns natural advantages in food safety including the biological expression and industrial production of recombinant food proteins, such as amylase (Chen et al , ), lipase (Lu et al , ) and proinsulin (Jorge & Rosalia, ).…”