Lovastatin Biosynthesis by Aspergillus terreus in a Chemically Defined Medium
01 Jun 2001-Applied and Environmental Microbiology (American Society for Microbiology)-Vol. 67, Iss: 6, pp 2596-2602
TL;DR: Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis.
Abstract: Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.
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TL;DR: History of the development of Aspergillus as an expression host, current state of the art and future directions are reviewed, touching on related research in other fungi when discussing the areas of greatest potential for future biotechnological applications.
262 citations
TL;DR: The aim of the present review is to describe important advances reported on mechanisms to explain the negative carbon catabolite effects on secondary metabolite production in bacteria and fungi, giving special emphasis to those reported for the genus Streptomyces.
Abstract: Microbial secondary metabolites are low molecular mass products, not essential for growth of the producing cultures, but very important for human health. They include antibiotics, antitumor agents, cholesterol-lowering drugs, and others. They have unusual structures and are usually formed during the late growth phase of the producing microorganisms. Its synthesis can be influenced greatly by manipulating the type and concentration of the nutrients formulating the culture media. Among these nutrients, the effect of the carbon sources has been the subject of continuous studies for both, industry and research groups. Different mechanisms have been described in bacteria and fungi to explain the negative carbon catabolite effects on secondary metabolite production. Their knowledge and manipulation have been useful either for setting fermentation conditions or for strain improvement. During the last years, important advances have been reported on these mechanisms at the biochemical and molecular levels. The aim of the present review is to describe these advances, giving special emphasis to those reported for the genus Streptomyces.
260 citations
Cites background from "Lovastatin Biosynthesis by Aspergil..."
...Several authors have reported the negative effect of glucose on lovastatin production in Monascus and A. terreus, where lactose is the preferred carbon source (Casa-López et al., 2003; Hajjaj et al., 2001; Lai et al., 2007)....
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...terreus, where lactose is the preferred carbon source (Casa-López et al., 2003; Hajjaj et al., 2001; Lai et al., 2007)....
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...The presence of CreA binding sites in two putative regulatory genes suggest that glucose repression of lovastatin biosynthesis could be mediated by CreA (Hajjaj et al., 2001)....
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TL;DR: This review focuses on C-10 mono-, C-15 sesqui-, and C-20 diterpenes, and the diverse functions of these molecules, including their ability to regulate expression of the β-HMG-CoA reductase and Ras-related proteins.
Abstract: The isoprenoid biosynthetic pathway is the source of a wide array of products. The pathway has been highly conserved throughout evolution, and isoprenoids are some of the most ancient biomolecules ever identified, playing key roles in many life forms. In this review we focus on C-10 mono-, C-15 sesqui-, and C-20 diterpenes. Evidence for interconversion between the pathway intermediates farnesyl pyrophosphate and geranylgeranly pyrophosphate and their respective metabolites is examined. The diverse functions of these molecules are discussed in detail, including their ability to regulate expression of the β-HMG-CoA reductase and Ras-related proteins. Additional topics include the mechanisms underlying the apoptotic effects of select isoprenoids, antiulcer activities, and the disposition and degradation of isoprenoids in the environment. Finally, the significance of pharmacological manipulation of the isoprenoid pathway and clinical correlations are discussed.
214 citations
TL;DR: Production of lovastatin and microbial biomass by Aspergillus terreus ATCC 20542 were influenced by the type of the carbon source and the nitrogen source used, and the use of spores gave a more consistent inoculum in the different runs.
212 citations
Cites background from "Lovastatin Biosynthesis by Aspergil..."
...034 mg g −1 h−1) has been reported [11] in cultures growing at high specific growth rate (e....
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...The selected carbon sources are considered not to cause catabolic repression as reported for glucose[11]....
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TL;DR: This review provides an overview of the characterized fungal PKS–NRPS hybrids, their manifold functionalities, and the diversity of the resulting secondary metabolites, as well as molecular engineering attempts that highly improved the understanding of their cryptic programming.
