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Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Chitin, a homopolymer of β (1-4)-linked N-acetylglucosamine, is one of the most abundant biopolymers in nature. It is widely distributed in the exoskeleton of crustaceans and insects, in the cell walls of most fungi and some algae. Chitin is an extremely insoluble material with limited industrial applicability. The deacetylated derivative of chitin, chitosan, is a water soluble cationic biopolymer having a broad range of applications (Hirano, 1999). Chitosan is naturally found in the cell wall of Zygomycetes, in the ascospore of Saccharomyces cerevisiae (Briza et al., 1988) and in the cyst wall of Entamoeba invadens (Das et al., 2006). Chitosan biosynthesis requires the coordinated action of chitin synthase (E.C.2.4.1.16) and chitin deacetylase (E.C.3.5.1.41) (Davis & Bartnicki, 1984). Chitin synthase polymerizes N-acetyl glucosamine precursor molecules into chitin and chitin deacetylase catalyzes the deacetylation of the nascent chitin chains. The chitin deacetylase enzymes are members of the family 4 of carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/~cazy/CAZY] (Couthino et al., 1999), which includes several members sharing a conserved region in the primary structure assigned as the NodB homology domain(Caufrier et al., 2003) or polysaccharide deacetylase domain. Chitin deacetylase was first identified and partially purified from extracts of the fungus Mucor rouxii. Since then, chitin deacetylase has been purified from several fungi and chitin deacetylase open reading frames have been cloned from a few microorganisms including M. rouxii (Kafetzopoulos et al., 1993), Colletotrichum lindemuthianum (Tokuyasu et al., 1999; Shresta et al., 2004), Phycomyces blakesleeanus (GenBank AB046690), Schizophillum commune (GenBank AF271216), Blumeria graminis (GenBank AAK84438), Saccharomyces cerevisiae (Christodoulidou et al., 1999) and Schizosaccharomyces pombe (Matsuo et al., 2005). The structure and the catalytic mechanism of chitin deacetylase from C. lindemuthianum were recently studied (Blair et al., 2006). Chitin deacetylase plays a role in the cell wall biosynthesis in M. rouxii and Absidia coerulea (Gao et al., 1995). In C. lindemuthianum and Aspergillus nidulans, it was suggested that chitin deacetylase participates in plant-pathogen interactions to promote plant invasion (Tsigos et al., 2000). In S. cerevisiae, chitin deacetylase is essential for the ascospore cell wall rigidity and the resistance against lytic enzymes (Christodoulidou et al., 1996). The use of chitin deacetylase enzyme for the industrial deacetylation of chitin awaked a great interest. Different fungal strains were screened and compared for their ability to produce a chitin deacetylase secreted, active on insoluble substrates and showing low inhibition with acetate, a product of reaction. Rhizopus circinans proved to be a good chitin deacetylase producer with the targeted characteristics. The second part of the work was to isolate the cDNA encoding for the chitin deacetylase of R. circinans. The native enzyme was purified to homogeneity for sequencing the N-Terminal extremity. The enzyme was purified in only two steps from the culture supernatant of R. circinans. Then, the purified enzyme was sequenced and the first nine amino acids were identified. In the same way, a R. circinans cDNA library was also constructed. The cDNA library was screened using two approaches: on the one hand with radiolabeled homologous probe and on the other hand by PCR with primers designed for the 5 extremity, on the basis of the deduced sequence of the N-Terminal extremity of the native enzyme and for the 3 extremity, from the deduced R. oryzae chitin deacetylase. Two cDNA sequences (D2 and I3/2) with homology to fungal chitin deacetylase genes were isolated with the radiolabeled probe and one sequence (RC+) by PCR approach. The sequences were analyzed and characterized. The three sequences possessed several characteristics of chitin deacetylase sequence: homology with known chitin deacetylase cDNA, the presence of the deacetylase polysaccharide domain, and the same potential glycosylation sites than M. rouxii chitin deacetylase. The cDNA D2, I3/2 and RC (RC sequence is the mature protein sequence of RC+ sequence) were expressed in the yeast Pichia pastoris to confirm their potential chitin deacetylase activity. Numerous constructions were tested. A poly-histidine tag was cloned to facilitate the further purification of the recombinant enzyme. Only the RC sequence showed a high chitin deacetylase activity. Several hypotheses were emitted to explain the low chitin deacetylase activity level measured with the inserts D2 and I3/2. The recombinant RC protein was purified to homogeneity in one step, and partially characterized.

