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.

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BackgroundBacillus subtilis, a Gram-positive organism, has been developed to be an attractive expression platform to produce both secreted and cytoplasmic proteins owing to its prominent biological characteristics. We previously developed an auto-inducible expression system containing the srfA promoter (PsrfA) which was activated by the signal molecules acting in the quorum-sensing pathway for competence. The PsrfA promoter exhibited the unique property of inducer-free activity that is closely correlated with cell density.ResultsTo improve the PsrfA-mediated expression system to the high-cell-density fermentation for industrial production in the B. subtilis mutant strain that is unable to sporulate, a spore mutant strain BSG1682 was developed, and the PsrfA promoter was enhanced by promoter engineering. Using green fluorescent protein (GFP) as the reporter, higher fluorescent intensity was observed in BSG1682 with expression from either plasmid or chromosome than that of the wild type B. subtilis 168. Thereafter, the PsrfA was engineered, yielding a library of PsrfA derivatives varied in the strength of GFP expression. The P23 promoter exhibited the best performance, almost twofold stronger than that of PsrfA. Two heterologous proteins, aminopeptidase (AP) and nattokinase (NK), were successfully overproduced under the control of P23 in BSG1682. Finally, the capacity of the expression system was demonstrated in batch fermentation in a 5-L fermenter.ConclusionsThe expression system demonstrates prominence in the activity of the auto-inducible promoter. Desired proteins could be highly and stably produced by integrating the corresponding genes downstream of the promoter on the plasmid or the chromosome in strain BSG1682. The expression system is conducive to the industrial production of pharmaceuticals and heterologous proteins in high-cell-density fermentation in BSG1682.

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In order to verify the cryoprotective effect of an antifreeze protein (BaAFP-1) obtained from barley on bread dough, the effect of BaAFP-1 on the rheological properties, microstructure, fermentation, and baking performance including the proofing time and the specific volume of bread dough and bread crumb properties during freezing treatment and freeze-thaw cycles were analysed. BaAFP-1 reduced the rate of decrease in storage modulus and loss modulus values during freezing treatment and freeze-thaw cycles. It influenced the formation and the shape of ice formed during freezing and inhibited ice recrystallization during freeze-thaw. BaAFP-1 maintained gas production ability and gas retention properties, protected gluten network and the yeast cells from deterioration caused by ice formation and ice crystals recrystallisation in dough samples during freezing treatment and freeze-thaw treatment. It slow down the increase rate of hardness of bread crumb. The average area of pores in bread crumbs decreased significantly (p < 0.05) as the total number of pores increased (p < 0.05), and the addition of BaAFP-1 inhibited this deterioration. These results confirmed the cryoprotective activity of BaAFP-1 in bread dough during freezing treatment and freeze-thaw cycles.