Coordination of glycerol utilization and clavulanic acid biosynthesis to improve clavulanic acid production in Streptomyces clavuligerus

Guo, DeKun; Zhao, Youbao; Yang, Keqian

doi:10.1007/s11427-013-4507-z

Cited by 6 publications

(6 citation statements)

References 26 publications

(26 reference statements)

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“…Moreover, overexpression and chromosomal integration of both ccaR and claR in a gap1-deleted mutant further improved CA yields by 2.6-fold and 5.9-fold, respectively (Jnawali et al, 2010a). In a study by Guo et al (2013), recombinant S. clavuligerus with extra ccaR under the control of the glycerol promoter in pSET152 provided 3.19-fold more CA production than recombinants with ccaR integration with its native promoter in pSET152.…”

Section: Discussionmentioning

confidence: 99%

“…Integrative and/or multicopy expressions of cas2, claR, and ccaR under the control of different promoters in wild-type S. clavuligerus have resulted in enhanced CA production (Pérez-Llarena et al, 1997;Pérez-Redondo et al, 1998;Hung et al, 2007;Baltz, 2011;Guo et al, 2013). Indeed, the use of previously reported gene manipulations on industrial strains obtained from random mutation strategies might yield more productive strains (Paradkar, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Genetic engineering of an industrial strain of Streptomyces clavuligerusfor further enhancement of clavulanic acid production

Kurt‐Kızıldoğan¹,

Jaccard²,

Mutlu³

et al. 2017

Turk J Biol

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An industrial clavulanic acid (CA) overproducer Streptomyces clavuligerus strain, namely DEPA, was engineered to further enhance its CA production. Single or multiple copies of ccaR, claR (pathway-specific activators), and cas2 (CA synthase) genes under the control of different promoters were introduced into this strain. CA titers of the resulting recombinants were analyzed by HPLC in a dynamic fashion and compared to the vector-only controls and a wild-type strain of S. clavuligerus while their growth was monitored throughout fermentation. The addition of an extra copy of ccaR, under control of its own promoter or constitutive ermE* promoter (P ermE* ), led to 7.6-and 2.3-fold increased volumetric levels of CA in respective recombinants, namely the AK9 and ID3 strains. Its highly stable multicopy expression by the glpF promoter (P glpF ) provided up to 25.9-fold enhanced volumetric CA titers in the respective recombinant, IDG3. claR expression controlled with its own promoter or ermE* and glpF-mediated amplification in an industrial strain brought about only about 1.2-fold increase in the volumetric CA titers. An extra copy of cas2 integration with P ermE* into the S. clavuligerus DEPA genome led to 3.8-fold higher volumetric CA production by GV61. Conclusively, multicopy expression of ccaR under P glpF can result in significantly improved industrial high-titer CA producers.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Genetic engineering of an industrial strain of Streptomyces clavuligerusfor further enhancement of clavulanic acid production

Kurt‐Kızıldoğan¹,

Jaccard²,

Mutlu³

et al. 2017

Turk J Biol

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“…Indeed, the integration of the tools of “classical” and “modern” approaches enables the researchers to stack multiple complex phenotypes [22] . For CA overproduction, the genetic and metabolic engineering approaches involving altering expression levels of biosynthetic or regulatory genes, increasing precursor flow into the pathway or eliminating competing reactions by oriented modifications have been applied mostly to the laboratory strains of S. clavuligerus , as summarized in Table S1 [11] , [12] , [13] , [23] , [24] , [25] , [26] , [27] , [28] , [29] . Because standard strains are able to produce only limited amounts of secondary metabolites, application of knowledge-based gene manipulations in industrial strains derived from random mutagenesis and selection might provide more productive strains [16] , [30] .…”

Section: Introductionmentioning

confidence: 99%

Proteome-wide alterations in an industrial clavulanic acid producing strain of Streptomyces clavuligerus

Ünsaldı

Kurt‐Kızıldoğan

Voigt

et al. 2017

Synthetic and Systems Biotechnology

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The usefulness of genetic/metabolic engineering for further improvement of industrial strains is subject of discussion because of the general lack of knowledge on genetic alterations introduced by iterative cycles of random mutagenesis in such strains. An industrial clavulanic acid (CA)-overproducer Streptomyces clavuligerus DEPA was assessed to understand proteome-wide changes that have occurred in a local industrial CA overproducer developed through succesive mutagenesis programs. The proteins that could be identified corresponded to 33 distinct ORFs for underrepresented ones and 60 ORFs for overrepresented ones. Three CA biosynthetic enzymes were overrepresented in S. clavuligerus DEPA; carboxyethylarginine synthase (Ceas2), clavaldehyde dehydrogenase (Car) and carboxyethyl-arginine beta-lactam-synthase (Bls2) whereas the enzymes of two other secondary metabolites were underrepresented along with two important global regulators [two-component system (TCS) response regulator (SCLAV_2102) and TetR-family transcriptional regulator (SCLAV_3146)] that might be related with CA production and/or differentiation. γ-butyrolactone biosynthetic protein AvaA2 was 2.6 fold underrepresented in S. clavuligerus DEPA. The levels of two glycolytic enzymes, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and phosophoglycerate kinase were found decreased while those of dihydrolipoyl dehydrogenase (E3) and isocitrate dehydrogenase, with two isoforms were found as significantly increased. A decrease of amino acid metabolism, methionine biosynthesis in particular, as well as S-adenosylmethionine synthetase appeared as one of the prominent mechanisms of success of S. clavuligerus DEPA strain as a prolific producer of CA. The levels of two enzymes of shikimate pathway that leads to the production of aromatic amino acids and aromatic secondary metabolites were also underrepresented. Some of the overrepresented stress proteins in S. clavuligerus DEPA included polynucleotide phosphorylase/polyadenylase (PNPase), ATP-dependent DNA helicase, two isoforms of an anti-sigma factor and thioredoxin reductase. Downregulation of important proteins of cell wall synthesis and division was recorded and a protein with β-lactamase domain (SCLAV_p1007) appeared in 12 isoforms, 5 of which were drastically overrepresented in DEPA strain. These results described herein provide useful information for rational engineering to improve CA production in Streptomyces clavuligerus.

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“…Moreover, the expression of additional copies of the glycerol utilization operon increased the CA yield by ∼4.5-fold, and further glycerol supplementation enhanced CA production by ∼7.5-fold (Baños et al., 2009 ). In S. clavuligerus , the genes responsible for glycerol utilization are organized in a glp operon consisting of gylR-glpF1-glpK1-glpD1 ; the gylR gene encodes an IclR family transcriptional regulator, and the glpF1, glpK1, and glpD1 genes code for a putative glycerol transporter, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively (Guo et al., 2013 ). It is known that gylR is transcribed as a single transcript, and glpF1-glpK1-glpD1 is polycistronically expressed under the control of a putative promoter located upstream of glpF1 (Baños et al., 2009 ) (Fig.…”

Section: Introductionmentioning

confidence: 99%

Enhanced production of clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

Shin

Cho

Won

et al. 2021

Journal of Industrial Microbiology and Biotechnology

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Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

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Coordination of glycerol utilization and clavulanic acid biosynthesis to improve clavulanic acid production in Streptomyces clavuligerus

Cited by 6 publications

References 26 publications

Genetic engineering of an industrial strain of Streptomyces clavuligerusfor further enhancement of clavulanic acid production

Genetic engineering of an industrial strain of Streptomyces clavuligerusfor further enhancement of clavulanic acid production

Proteome-wide alterations in an industrial clavulanic acid producing strain of Streptomyces clavuligerus

Enhanced production of clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

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