Bacterial polyketides possess an enormous range of chemical diversity and biological function. Many polyketides such as tetracycline, epothilone, and rapamycin have been developed into key clinical pharmaceuticals in a broad range of therapeutic areas. Sequencing of bacterial genomes has shown that there are many more polyketide biosynthetic pathways than there are polyketides isolated from standard cultivation techniques. These genetically encoded polyketide natural products from cultivatable and uncultivatable bacteria represent one of the greatest remaining untapped reservoirs of new natural product diversity. To access this untapped diversity of polyketide products, a general method for heterologous expression of these pathways is needed. Heterologous expression has proven to be a valuable asset in the discovery, production, engineering, and characterization of bacterial secondary metabolites and the complex enzymology involved in their biosynthesis. Herein we discuss the development and investigation of two unique heterologous expression platforms utilizing host strains of Myxococcus xanthus and Escherichia coli. Using our developed heterologous hosts, we were able to produce the Streptomyces rimosus polyketide oxytetracycline. Through production of oxytetracycline in E .coli we have identified the potential of alternative transcription factors as regulators of secondary metabolism. Further investigation and development of alternative transcription factors as regulators of secondary metabolism in heterologous hosts could benefit the development of robust general methodology for the heterologous expression of polyketides.

Bacterial polyketides possess an enormous range of chemical diversity and biological function. Many polyketides such as tetracycline, epothilone, and rapamycin have been developed into key clinical pharmaceuticals in a broad range of therapeutic areas. Sequencing of bacterial genomes has shown that there are many more polyketide biosynthetic pathways than there are polyketides isolated from standard cultivation techniques. These genetically encoded polyketide natural products from cultivatable and uncultivatable bacteria represent one of the greatest remaining untapped reservoirs of new natural product diversity. To access this untapped diversity of polyketide products, a general method for heterologous expression of these pathways is needed. Heterologous expression has proven to be a valuable asset in the discovery, production, engineering, and characterization of bacterial secondary metabolites and the complex enzymology involved in their biosynthesis. Herein we discuss the development and investigation of two unique heterologous expression platforms utilizing host strains of Myxococcus xanthus and Escherichia coli. Using our developed heterologous hosts, we were able to produce the Streptomyces rimosus polyketide oxytetracycline. Through production of oxytetracycline in E .coli we have identified the potential of alternative transcription factors as regulators of secondary metabolism. Further investigation and development of alternative transcription factors as regulators of secondary metabolism in heterologous hosts could benefit the development of robust general methodology for the heterologous expression of polyketides.

Bacterial polyketides possess an enormous range of chemical diversity and biological function. Many polyketides such as tetracycline, epothilone, and rapamycin have been developed into key clinical pharmaceuticals in a broad range of therapeutic areas. Sequencing of bacterial genomes has shown that there are many more polyketide biosynthetic pathways than there are polyketides isolated from standard cultivation techniques. These genetically encoded polyketide natural products from cultivatable and uncultivatable bacteria represent one of the greatest remaining untapped reservoirs of new natural product diversity. To access this untapped diversity of polyketide products, a general method for heterologous expression of these pathways is needed. Heterologous expression has proven to be a valuable asset in the discovery, production, engineering, and characterization of bacterial secondary metabolites and the complex enzymology involved in their biosynthesis. Herein we discuss the development and investigation of two unique heterologous expression platforms utilizing host strains of Myxococcus xanthus and Escherichia coli. Using our developed heterologous hosts, we were able to produce the Streptomyces rimosus polyketide oxytetracycline. Through production of oxytetracycline in E .coli we have identified the potential of alternative transcription factors as regulators of secondary metabolism. Further investigation and development of alternative transcription factors as regulators of secondary metabolism in heterologous hosts could benefit the development of robust general methodology for the heterologous expression of polyketides.

