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Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans.

ABSTRACT: We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces lividans. This was accomplished by using the vector pIJ702 and BglII-BclI libraries of M. viridifaciens chromosomal inserts created in S. lividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BglII fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M(r) of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. lividans.

SUBMITTER: Sakurada K

PROVIDER: S-EPMC207368 | biostudies-other | 1992 Nov

REPOSITORIES: biostudies-other

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Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans.

Sakurada K K Ohta T T Hasegawa M M

Journal of bacteriology 19921101 21

We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces lividans. This was accomplished by using the vector pIJ702 and BglII-BclI libraries of M. viridifaciens chromosomal inserts created in S. lividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the ...[more]

PMID: 1400240

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Project description:BACKGROUND:Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS:In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION:The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

Project description:Streptomyces lividans is a suitable host for the heterologous expression of biosynthetic gene clusters (BGCs) from actinomycetes to discover "cryptic" secondary metabolites. To improve the heterologous expression of BGCs, herein we optimized S. lividans strain SBT5 via the stepwise integration of three global regulatory genes and two codon-optimized multi-drug efflux pump genes and deletion of a negative regulatory gene, yielding four engineered strains. All optimization steps were observed to promote the heterologous production of polyketides, non-ribosomal peptides, and hybrid antibiotics. The production increments of these optimization steps were additional, so that the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the final optimized strain, S. lividans LJ1018, was used as the heterologous expression host. The heterologous production of these antibiotics in S. lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated. S. lividans LJ1018 and GX28 markedly promoted the heterologous production of secondary metabolites, without requiring manipulation of gene expression components such as promoters on individual gene clusters. Therefore, these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs. In addition, we successfully conducted high-throughput library expression and functional screening (LEXAS) of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S. lividans GX28 as the library-expression host. The LEXAS experiments identified clones carrying intact BGCs sufficient for the heterologous production of piericidin A1, murayaquinone, actinomycin D, and dehydrorabelomycin. Notably, due to lower antibiotic production, the piericidin A1 BGC had been overlooked in a previous LEXAS screening using S. lividans SBT5 as the expression host. These results demonstrate the feasibility and superiority of S. lividans GX28 as a host for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds.

Dataset Information

Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans.

Publications

Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans.

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