Improvement of protein production in Streptomyces

Improvement of protein production in Streptomyces

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 [P-I.90] The effect of L-arginine on the heat induced unfolding of proteins A. Cirko...

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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-I.90] The effect of L-arginine on the heat induced unfolding of proteins A. Cirkovas, J. Sereikaite ∗ Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Lithuania Keywords: L-arginine; thermal unfolding; aggregation L-Arginine is widely used in biotechnology. The amino acid is one of the most effective chemical additives in the refolding process of recombinant proteins from E. coli inclusion bodies. LArginine suppresses the aggregation and enhances the yield of protein renaturation. Moreover, L-arginine is an excipient in protein pharmaceuticals and is used for the stabilization of human tissue plasminogen activator and fibroblast growth factor 20. Circular dichroism spectroscopy was used to study the effect of L-arginine on the temperature related unfolding and aggregation of three growth hormones, i.e. human, porcine and mink growth hormones, and human interferon-␣2b. The thermal unfolding of the proteins was monitored by registering the changes of ellipticity at an appropriate wavelength for each protein. The changes of high tension voltage applied to the photomultiplier tube of detector reported on the increase in the particle size due to the aggregation of unfolded protein. The onset temperatures of melting and aggregation were determined in the absence and in the presence of L-arginine. We found that the onset temperature of the unfolding and aggregation of both porcine and mink growth hormones increased with increasing L-arginine concentration. However, even the low concentration of L-arginine (0.025-0.25 M) had the destabilizing effect on human growth hormone. The onset temperature of aggregation decreased in the presence of L-arginine. This effect was observed at two different pH values. Similar destabilizing effect was found on interferon-␣2b. The analysis of the tertiary structure of human growth hormone and interferon-␣2b revealed some perturbations in the presence of L-arginine while the near UV CD spectra of porcine and mink growth hormones in the presence of L-arginine were similar to the spectra in the absence of L-arginine. In conclusion, L-arginine is not an universal excipient for preventing temperature related aggregation of proteins as we exemplified by human growth hormone and interferon-␣2b. doi:10.1016/j.jbiotec.2010.09.456 [P-I.91] Improvement of protein production in Streptomyces L. Sevillano 1 , M. Benito 2 , M. Diaz 2 , R.I. Santamaria 1,∗ 1

Consejo Superior de Investigaciones Cientificas, Spain Universidad de Salamanca, Spain Keywords: protein; expression; Streptomyces 2

Streptomycetes are interesting hosts for heterologous expression of proteins in bacteria and could overcome some of the problems encountered when other systems, such as Escherichia coli or Bacillus subtilis, are used. Streptomyces as a host prevents the formation of inclusion bodies, displays a relatively low level of endogenous extracellular protease activity, is more suited to express genes rich in GC, and shows a high secretion capacity which would allow obtain the proteins of interest in the culture supernatant. The goal of this work is to develop a set of stable vectors for Streptomyces that allow a high expression of proteins of interest. We have

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used different S. lividans strains as hosts and tested several promoters and signal peptides upstream the xylanase xysA gene from S. halstedii as a reporter (Rodríguez et al., 2005). The higher xylanase activity was obtained with the own xysA promoter, a strong promoter that is repressed by glucose and induced by xylose or xylan, and with the pstS promoter from S. lividans that is strongly induced by different carbon sources (Esteban et al., 2008). Changes in signal peptides demonstrated that the best results were obtained when replacing xylanase signal peptide by the signal peptide of an S. griseus alpha-amylase (Vigal et al., 1991). Modified S. lividans strains were also tested to obtain the best host. Among them, we tested strains with a fragmented morphology, strains altered in phosphate metabolism and others altered in catabolite control. Protein production and secretion was slightly increased in some of these strains compared to the wild type. In short, with the use of strong promoters, modifications in signal peptides and the use of different strains as host, we have achieved an improvement in the production of recombinant proteins in Streptomyces. This improvement could be very advantageous for the use of Streptomyces as industrial production platform. References Rodríguez, et al., 2005. Gene 351, 1–9. Esteban, et al., 2008. BMC Microbiology 8, 201. Vigal, et al., 1991. Mol. Gen. Genet 225, 278–288.

doi:10.1016/j.jbiotec.2010.09.457 [P-I.92] Biological production of vanillin using enzyme and whole-cell biocatalysts P. Di Matteo 1 , A.G. Ficca 2 , F. Luziatelli 1 , D. Di Gioia 4 , F. Fava 3 , M. Ruzzi 1,∗ 1

DABAC, University of Tuscia, Italy DISA, University of Tuscia, Italy 3 DICASM, University of Bologna, Italy 4 DISTA, University of Bologna, Italy Keywords: Vanillin; Biocatalyst; Ferulic acid; Capsaicin 2

Phytochemicals, such as phenylpropenoids (ferulic acid) and capsaicinoids (capsaicin), can be used as natural substrates for the biotechnological production of vanillin: one of the world’s most important flavor compounds. The goal of this work was to develop improved biocatalysts for the production of vanillin or of vanillin precursors, such as vanillylamine. We have developed a chromosomal integration system that allowed us to express a catabolic cassette for the conversion of ferulic acid into vanillin in different strains of Escherichia coli. The better recombinant strains were obtained transforming two different hosts, namely E. coli JM109 and BL21, with a single copy of the recombinant genes. The resulting strains were very stable and more efficient in vanillin production than strains carrying the catabolic cassette on a low-copy vector. Moreover, the efficiency and selectivity of vanillin recovery from aqueous solutions by adsorption onto Amberlite XAD-4 was studied. Experiments carried out with mixed aqueous solutions of vanillin and ferulic acid demonstrated that this resin has the best capacity to adsorb vanillin (5.6 mg vanillin/g resin) at moderate acidic pH (5.5–6.0), when the vanillin molecule is uncharged, and the highest selectivity at alkaline pH (9.0–10.50), when vanillin molecules become deprotonated and are negatively charged. Finally, the possibility of using penicillin V acylase (PVA) from Streptomyces mobaraensis for the production of vanillin precursors from capsaicin was explored. The results obtained demonstrated