Self-inducible Bacillus subtilis expression system for reliable and inexpensive protein production by high-cell-density fermentation

Abstract

A novel technically compliant expression system was developed for heterologous protein production in Bacillus subtilis with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated manP promoter of the mannose operon, which led to relatively high product yields of 5.3% (5.3 g enhanced green fluorescent protein [eGFP] per 100 g cell dry weight [CDW]) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant B. subtilis strains were used: the ΔmanA (mannose metabolism) strain TQ281 and the ΔmanP (mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6%. The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.

Fig. 1.

Schematic representation of the B. subtilis mannose operon. The genes are shown as thick arrows, the two promoters as thin arrows above the genes, and the cre site near the P_manR as a box. The positive regulatory effect (plus sign) of ManR on the two promoters is indicated.

Fig. 2.

Model of PTS-mediated control of ManR. The domain structure of ManR is shown along with its putative phosphorylation sites (open circles) and the functions of these domains (?, unknown; +, activating; −, deactivating). Phosphoryl groups are indicated as black circles with a white “P.” The mannose operon, including the binding sites for ManR (o, operator), CcpA-HPr-His-P (cre), and the promoters (p), is shown. (A) Glucose (glc) is taken up and phosphorylated by EII^Glc. The PRD2 of ManR becomes dephosphorylated by HPr, while the EII domains remain phosphorylated. ManR is inactive and cannot bind to its operator sequences. Rising intracellular levels of glucose 6-phosphate (glc-6-P) and other metabolites lead to the formation of CcpA-HPr-His-P, a complex that acts as a repressor by binding to the cre site. No expression from the mannose operon promoters takes place. (B) When the inducer mannose is available, it is taken up and phosphorylated by its cognate EII^Man. HPr phosphorylates the PRD2, and the EII domains transfer their phosphoryl groups to EII^Man. ManR is active, and expression from the mannose promoters takes place. (C) When no sugar is available, all PTS components, as well as the PRD2 and EIIB- and EIIA-like domains of ManR, are present in a phosphorylated state. ManR is not active, and no expression from the mannose operon promoters takes place.

Fig. 3.

Physical map of the expression vector pMW168.1. The following genes (arrows) and regions (boxes) are shown: SpcR (aad9), spectinomycin resistance gene; ter, transcriptional terminator from tufA; ′manR, 5′-truncated form of manR; P_manP, promoter region from manPA operon; TIR_gsiB, translational initiation region from gene gsiB; eGFP, enhanced green fluorescent protein gene; ori, origin of replication from pUC18; rep and ori⁺, rep gene (initiator protein) and ori⁺ from pUB110.

Fig. 4.

Fed-batch fermentation of B. subtilis TQ356/pMW168.1. Cell dry weight (X, black circles), total amount of eGFP produced (P_eGFP, black squares), and product yield (P/X, black triangles) are plotted over the fermentation time. The beginning of the fed-batch phase is indicated by a vertical line.

Fig. 5.

SDS-PAGE of cell samples taken from the fermentation with B. subtilis TQ356/pMW168.1. Lanes: M, Roti-Mark Standard protein molecular mass marker; 1, soluble fraction after 12.5 h (batch); 2, soluble fraction after 20.5 h (batch); 3, soluble fraction after 36 h (13.5 h fed-batch); 4, soluble fraction after 41 h (18.5 h fed-batch); 5, soluble fraction after 44 h (21.5 h fed-batch); 6, soluble fraction after 46 h (23.5 h fed-batch, end of process); 7, insoluble fraction after 46 h; 8, culture supernatant after 46 h.