Improved Expression of a Hybrid <i>Streptomyces clavuligerus cef</i>E Gene in <i>Penicillium chrysogenum</i>

Queener, Stephen W.; Beckmann, Robert J.; Cantwell, Cathleen A.; Hodges, Roland; Fisher, Deborah L.; Dotzlaf, Joe E.; Yeh, Wu-Kuang; McGilvray, D. I.; Greaney, Michael F.; Rosteck, Paul R.

doi:10.1111/j.1749-6632.1994.tb47391.x

Cited by 9 publications

(3 citation statements)

References 19 publications

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“…Data are presented as averages 7 mean deviation from at least two independent replicate cultures. Harris et al, 2009b;Queener et al, 1994;Velasco et al, 2000) has transformed this fungus into a platform organism for production of b-lactam antibiotics and their precursors.…”

Section: Metabolic Engineering Of Side-chain Precursor Degradation Inmentioning

confidence: 99%

Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis

Veiga¹,

Gombert²,

Landes³

et al. 2012

Metabolic Engineering

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Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporin-related products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. chrysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via β-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of β-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of β-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis.

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Section: Metabolic Engineering Of Side-chain Precursor Degradation Inmentioning

confidence: 99%

Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis

Veiga¹,

Gombert²,

Landes³

et al. 2012

Metabolic Engineering

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“…Although recent years have seen few examples of the use of recombinant DNA techniques to improve yields of small‐molecule natural products (14–16), this work showcases the synergy of combining all techniques of process improvement. Mutagenesis techniques have historically been used on the natural producers of polyketides; however, we demonstrated that mutagenesis is also effective on plasmid‐based recombinant organisms.…”

Section: Discussionmentioning

confidence: 97%

Combining Classical, Genetic, and Process Strategies for Improved Precursor-Directed Production of 6-Deoxyerythronolide B Analogues

Desai¹,

Leaf²,

Hu³

et al. 2008

Biotechnol Progress

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A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1 degrees mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25-0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.

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“…The secondary structures of residues 81-90 and 167-177 were highly disordered and were not determined [84]. The cylinders, arrows, and black lines represent -helices, -sheets, and coil structures, respectively expression in E. coli [28,34,49], S. lividans [22,33], Penicillium chrysogenum [13,24,68,[70][71][72][73], Pseudomonas putida [47], and Pichia pastoris [3]. High level expression of recombinant scDAOCS has often been carried out in E. coli cells together with the use of traditional extraction to purify scDAOCS from E. coli cell lysate for biochemical studies [57,58,90].…”

Section: Expression Of Recombinant S Clavuligerus Daocsmentioning

confidence: 99%

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

Goo

Chua

Sim

2009

J Ind Microbiol Biotechnol

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It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies, its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate and molecular oxygen as co-substrates. It has highly conserved HXDX( n ) H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application.

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Improved Expression of a Hybrid Streptomyces clavuligerus cefE Gene in Penicillium chrysogenum

Cited by 9 publications

References 19 publications

Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis

Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis

Combining Classical, Genetic, and Process Strategies for Improved Precursor-Directed Production of 6-Deoxyerythronolide B Analogues

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

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