Less is more: Genome-reduced Bacillus subtilis for protein production

Bacteria are nowadays widely used as cell factories for production of enzymes and pharmaceutical products that, respectively, replace wasteful chemical processes and support human health and wellbeing. However, not all societal demands can be adequately met with current bacterial production strains. Genome engineering could offer the possibility to create novel strains with the desired properties. The present PhD thesis describes research on genome-minimised strains of the popular bacterial cell factory Bacillus subtilis, a non-pathogenic soil bacterium that is often used to produce enzymes and vitamins at industrial scale. The results show that strains lacking 26%, 31% or even 35% of the genome gained the ability to efficiently secrete antigens of the major human pathogen Staphylococcus aureus. This is an important observation, as it opens up the possibility to apply engineered strains for large-scale vaccine production, which is not possible with current production strains. To pinpoint the cellular changes underlying the enhanced properties, the total protein composition and metabolic features of genome-reduced Bacillus strains were investigated. This revealed that the new strains have a higher capacity for the synthesis and secretion of proteins, while suffering less from the detrimental effects of so-called ‘protein production stress’. The enhanced protein secretion allows for easy downstream product purification. Moreover, some of the genome-minimised strains show enhanced fermentation properties. To conclude, the results described in this thesis highlight the great potential of genome engineering to develop new-generation bacterial cell factories for industrial applications that satisfy the ever-increasing demands for high quality enzymes and pharmaceutical proteins

The cyanobacterial rhomboid protease is a regulator of the CCM

Cyanobacteria are aquatic photosynthetic bacteria and useful models for study of the chloroplast and photosynthesis. We are studying a ‘rhomboid’ membrane-located proteases in Synechocystis sp. PCC 6803, which appears to function as a previously undiscovered regulator of the carbon concentrating mechanism (CCM) of this phototroph. Rhomboids are almost ubiquitous across evolution, and are known to activate diverse cellular processes via proteolysis of their specific, membrane-sequestered substrates. Although this well-conserved family has solved crystal structures of bacterial enzymes such as Escherichia coli GlpG, ironically, most work has been carried out on eukaryotic representatives. Following our study of the Arabidopsis thaliana chloroplast RBL10 protease, we identified cyanobacterial orthologues with the aim of discovering if roles might be conserved between these and organellar rhomboids. Molecular biology and reverse-genetics studies were made on slr1461, a mutant in the single rhomboid protease of Synechocystis. When photosynthetic parameters were investigated, it could be seen that inactivation of slr1461 did not affect nonphotochemical quenching, unlike the chloroplast rbl10 mutant, but Slr1461 was required for reduction of photosynthetic activity in mixotrophic conditions. This reduction allows cyanobacteria to avoid expending energy on the uptake of CO2 when an organic carbon source can be utilised: as might be expected, therefore, Slr1461 transcription was linked with downregulation of genes encoding proteins facilitating high-affinity CO2 import under high CO2 and mixotrophic conditions. Quantitative RT-PCR of CCM network genes suggested that Slr1461 is located upstream of known regulators, including another membrane protease, the Slr0228 FtsH, and a central, controlling transcription factor NdhR

Imaging polyphenolic therapeutic compounds in a eukaryotic model microbe

Flavonoids are polyphenolic metabolites that have a range of physiological and developmental functions in plants. They are the focus of much work as potential therapeutics, although investigation of specific mode of action remains a notably under-researched area. Monitoring transport and location of flavonoids in cells is difficult because, despite a role in UV-absorption in plants, they emit only low levels of fluorescence. Visualising them in plants is possible using the Naturstoff reagent (NA), reported historically to be a polyphenol-fluorescence-enhancing stain. We explored therefore whether this agent was effective during preclinical assessment of polyphenolic therapeutics in a microbial-model. The eukaryote Dictyostelium discoideum has been shown to be a useful model when identifying novel drug targets for treating various diseases. For example, in the case of polycystic kidney disease, naringenin decreased Dictyostelium cell division whereas a polycystin-2-null Dictyostelium line was resistant to the flavonoid, and, subsequently, naringenin treatment proved to reduce cyst-formation in mammalian-kidney model cell lines1. To monitor transport and site of action of the drugs investigated in such studies, we developed a method using NA-staining in this model organism. A range of polyphenolics were assayed in cells, cell-extracts and the cell-washes, and NA-enhanced imaging was evaluated in parallel with LCMS-quantification. NA-enhanced fluorescence of compounds at therapeutically relevant concentrations proved an effective and qualitative measure of transport and localisation in Dictyostelium, and could be used in concert with localisation dyes. Fluorescence-enhancement is limited to a subset of flavonoids, however, and not more widely applicable in our studies to date

Establishment of maize resistance to fungal diseases by host-induced gene silencing and site-directed mutagenesis

