Engineering Bacillus subtilis Cells as Factories:Enzyme Secretion and Value-added Chemical Production

Bacillus subtilis has been studied for more than half a century, ever since the dawn of molecular biology, as a representative Gram-positive bacterium and cell factory. Two characteristic capacities of B. subtilis, namely its natural competence for DNA uptake and high-level enzyme secretion, have been investigated and exploited intensively during these long years. As a consequence, this bacterium has evolved into an excellent platform for synthetic biological research and development. In this review, we outline basic concepts for B. subtilis cell factory engineering, and we describe several examples of its applications in the production of proteins and high-value metabolites. In particular, we highlight engineering approaches that can make the already very efficient Bacillus protein secretion pathways even more efficient for the production of enzymes and pharmaceutical proteins. We further showcase examples of metabolic engineering in B. subtilis based on synthetic biology principles to produce various high-value or health-promoting substances, especially inositol stereoisomers. We conclude that the versatile traits of B. subtilis, combined with multi-omics approaches and rapidly developing technologies for genome engineering and high-throughput screening enable us to modify and optimize this bacterium's metabolic circuits to deliver compounds that are needed for a green and sustainable society as well as a healthy population

Staphylococcus aureus cell wall maintenance:the multifaceted roles of peptidoglycan hydrolases in bacterial growth, fitness, and virulence

Staphylococcus aureus is an important human and livestock pathogen that is well-protected against environmental insults by a thick cell wall. Accordingly, the wall is a major target of present-day antimicrobial therapy. Unfortunately, S. aureus has mastered the art of antimicrobial resistance, as underscored by the global spread of methicillin-resistant S. aureus (MRSA). The major cell wall component is peptidoglycan. Importantly, the peptidoglycan network is not only vital for cell wall function, but it also represents a bacterial Achilles' heel. In particular, this network is continuously opened by no less than 18 different peptidoglycan hydrolases (PGHs) encoded by the S. aureus core genome, which facilitate bacterial growth and division. This focuses attention on the specific functions executed by these enzymes, their subcellular localization, their control at the transcriptional and post-transcriptional levels, their contributions to staphylococcal virulence and their overall importance in bacterial homeostasis. As highlighted in the present review, our understanding of the different aspects of PGH function in S. aureus has been substantially increased over recent years. This is important because it opens up new possibilities to exploit PGHs as innovative targets for next-generation antimicrobials, passive or active immunization strategies, or even to engineer them into effective antimicrobial agents. Here, we review the roles of peptidoglycan hydrolases of the Gram-positive bacterial pathogen Staphylococcus aureus in bacterial growth and division, cell wall maintenance, protein localization, pathogenesis and antimicrobial susceptibility

Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis:pathways, applications, and innovation potential

Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for efficient production platforms to deliver such proteins in high amounts and high quality. Gram-positive bacteria, particularly bacilli such as Bacillus subtilis, are favored for the production of secreted industrial enzymes. Nevertheless, recombinant protein production in the B. subtilis cell factory can be very challenging due to bottlenecks in the general (Sec) secretion pathway as well as this bacterium's intrinsic capability to secrete a cocktail of highly potent proteases. This has placed another Gram-positive bacterium, Lactococcus lactis, in the focus of attention as an alternative, non-proteolytic, cell factory for secreted proteins. Here we review our current understanding of the secretion pathways exploited in B. subtilis and L. lactis to deliver proteins from their site of synthesis, the cytoplasm, into the fermentation broth. An advantage of this cell factory comparison is that it identifies opportunities for protein secretion pathway engineering to remove or bypass current production bottlenecks. Noteworthy new developments in cell factory engineering are the mini-Bacillus concept, highlighting potential advantages of massive genome minimization, and the application of thus far untapped 'non-classical' protein secretion routes. Altogether, it is foreseen that engineered lactococci will find future applications in the production of high-quality proteins at the relatively small pilot scale, while engineered bacilli will remain a favored choice for protein production in bulk

Empowering antimicrobial photodynamic therapy of Staphylococcus aureus infections with potassium iodide

Infections caused by the Gram-positive bacterium Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), impose a great burden on global healthcare systems. Thus, there is an urgent need for alternative approaches to fight staphylococcal infections, such as targeted antimicrobial photodynamic therapy (aPDT). We recently reported that targeted aPDT with the S. aureus-specific immunoconjugate 1D9-700DX can be effectively applied to eradicate MRSA. Nonetheless, the efficacy of aPDT in the human body may be diminished by powerful antioxidant activities. In particular, we observed that the efficacy of aPDT with 1D9-700DX towards MRSA was reduced in human plasma. Here we show that this antagonistic effect can be attributed to human serum albumin, which represents the largest pool of free thiols in plasma for trapping reactive oxygen species. Importantly, we also show that our targeted aPDT approach with 1D9-700DX can be empowered by the non-toxic inorganic salt potassium iodide (KI), which reacts with the singlet oxygen produced upon aPDT, resulting in the formation of free iodine. The targeted iodine formation allows full eradication of MRSA (more than 6-log reduction) without negatively affecting other non-targeted bacterial species or human cells. Altogether, we show that the addition of KI allows a drastic reduction of both the amount of the immunoconjugate 1D9-700DX and the irradiation time needed for effective elimination of MRSA by aPDT in the presence of human serum albumin

GP4:an integrated Gram-Positive Protein Prediction Pipeline for subcellular localization mimicking bacterial sorting

Subcellular localization is a critical aspect of protein function and the potential application of proteins either as drugs or drug targets, or in industrial and domestic applications. However, the experimental determination of protein localization is time consuming and expensive. Therefore, various localization predictors have been developed for particular groups of species. Intriguingly, despite their major representation amongst biotechnological cell factories and pathogens, a meta-predictor based on sorting signals and specific for Gram-positive bacteria was still lacking. Here we present GP(4), a protein subcellular localization meta-predictor mainly for Firmicutes, but also Actinobacteria, based on the combination of multiple tools, each specific for different sorting signals and compartments. Novelty elements include improved cell-wall protein prediction, including differentiation of the type of interaction, prediction of non-canonical secretion pathway target proteins, separate prediction of lipoproteins and better user experience in terms of parsability and interpretability of the results. GP(4) aims at mimicking protein sorting as it would happen in a bacterial cell. As GP(4) is not homology based, it has a broad applicability and does not depend on annotated databases with homologous proteins. Non-canonical usage may include little studied or novel species, synthetic and engineered organisms, and even re-use of the prediction data to develop custom prediction algorithms. Our benchmark analysis highlights the improved performance of GP(4) compared to other widely used subcellular protein localization predictors. A webserver running GP(4) is available at http://gp4.hpc.rug.nl