Browsing by Subject "Heterologe Genexpression"

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Investigating different Bacillus subtilis expression systems for recombinant enzyme production
(2025) Senger, Jana; Fischer, Lutz
Enzymes play an indispensable role in the food industry by improving texture, color, shelf life, or nutritional value of food products. A prerequisite for the application of food enzymes is their high-yield and cost-effective production in a suitable host. The Gram-positive bacterium Bacillus subtilis is a promising host due to the organism's qualified presumption of safety status, its genetic accessibility, and short cultivation times. In addition, B. subtilis can efficiently secrete heterologous enzymes into the extracellular medium, which simplifies downstream processing. This thesis explored different B. subtilis expression systems for the recombinant production and secretion of the β-galactosidase (EC 3.2.1.23) from Paenibacillus wynnii (β-gal-Pw) and the protein-glutamine glutaminase (EC 3.5.1.44) from Bacteroides helcogenes (PGB). Both enzymes have a potential application in the food industry. The β-gal-Pw offers favorable kinetic properties for application in lactose-depleted dairy products. The PGB is used to modify the techno-functional properties of proteins, thereby improving their application in food products. The first study investigated the secretion of the cytoplasmic 120 kDa β-gal-Pw using B. subtilis SCK6. Different expression conditions were tested to find proficient conditions for secretion. Codon-optimization of the native β-gal-Pw gene and cultivation temperature reduction from 37°C to 30°C increased secretory β-gal-Pw production. Furthermore, two promoters and four signal peptides were tested in multiple combinations. Signal peptides of the Sec-pathway and the Tat-pathway enabled efficient secretion, which, however, depended on the specific combination of promoter and signal peptide used. The highest extracellular activity of 55 µkat/Lculture was achieved with the PaprE promoter and the Tat-signal peptide PhoD in shake flask cultivations. The subsequent bioreactor cultivation further improved secretory β-gal-Pw production by 1.4-fold and resulted in 21 mg/Lculture purified β-gal-Pw. The second study explored the intracellular β-gal-Pw production in the undomesticated strain B. subtilis 007. Great differences in production were observed by testing the PaprE and P43 promoter with each corresponding 5’ untranslated region (5’UTR). The use of the PaprE promoter led to an intracellular β-gal-Pw activity of 2515 µkat/Lculture, which was 45-fold higher compared to the P43 promoter. Further modification of the core PaprE promoter or the spacer sequence in the 5’UTR did not improve β-gal-Pw production. The 5’UTR in the P43 construct was replaced with the aprE 5’UTR, which significantly improved mRNA stability. In addition, β-gal-Pw production was enhanced from 56 µkat/Lculture to 2756 µkat/Lculture. The crucial role of the 5’UTR and the corresponding mRNA stability was subsequently confirmed by producing the β-glucosidase from Pyrococcus furiosus and the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus in B. subtilis 007. The third study focused on B. subtilis 007 for secretory and antibiotic-free PGB production. The genome of the undomesticated B. subtilis 007 was sequenced and provided the basis for multiple genomic integrations of the PGB expression cassette via CRISPR/Cas9. By selecting the specific integration sites, genes were simultaneously deleted to optimize the production strain and process. Four genes were targeted for the elimination of sporulation (sigF), foaming (sfp), motility (flgE), and α-amylase production (amyE). The first PGB expression cassette was integrated into sigF, which resulted in the expected asporogenic strain. An extracellular PGB activity of 4.1 µkat/Lculture was reached in bioreactor cultivations. The second expression cassette was integrated into sfp, which reduced foaming and increased the secretory PGB production by 1.3-fold. Since integration into the flgE locus did not enhance PGB production, the third PGB expression cassette was inserted into the amyE locus. The extracellular PGB activity of the respective strain was significantly increased from 5.4 µkat/Lculture to 9.5 µkat/Lculture.
Tth-IM60, eine Membraninsertase aus Thermus thermophilus
(2011) Meyer, Susanne H.; Kuhn, Andreas
The evolutionarily conserved YidC/Oxa1/Alb3 family of proteins catalyzes the insertion of integral membrane proteins in bacteria, mitochondria, and chloroplasts. In this work Tth-IM60 from Thermus thermophilus was identified as a member of this family. The function and structure of the protein was analysed in detail. Complementation studies in a Escherichia coli YidC-depletion strain show a functional replacement of the essential YidC by Tth-IM60 in vivo. A heterologous expression of the his-tagged Tth-IM60 protein was achieved in the E. coli strain C43 and pMS as a plasmid vector. It was shown that Tth-IM60 protein is located in the inner membrane probably in a dimeric state. After purification the protein tends to oligomerize in a higher, but very stable oligomeric state. The Tth-IM60 oligomeric protein was stable for 50 days at least. By size-exclusion chromatography the zwitterionic detergent LDAO and a buffer containing 20 mM TrisHCl pH 8,5, 500 mM NaCl and 10 % glycerol were identified as the best conditions for purification and stability. With this buffer, Tth-IM60 was purified as a dimer via its C-terminal histag by two Ni-IMACs (immobilized metal affinity chromatography) and a size-exclusion chromatography. A final protein concentration up to 10 mg/ml was feasible. The purified Tth-IM60 protein was used for functional and structural studies. A weak binding of Tth-IM60 to the first periplasmic loop of SecF as shown by pulldown assays, suggests a Sec-dependent function of Tth-IM60. In addition, the essential Sec-independent function of Tth-IM60 was demonstrated by the translocation of the YidC substrate Pf3 coat into Tth-IM60 proteoliposomes. Moreover, the results of the single molecule spectroscopy measurements implicate that the translocation of Pf3 coat proteins occurs with a fast kinetics within 5 minutes. The secondary structure of the Tth-IM60 protein was analysed by circular dichroism spectroscopy: 49 ? 55 % alpha-helical, 13 - 14 % beta-sheet, 14 ? 17 % beta-turns and 18 - 21% unordered structures were calculated. Tth-IM60 comprises more alpha-helical structures, but less beta-sheets than YidC, probably because of the first periplasmic loop of Tth-IM60 being shorter. The melting point of Tth-IM60 was determined to 68 °C, which is 10 degrees higher than the melting point of YidC. Furthermore, the Tth-IM60 protein was crystallized and X-ray analysis was performed. However, due to the low resolution the structure of the Tth-IM60 protein could not be determined so far. The second part of this thesis concerns the ?translocating chain-associated membrane? (TRAM) protein. The TRAM protein is involved in the insertion of integral membrane proteins of the endoplasmic reticulum (ER) together with the Sec61-complex. In contrast to the Sec-components, no homologous YidC protein exists in the ER-membrane. Therefore, it was postulated that TRAM and YidC could have functional similarities. For functional studies the TRAM protein from X. laevis was expressed in E. coli as a fusion protein with a N-terminal MBP (maltose-binding protein) and a C-terminal histag. TRAM was purified via two different affinity matrices: Ni sepharose and an amylose resin. It was possible to reconstitute the fusion protein into liposomes. However a translocation of the YidC-substrate Pf3 coat into these proteoliposomes was not detectable. In addition, complementation studies with a YidC-depletion strain did not show that the essential YidC function can be replaced by TRAM in vivo.