Bacterial polyketides possess an enormous range of chemical diversity and biological function. Many polyketides such as tetracycline, epothilone, and rapamycin have been developed into key clinical pharmaceuticals in a broad range of therapeutic areas. Sequencing of bacterial genomes has shown that there are many more polyketide biosynthetic pathways than there are polyketides isolated from standard cultivation techniques. These genetically encoded polyketide natural products from cultivatable and uncultivatable bacteria represent one of the greatest remaining untapped reservoirs of new natural product diversity. To access this untapped diversity of polyketide products, a general method for heterologous expression of these pathways is needed. Heterologous expression has proven to be a valuable asset in the discovery, production, engineering, and characterization of bacterial secondary metabolites and the complex enzymology involved in their biosynthesis. Herein we discuss the development and investigation of two unique heterologous expression platforms utilizing host strains of Myxococcus xanthus and Escherichia coli. Using our developed heterologous hosts, we were able to produce the Streptomyces rimosus polyketide oxytetracycline. Through production of oxytetracycline in E .coli we have identified the potential of alternative transcription factors as regulators of secondary metabolism. Further investigation and development of alternative transcription factors as regulators of secondary metabolism in heterologous hosts could benefit the development of robust general methodology for the heterologous expression of polyketides.

>Magister Scientiae - MSc / The yeast Saccharomyces cerevisiae is considered a good host used for heterologous protein production due to the organism’s microbial safety, rapid growth and eukaryotic post- translational processing. As a fermentative organism, S. cerevisiae is thus not only a useful platform for the production of biopharmaceuticals and industrial enzymes, but also a promising organism for second-generation biofuel production. Substantial effort has been focused on alleviating the many bottlenecks in recombinant gene expression, as well as in the secretory pathway to enhance heterologous protein titres. It was recently shown that highly active endocytosis could decrease the overall secreted protein titre in the supernatant. In this study, we aimed to block endocytotic and vacuolar complexes to ultimately disrupt, or impair, the endocytotic and vacuolar mechanisms of proteolysis and test the effect that this would have on secreted heterologous protein titres. This was accomplished by knocking out various genes involved in endocytosis and transforming the strains with genes encoding various hydrolases including β-glucosidase (Bgl), xylanase (Xyn2), endoglucanase (Eg2) and cellobiohydrylase (Cbh1). Our study demonstrated that genetic blocking of endocytotic mechanisms as well as vacuolar complexes could theoretically improve heterologous protein secretion in S. cerevisiae. Endoglucanase (Eg2) titres displayed improvement of 26% and 30% in strains which had the RVS161 and VRP1 genes deleted and xylanase titres displayed an improvement of 71% and 143% in strains with the END3 and SSA4 gene deletions. Several of the gene knockouts tested improved Xyn2 and Eg2 titres but the effect of the different gene targets varied widely. A double knock-out strain with deletions in CLC1 and RVS161 secreted 104% more Eg2 than its parental control strain on a per dry cell weight basis, a significant synergistic improvement. Other double knock-out strains displayed additive or similar activities when compared to their controls. Cbh1 secretion could not be improved through the gene deletions tested in our study and Bgl activity could not be measured in our transformants. These results demonstrate the different relationships of various heterologous proteins with various components of the secretion machinery and may also imply how endocytosis as well as vacuolar complexes affect the level of secreted protein.

