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Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan.

ABSTRACT: BACKGROUND: Chitosanases are enzymes hydrolysing chitosan, a ?-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the genetic regulation of a heterologous chitosanase gene (csnN106) in Streptomyces lividans. RESULTS: Two S. lividans strains were used for induction experiments: the wild type strain and its mutant (?csnR), harbouring an in-frame deletion of the csnR gene, encoding a negative transcriptional regulator. Comparison of chitosanase levels in various media indicated that CsnR regulates negatively the expression of the heterologous chitosanase gene csnN106. Using the ?csnR host and a mutated csnN106 gene with a modified transcription operator, substantial levels of chitosanase could be produced in the absence of chitosan, using inexpensive medium components. Furthermore, chitosanase production was of higher quality as lower levels of extracellular protease and protein contaminants were observed. CONCLUSIONS: This new chitosanase production system is of interest for biotechnology as only common media components are used and enzyme of high degree of purity is obtained directly in the culture supernatant.

SUBMITTER: Dubeau MP

PROVIDER: S-EPMC3048496 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan.

Dubeau Marie-Pierre MP Guay Isabelle I Brzezinski Ryszard R

Microbial cell factories 20110210

<h4>Background</h4>Chitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the g ...[more]

PMID: 21310076

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Project description:BackgroundThe Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins-which favors correct folding and facilitates downstream processing-as well as its acceptance of methylated DNA and its low endogeneous protease activity. However, current inconsistencies in protein yields urge for a deeper understanding of the burden of heterologous protein production on the cell. In the current study, transcriptomics and [Formula: see text]-based fluxomics were exploited to uncover gene expression and metabolic flux changes associated with heterologous protein production. The Rhodothermus marinus thermostable cellulase A (CelA)-previously shown to be successfully overexpressed in S. lividans-was taken as an example protein.ResultsRNA-seq and [Formula: see text]-based metabolic flux analysis were performed on a CelA-producing and an empty-plasmid strain under the same conditions. Differential gene expression, followed by cluster analysis based on co-expression and co-localization, identified transcriptomic responses related to secretion-induced stress and DNA damage. Furthermore, the OsdR regulon (previously associated with hypoxia, oxidative stress, intercellular signaling, and morphological development) was consistently upregulated in the CelA-producing strain and exhibited co-expression with isoenzymes from the pentose phosphate pathway linked to secondary metabolism. Increased expression of these isoenzymes matches to increased fluxes in the pentose phosphate pathway. Additionally, flux maps of the central carbon metabolism show increased flux through the tricarboxylic acid cycle in the CelA-producing strain. Redirection of fluxes in the CelA-producing strain leads to higher production of NADPH, which can only partly be attributed to increased secretion.ConclusionsTranscriptomic and fluxomic changes uncover potential new leads for targeted strain improvement strategies which may ease the secretion stress and metabolic burden associated with heterologous protein synthesis and secretion, and may help create a more consistently performing S. lividans strain. Yet, links to secondary metabolism and redox balancing should be further investigated to fully understand the S. lividans metabolome under heterologous protein production.

Dataset Information

Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan.

Publications

Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan.

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