Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome

<p>Abstract</p> <p>Background</p> <p>Plasmid-less, engineered <it>Bacillus </it>strains have several advantages over plasmid-carrier variants. Specifically, their stability and potential ecological safety make them of use in industrial applications. As a rule, however, it is necessary to incorporate many copies of a key gene into a chromosome to achieve strain performance that is comparable to that of cells carrying multiple copies of a recombinant plasmid.</p> <p>Results</p> <p>A plasmid-less <it>B. subtilis </it>JE852-based strain secreting glutamyl-specific protease (GSP-the protein product of the <it>mpr </it>gene from <it>B. amyloliquefaciens</it>) was constructed that exhibits decreased levels of other extracellular proteases. Ten copies of an <it>mpr^B.amy</it>cassette in which the GSP gene was placed between the promoter of the <it>B. amyloliquefaciens rplU-rpmA </it>genes and the Rho-independent transcription terminator were ectopically inserted into designated (3 copies) and random (7 copies) points in the recipient chromosome. The resulting strain produced approximately 0.5 g/L of secreted GSP after bacterial cultivation in flasks with starch-containing media, and its performance was comparable to an analogous strain in which the <it>mpr^B.amy</it>cassette was carried on a multi-copy plasmid.</p> <p>Conclusion</p> <p>A novel strategy for ectopically integrating a cassette into multiple random locations in the <it>B. subtilis </it>chromosome was developed. This new method is based on the construction of DNA fragments in which the desired gene, marked by antibiotic resistance, is sandwiched between "front" and "back" portions of random chromosomal DNA restriction fragments. These fragments were subsequently inserted into the targeted sites of the chromosome using double-cross recombination. The construction of a marker-free strain was achieved by gene conversion between the integrated marked gene and a marker-less variant carried by plasmid DNA, which was later removed from the cells.</p

From gut dysbiosis to altered brain function and mental illness: mechanisms and pathways

The human body hosts an enormous abundance and diversity of microbes, which perform a range of essential and beneficial functions. Our appreciation of the importance of these microbial communities to many aspects of human physiology has grown dramatically in recent years. We know, for example, that animals raised in a germ-free environment exhibit substantially altered immune and metabolic function, while the disruption of commensal microbiota in humans is associated with the development of a growing number of diseases. Evidence is now emerging that, through interactions with the gut-brain axis, the bidirectional communication system between the central nervous system and the gastrointestinal tract, the gut microbiome can also influence neural development, cognition and behaviour, with recent evidence that changes in behaviour alter gut microbiota composition, while modifications of the microbiome can induce depressive-like behaviours. Although an association between enteropathy and certain psychiatric conditions has long been recognized, it now appears that gut microbes represent direct mediators of psychopathology. Here, we examine roles of gut microbiome in shaping brain development and neurological function, and the mechanisms by which it can contribute to mental illness. Further, we discuss how the insight provided by this new and exciting field of research can inform care and provide a basis for the design of novel, microbiota-targeted, therapies.GB Rogers, DJ Keating, RL Young, M-L Wong, J Licinio, and S Wesseling