Filamentous fungi are commonly used as production hosts for bulk enzymes in biotechnological applications. Their robust and quick growth combined with their ability to secrete large amounts of ...protein directly into the culture medium makes fungi appealing organisms for the generation of novel production systems. The red bread mold Neurospora crassa has long been established as a model system in basic research. It can be very easily genetically manipulated and a wealth of molecular tools and mutants are available. In addition, N. crassa is very fast growing and non-toxic. All of these features point to a high but so far untapped potential of this fungus for biotechnological applications. In this study, we used genetic engineering and bioprocess development in a design-build-test-cycle process to establish N. crassa as a production host for heterologous proteins.
The human antibody fragment HT186-D11 was fused to a truncated version of the endogenous enzyme glucoamylase (GLA-1), which served as a carrier protein to achieve secretion into the culture medium. A modular expression cassette was constructed and tested under the control of different promoters. Protease activity was identified as a major limitation of the production strain, and the effects of different mutations causing protease deficiencies were compared. Furthermore, a parallel bioreactor system (1 L) was employed to develop and optimize a production process, including the comparison of different culture media and cultivation parameters. After successful optimization of the production strain and the cultivation conditions an exemplary scale up to a 10 L stirred tank reactor was performed.
The data of this study indicate that N. crassa is suited for the production and secretion of heterologous proteins. Controlling expression by the optimized promoter Pccg1nr in a fourfold protease deletion strain resulted in the successful secretion of the heterologous product with estimated yields of 3 mg/L of the fusion protein. The fungus could easily be cultivated in bioreactors and a first scale-up was successful. The system holds therefore much potential, warranting further efforts in optimization.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization ...approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5–100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft‐sensor for biomass estimation to establish a μ‐oscillating time‐dependent fed‐batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH4)2SO4, and inductor concentrations. For bioprocess design, an adapted oscillating fed‐batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed‐batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale‐free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The filamentous fungus
Aspergillus niger is widely used as a host organism for homologous and heterologous protein production. A thorough characterization of product formation is needed to optimize ...the cultivation process. To evaluate the success of product formation during the cultivation, it is necessary to draw conclusions about the product yield on the genetic level. The method presented here includes the disruption of fungal cells, total RNA purification, reverse transcription of mRNA into cDNA and specific mRNA quantification by means of real-time PCR (polymerase chain reaction) using iCycler iQ and Light Upon eXtension (LUX™) primer technology. Specific mRNA quantification is based on the application of external standards, using total cellular RNA concentration as a reference. Quantitative real-time PCR has not been used for such purposes before. The method has been developed to monitor bioprocesses related to product formation. For the investigation presented, the protein glucoamylase has been used as a model product. The method allows the specific genetic activity of the cultivated pellets to be determined and can be modified for every other product. The results, which were generated during different fermentation experiments using the established method, very convincingly show the time at which glucoamylase was produced and corresponding mRNA was present in the fungal pellets.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Non-alcoholic steatohepatitis (NASH) represents a risk factor for the development of hepatocellular carcinoma (HCC) and is characterized by quantitative and qualitative changes in hepatic lipids. ...Since elongation of fatty acids from C16 to C18 has recently been reported to promote both hepatic lipid accumulation and inflammation we aimed to investigate whether a frequently used mouse NASH model reflects this clinically relevant feature and whether C16 to C18 elongation can be observed in HCC development. Feeding mice a methionine and choline deficient diet to model NASH not only increased total hepatic fatty acids and cholesterol, but also distinctly elevated the C18/C16 ratio, which was not changed in a model of simple steatosis (ob/ob mice). Depletion of Kupffer cells abrogated both quantitative and qualitative methionine-and-choline deficient (MCD)-induced alterations in hepatic lipids. Interestingly, mimicking inflammatory events in early hepatocarcinogenesis by diethylnitrosamine-induced carcinogenesis (48 h) increased hepatic lipids and the C18/C16 ratio. Analyses of human liver samples from patients with NASH or NASH-related HCC showed an elevated expression of the elongase ELOVL6, which is responsible for the elongation of C16 fatty acids. Taken together, our findings suggest a detrimental role of an altered fatty acid pattern in the progression of NASH-related liver disease.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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