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.

The semipinacol rearrangement (SPR) is highly useful in the asymmetric synthesis of complex compounds. In biological systems, only a few semi-pinacolases belonging to a few families have been identified to catalyze the SPR on alkaloids. Here, based on the biosynthesis of a fungal mycotoxin asteltoxin (1), two semi-pinacolases AstD/MrvD were identified from the epoxide hydrolase family to catalyze type III SPR on the polyketide backbone. They were proposed to catalyze efficient regio-selective hydrolysis on the bis-epoxide and 2,3-migration on the epoxide alcohol for the rearrangement. Based on the comprehensive mutations and chemical calculations, a critical Asp residue was identified as an acid for the coupled catalysis of selective epoxide collapse and subsequent SPR, while other critical residues facilitated efficient hydrolysis and protected carbocation for SPR. Thus, this study expanded the SPR biocatalyst family and provided an understanding of the catalytic mechanisms of these bifunctional semi-pinacolases.

BackgroundSynthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.ResultsA novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43′-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43′-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.ConclusionThe constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.

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Background: Secondary metabolites as natural products from endophytic fungi are important sources of pharmaceuticals. However, there is currently little understanding of endophytic fungi at the omics levels about their potential in secondary metabolites. Calcarisporium arbuscula, an endophytic fungus from the fruit bodies of Russulaceae, produces a variety of secondary metabolites with anti-cancer, anti-nematode and antibiotic activities. A comprehensive survey of the genome and transcriptome of this endophytic fungus will help to understand its capacity to biosynthesize secondary metabolites and will lay the foundation for the development of this precious resource. Results: In this study, we reported the high-quality genome sequence of C. arbuscula NRRL 3705 based on Single Molecule Real-Time sequencing technology. The genome of this fungus is over 45 Mb in size, larger than other typical filamentous fungi, and comprises 10,001 predicted genes, encoding at least 762 secretory-proteins, 386 carbohydrate-active enzymes and 177 P450 enzymes. 398 virulence factors and 228 genes related to pathogen-host interactions were also predicted in this fungus. Moreover, 65 secondary metabolite biosynthetic gene clusters were revealed, including the gene cluster for the mycotoxin aurovertins. In addition, several gene clusters were predicted to produce mycotoxins, including aflatoxin, alternariol, destruxin, citrinin and isoflavipucine. Notably, two independent gene clusters were shown that are potentially involved in the biosynthesis of alternariol. Furthermore, RNA-Seq assays showed that only expression of the aurovertin gene cluster is much stronger than expression of the housekeeping genes under laboratory conditions, consistent with the observation that aurovertins are the predominant metabolites. Gene expression of the remaining 64 gene clusters for compound backbone biosynthesis was all lower than expression of the housekeeping genes, which partially explained poor production of other secondary metabolites in this fungus. Conclusions: Our omics data, along with bioinformatics analysis, indicated that C. arbuscula NRRL 3705 contains a large number of biosynthetic gene clusters and has a huge potential to produce a profound number of secondary metabolites. This work also provides the basis for development of endophytic fungi as a new resource of natural products with promising biological activities.

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