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Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544.

ABSTRACT: We have purified a fructosyltransferase from conidia of the inulin-producing fungus Aspergillus sydowi IAM 2544 and obtained peptide sequences from proteolytic fragments of the protein. With degenerated primers, we amplified a PCR fragment that was used to screen a cDNA library. The fructosyltransferase gene from Aspergillus sydowi (EMBL accession no. AJ289046) is expressed in conidia, while no expression could be detected in mycelia by Northern blot analysis of mycelial RNA. The gene encodes a protein with a calculated molecular mass of 75 kDa that is different from all fructosyltransferases in the databases. The only homology that could be detected was to the invertase of Aspergillus niger (EMBL accession no. L06844). The gene was functionally expressed in Escherichia coli, yeast, and potato plants. With protein extracts from transgenic bacteria and yeast, fructooligosaccharides could be produced in vitro. In transgenic potato plants, inulin molecules of up to 40 hexose units were synthesized in vivo. While in vitro experiments with protein extracts from conidia of Aspergillus sydowi yielded the same pattern of oligosaccharides as extracts from transformed bacteria and yeast, in vivo inulin synthesis with fungal conidia leads to the production of a high-molecular-weight polymer.

SUBMITTER: Heyer AG

PROVIDER: S-EPMC92586 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544.

Heyer A G AG Wendenburg R R

Applied and environmental microbiology 20010101 1

We have purified a fructosyltransferase from conidia of the inulin-producing fungus Aspergillus sydowi IAM 2544 and obtained peptide sequences from proteolytic fragments of the protein. With degenerated primers, we amplified a PCR fragment that was used to screen a cDNA library. The fructosyltransferase gene from Aspergillus sydowi (EMBL accession no. AJ289046) is expressed in conidia, while no expression could be detected in mycelia by Northern blot analysis of mycelial RNA. The gene encodes a ...[more]

PMID: 11133467

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Project description:Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that CamCPR was expressed in the roots, stems, and leaves of C. acuminata seedlings. The relative transcript level of CamCPR in leaves was 2.2-fold higher than that of roots and the stems showed 1.5-fold transcript level higher than the roots. The functional characterization of CamCPR will be helpful to disclose the mysterious mechanisms of the biosynthesis of CAM. The present study established a platform to characterize the P450 enzymes involved in the growth, development, and metabolism of eukaryotic organisms.

Project description:BACKGROUND: Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. RESULTS: A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn???) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp???-Val??? peptide was shortened to Asp???-Arg??? in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution. CONCLUSIONS: The nitrilase from Aspergillus niger K10 is highly homologous (?86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.

Dataset Information

Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544.

Publications

Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544.

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