Hyperthermophiles grow optimally at 80 ??C and above, and many of them have the ability to utilize various carbohydrates as carbon source and produce ethanol as an end product. Alcohol dehydrogenase (ADH) is a key enzyme responsible for alcohol production, catalyzing interconversions between alcohols and corresponding ketones or aldehydes. ADHs from hyperthermophiles are of great interests due to their thermostability, high activity and enantioselectivity. The gene encoding ADH from hyperthermophilic archaeon Thermococcus guaymasensis was cloned, sequenced and over-expressed. DNA fragments of the genes encoding the ADHs were amplified directly from the corresponding genomic DNA by combining the use of conventional and inverse PCRs. The entire gene was detected to be 1092 bp and the deduced amino acid sequence had a total of 364 amino acids with a calculated molecular mass of 39463 Dalton. The enzyme belonged to the family of zinc-containing ADHs with catalytic zinc only. It was verified that the enzyme had binding motifs of catalytic zinc only (GHEX2GX5GX2V, residues 62-76) and coenzyme NADP (GXGX2G, residues 183-188). The tertiary structural modeling showed two typical domains, one catalytic domain close to amino-terminal (N-terminal) end and one coenzyme-binding domain close to carboxy-terminal (C-terminal) end. Since its codon usage pattern seemed to be different from that of Escherichia coli, the enzyme was over-expressed in the E. coli codon plus strain using pET-30a vector. The recombinant enzyme was detected to be soluble and active (1073 U/mg), which was virtually the same to the native enzyme (1049 U/mg). The recombinant ADH possessed almost identical properties with the native enzyme. The optimal pHs for ethanol oxidation and acetaldehyde reduction were 10.5 and 7.5 respectively, while the activity for alcohol oxidation was much higher than that of aldehyde reduction. The enzyme activity was inhibited in the presence of 100 ??M Zn2+ in the assay mixture and it has a half-life of 6 hours after exposure to air. Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 ??C. The gene encoding an alcohol dehydrogenase from T. hypogea was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained successfully by sequencing all the DNA fragments amplified from PCR. The deduced amino acid sequence was found to have high degrees of identity (~72%) to iron-containing ADHs from Thermotoga species and harbored typical iron and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. The structural modeling showed that N-terminal domain of ThADH contained ??/??-dinucleotide-binding motif and its C-terminal domain was ??-helix-rich region including iron-binding motif. The gene encoding T. hypogea ADH was functionally expressed in E. coli using the vector pET-30a. The recombinant protein was expressed optimally in E. coli grown in the presence of 1 mM ferrous and induced by 0.4-0.6 mM IPTG. The recombinant enzyme was found to be soluble, active and thermostable, and had a subunit size of 43 kDa revealed by SDS-PAGE analyses. The native ADH from T. hypogea was purified to homogeneity for comparative analysis using a three-step liquid chromatography while the recombinant ADH over-expressed in E. coli was isolated by a simpler procedure including one-hour heat treatment. The activity of the purified recombinant enzyme was 69 U/mg and presented almost identical properties with the native enzyme. The optimal pHs for ethanol oxidation and acetaldehyde reduction were 11.0 and 8.0 respectively, while activity for alcohol oxidation were higher than that of aldehyde reduction. The enzyme was oxygen sensitive and it had a half-life (t1/2) of 20 minutes after exposed to air. The enzyme remained 50% activity after incubation at 70 ??C for 2 hours. Successful high-level expression of T. hypogea ADH in E. coli will significantly facilitate further study on the catalytic mechanism of iron-containing ADHs. In summary, both zinc- and iron-containing ADHs from two hyperthermophiles were successfully cloned, sequenced and overexpressed in mesophilic host E. coli, and such a high-level expression of ADH genes provides possibilities for three dimensional structural analysis by X-ray crystallography and enzyme modification by mutagenesis, which will help further explore mechanisms of catalysis and protein thermostability of iron and zinc-containing ADHs and their potential applications in biotechnology.

Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant. / Ph. D.