Corynebacterium glutamicum is a prominent production host for various value-added compounds in white biotechnology. Gene repression by dCas9/clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) allows for the identification of target genes for metabolic engineering. In this study, a CRISPRi-based library for the repression of 74 genes of C. glutamicum was constructed. The chosen genes included genes encoding enzymes of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, regulatory genes, as well as genes of the methylerythritol phosphate and carotenoid biosynthesis pathways. As expected, CRISPRi-mediated repression of the carotenogenesis repressor gene crtR resulted in increased pigmentation and cellular content of the native carotenoid pigment decaprenoxanthin. CRISPRi screening identified 14 genes that affected decaprenoxanthin biosynthesis when repressed. Carotenoid biosynthesis was significantly decreased upon CRISPRi-mediated repression of 11 of these genes, while repression of 3 genes was beneficial for decaprenoxanthin production. Largely, but not in all cases, deletion of selected genes identified in the CRISPRi screen confirmed the pigmentation phenotypes obtained by CRISPRi. Notably, deletion of pgi as well as of gapA improved decaprenoxanthin levels 43-fold and 9-fold, respectively. The scope of the designed library to identify metabolic engineering targets, transfer of gene repression to stable gene deletion, and limitations of the approach were discussed.

Astaxanthin, a highly valuable natural pigment with potent antioxidant properties, is widely used in various industries, including food, pharmaceuticals, and cosmetics. The demand for astaxanthin has led to the development of multiple production methods, including algal, bacterial, and synthetic approaches. Understanding the environmental implications of these production processes is crucial for sustainable decision-making in the astaxanthin industry. This research paper presents a comprehensive Life Cycle Assessment (LCA) comparing the environmental impacts of algal, bacterial, and synthetic astaxanthin production methods. Moreover, a sensitivity analysis is conducted, focusing on the source of energy used during production, and the shift from conventional to renewable energy sources is explored. The environmental importance of natural astaxanthin production is highlighted, considering the potential benefits of reducing dependence on synthetic production methods that rely on non-renewable resources. The results of the LCA, for the production of 1 g astaxanthin, reveal the relative environmental performance of each production process, identifying hotspots and potential areas for improvement. On average, the environmental impacts indicate that chemical synthesis is the best solution for the production of the pigment, followed by the bacterial extraction process and lastly by algae extraction. Furthermore, the sensitivity analysis demonstrates the significant influence of energy sources on the overall environmental footprint. Shifting towards renewable energy sources presents an opportunity for mitigating environmental impacts and enhancing the sustainability of astaxanthin production.

Circular economy holds great potential to minimize the use of finite resources, and reduce waste formation by the creation of closed-loop systems. This also pertains to the utilization of sidestreams in large-scale biotechnological processes. A flexible feedstock concept has been established for the industrially relevant Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin. In this study, we aimed to use a preprocessed aquaculture sidestream for production of carotenoids, including the fish feed ingredient astaxanthin by C. glutamicum. The addition of a preprocessed aquaculture sidestream to the culture medium did not inhibit growth, obviated the need for addition of several components of the mineral salt’s medium, and notably enhanced production of astaxanthin by an engineered C. glutamicum producer strain. Improved astaxanthin production was scaled to 2 L bioreactor fermentations. This strategy to improve astaxanthin production was shown to be transferable to production of several native and non-native carotenoids. Thus, this study provides a proof-of-principle for improving carotenoid production by C. glutamicum upon supplementation of a preprocessed aquaculture sidestream. Moreover, in the case of astaxanthin production it may be a potential component of a circular economy in aquaculture.

Corynebacterium glutamicum is prominent in the industrial production of secreted amino acids. Notably, it naturally accumulates the carotenoid pigment decaprenoxanthin in its membranes. Metabolic engineering enabled the production of astaxanthin. Here, a bioprocess for astaxanthin production in lab-scale stirred bioreactors was established by a DoE-guided approach to optimize the basic process parameters pH, rDOS, aeration rate as well as inoculation cell density. The DoE-guided approach to characterize 2 L scale cultivation revealed that the pH showed the strongest effect on the product formation. Subsequently, an optimum at pH 8, an aeration rate of 0.25 vvm, 30% rDOS and an initial optical density of 1 was established that allowed production of 7.6 ± 0.6 mg L⁻¹ astaxanthin in batch mode. These process conditions were successfully transferred to a fed-batch process resulting in a high cell density cultivation with up to 60 g CDW L⁻¹ biomass and 64 mg L⁻¹ astaxanthin and thus demonstrating an about 9-fold improvement compared to optimal batch conditions. Moreover, pH-shift experiments indicate that the cells can quickly adapt to a change from pH 6 to 8 and start producing astaxanthin, showing the possibility of biphasic bioprocesses for astaxanthin production.

Corynebacterium glutamicum is prominent for the industrial production of secreted amino acids. Notably, it naturally accumulates the carotenoid pigment decaprenoxanthin in its membranes. Metabolic engineering enabled production of astaxanthin. Here, a bioprocess for astaxanthin production in lab-scale stirred bioreactors was established by a DoE-guided approach to optimize the basic process parameters pH, rDOS, aeration rate as well as inoculation cell density. The DoE-guided approach from 2 L scale cultivation revealed that the pH showed the strongest effect on the product formation. Subsequently, an optimum at pH 8, an aeration rate of 0.25 vvm, 30 % rDOS and an initial optical density of 1 was established that allowed production of 7.6±0.6mg L-1 astaxanthin in batch mode. These process conditions were successfully transferred to a fed-batch process resulting in a high cell density cultivation with up to 60 g CDW L-1 biomass and 64 mg L-1 astaxanthin and thus demonstrating an about 9-fold improvement compared to optimal batch conditions. Moreover, pH shifts experiments indicate that the cells can quickly adapt to a change from pH 6 to 8 and start producing astaxanthin showing the possibility of a biphasic bioprocesses for astaxanthin production.

Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph-Disease (MJD) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG stretch in the MJD1 gene encoding a polyglutamine repeat in the respective ataxin-3-protein. In order to study the course of the disease we generated an inducible transgenic mouse model using the „Tet-Off-System“ developed by Dr. Bujard (Heidelberg). This system is based on two constructs: The promoter construct controls the expression of the so called tTA (Tetracycline transactivator) gene product. The binding of this protein to a Tetracycline responsive element (TRE) in the responder construct induces the transcription of the gene of interest. The expression can be blocked by the addition of Tetracycline which allosterically inhibits the tTA protein. For the ataxin-3-responder mouse lines a full length constructs containing an expanded repeat with the pathological length of 77 glutamine repeats was used. The use of two different promoter mouse lines with known expression in the brain (Prion protein (Prp) promoter, Ca2+/Calmoduline-dependent protein kinase II (CamKII) promoter) allows us to target the transgene expression to different brain regions. In preceding promoter analyses we identified and characterized these brain regions in detail. Double transgenic SCA3 mice were analysed at different levels: We first analysed the phenotype in behavioural studies. Afterwards we performed immunohistochemical analyses of brain sections to study the expression of the transgene in the brain. In addition the Tet-Off-System allows us to turn the expression of ataxin-3 on or off at different developmental stages. Using this model we therefore will be enabled to study whether or not the disease phenotype is reversible.