Maize is one of the most cultivated crops in the world. A disease called anthracnose accounts for up to 80% of the loss in maize production. It is caused by the hemibiotrophic fungus Colletotrichum graminicola. Unfortunately, the disease is notoriously difficult to combat, since host resistance mechanisms are hardly available. In the present investigation, the principle of host-induced gene silencing (HIGS) was employed to protect maize plants from C. graminicola infection. HIGS is an RNA-interefence (RNAi)-based process, wherein plant-produced short interfering RNAs (siRNA) are taken up by the fungus and trigger the silencing of cognate genes of the latter. In the present study, genes encoding fungicide targets were chosen as HIGS targets, namely C. graminicola -Tubulin 2 and Succinate dehydrogenase 1. RNAi vectors were designed using appropriate regions of these target genes. Transgenic plants expressing RNAi constructs were infected with C. graminicola, whereby the plants showed quantitative resistance. In addition to the HIGS approach, a further strategy was pursued, which consisted in knocking out a susceptibility factor against C. graminicola by means of targeted mutagenesis. This factor was the 9-LIPOXYGENASE LOX3 gene from maize, for which several mutated plants were generated by expression of RNA-directed Cas9 endonuclease. Homozygous lox3 mutants were tested in C. graminicola infection assays to analyze the consequences of their mutations. Quantification of fungal biomass revealed that the lox3 mutants were significantly less colonized by C. graminicola compared to the non-mutated wild-type. Corn common smut, another important fungal disease, is caused by the biotrophic pathogen Ustilago maydis. Transcriptional data (Doehlemann et al., 2008) collected during the course of infection with U. maydis showed that, depending on the infection, several members of the LOX gene family are upregulated, one of which is LOX3. Therefore, the available lox3 mutants were tested for their response to infection with U. maydis. The quantification of the disease symptoms showed that the lox3 mutants showed a moderate resistance against U. maydis infections. Furthermore, the quantification of the biomass of U. maydis revealed that the lox3 mutants were colonized by the fungus to a lesser extent compared to the wild-type. Furthermore, infection tests were performed using lox3 mutants independently produced by transposon insertion mutagenesis. These lines showed a resistance behavior similar to that of Cas9-induced mutants, by which the anticipated role of LOX3 for the interaction of maize and U. maydis was corroborated. From the literature it is known that U. maydis suppresses the accumulation of reactive oxygen species (ROS) to establish its biotrophic mode of pathogenesis. A ROS accumulation test revealed that lox3 mutants feature increased ROS accumulationcompared to the wild-type, suggesting that the immunity of the mutants triggered by pathogen-associated molecular pattern (PAMP) led to a reduction in the severity of fungal infection. This is the first study showing that lox3 mutants show moderate resistance to U. maydis. In view of these results, it is concluded that LOX3 is a susceptibility factor for U. maydis as wel

Modern meat: the next generation of meat from cells

Modern Meat is the first textbook on cultivated meat, with contributions from over 100 experts within the cultivated meat community. The Sections of Modern Meat comprise 5 broad categories of cultivated meat: Context, Impact, Science, Society, and World. The 19 chapters of Modern Meat, spread across these 5 sections, provide detailed entries on cultivated meat. They extensively tour a range of topics including the impact of cultivated meat on humans and animals, the bioprocess of cultivated meat production, how cultivated meat may become a food option in Space and on Mars, and how cultivated meat may impact the economy, culture, and tradition of Asia

Advanced Knowledge Application in Practice

The integration and interdependency of the world economy leads towards the creation of a global market that offers more opportunities, but is also more complex and competitive than ever before. Therefore widespread research activity is necessary if one is to remain successful on the market. This book is the result of research and development activities from a number of researchers worldwide, covering concrete fields of research

Production of Single Cell Protein and Astaxanthin Using Methanol as Carbon Source

Singlecell protein (SCP) is the biomass of unicellular organisms, such as bacteria or yeast, which is used commonly as a food source for animals. With a high protein content, a broad amino acid profile, and the ability to produce essential organic compounds and vitamins, SCP is a promising alternative to other classical sources of animal feed. Several processes have been developed to manufacture SCP for use in feedstocks for the sustainable farming of fish and other aquatic life, or aquaculture, which is one of the fastest growing food markets in the world. Here, a process is presented for the production of 8,800 MT of SCP per year using methanotrophic bacteria with methanol as the carbon source. To increase process profitability, the cells will be genetically engineered to produce astaxanthin, a carotenoid pigment found naturally in aquatic algae. When used as a feed supplement for farmed salmon, these SCP will serve as a nutritional additive and ensure that the salmon possess the pink pigmentation consumers expect. The final product is SCP with 0.3% by weight astaxanthin sold for $16,500/MT. The high market price of astaxanthin significantly improves the profitability of the process. According to a 10year profitability analysis, the predicted IRR is 41.9$129,000,000. In the third production year, the ROI will be 56.0%