Abstract: Fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrids manufacture a wide range of structurally diverse secondary metabolites that play an eminent role in the environment, as molecular tools and leads for therapeutic development. To date, a dozen PKS-NRPS megasynthetases can be linked to the corresponding secondary metabolites, which stand out because of their structural complexity. The diversity of their structures, biological activities, and biosynthetic routes are particularly intriguing considering the iterative use of the catalytic domains of the biosynthetic enzymes-implying an enigmatic biosynthetic code. This review provides an overview of the characterized fungal PKS-NRPS hybrids, their manifold functionalities, and the diversity of the resulting secondary metabolites, as well as molecular engineering attempts that highly improved the understanding of their cryptic programming.
165 citations
References
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TL;DR: It was shown that mevinolin was an orally active cholesterol-lowering agent in the dog and orally administered sodium mevinolinate was an active inhibitor of cholesterol synthesis in an acute assay.
Abstract: Mevinolin, a fungal metabolite, was isolated from cultures of Aspergillus terreus. The structure and absolute configuration of mevinolini and its open acid form, mevinolinic acid, were determined by a combination of physical techniques. Mevinolin was shown to be 1,2,6,7,8,8a-hexahydro-beta, delta-dihydroxy-2,6-dimethyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-hepatanoic acid delta-lactone. Mevinolin in the hydroxy-acid form, mevinolinic acid, is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]; its Ki of 0.6 nM can be compared to 1.4 nM for the hydroxy acid form of the previously described related inhibitor, ML-236B (compactin, 6-demethylmevinolin). In the rat, orally administered sodium mevinolinate was an active inhibitor of cholesterol synthesis in an acute assay (50% inhibitory dose = 46 microgram/kg). Furthermore, it was shown that mevinolin was an orally active cholesterol-lowering agent in the dog. Treatment of dogs for 3 weeks with mevinolin at 8 mg/kg per day resulted in a 29.3 +/- 2.5% lowering of plasma cholesterol.
1,517 citations
"Lovastatin Biosynthesis by Aspergil..." refers background or methods in this paper
...Reported growth and production conditions for lovastatin are from batch fermentations performed on media with glucose and a complex nitrogen source (1, 4, 13, 22, 28)....
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...FeSO4 z 7H2O, 1 g of MnSO4 z 4H2O, 200 mg of ZnSO4 z 7H2O, 100 mg of CaCl2 z 2H2O, 25 mg of CuCl2 z 2H2O, 56 mg of H3BO3, and 19 mg of (NH4)6Mo7O24 z 4H2O—per liter of solution (1)] was inoculated with 2 z 10(6) conidiospores....
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...Lovastatin, monacolin J, monacolin L, and mevastatin can be produced by Monascus ruber (7), Penicillium brevicompactum, and Aspergillus terreus (1, 36)....
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637 citations
TL;DR: Synthesis of the main nonaketide-derived skeleton was found to require the previously known iterative lovastatin nonAKetide synthase (LNKS), plus at least one additional protein that interacts with LNKS and is necessary for the correct processing of the growing polyketide chain and production of dihydromonacolin L.
Abstract: Polyketides, the ubiquitous products of secondary metabolism in microorganisms, are made by a process resembling fatty acid biosynthesis that allows the suppression of reduction or dehydration reactions at specific biosynthetic steps, giving rise to a wide range of often medically useful products. The lovastatin biosynthesis cluster contains two type I polyketide synthase genes. Synthesis of the main nonaketide-derived skeleton was found to require the previously known iterative lovastatin nonaketide synthase (LNKS), plus at least one additional protein (LovC) that interacts with LNKS and is necessary for the correct processing of the growing polyketide chain and production of dihydromonacolin L. The noniterative lovastatin diketide synthase (LDKS) enzyme specifies formation of 2-methylbutyrate and interacts closely with an additional transesterase (LovD) responsible for assembling lovastatin from this polyketide and monacolin J.
630 citations
"Lovastatin Biosynthesis by Aspergil..." refers background in this paper
...The lovastatin biosynthetic gene cluster consists of 18 putative open reading frames (ORFs) (18), among which 2 were annotated to encode regulatory proteins, lovE and ORF 13....
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TL;DR: It is found that loss‐of‐function mutations in flbA, which encodes a RGS domain protein, or dominant activating mutations in fadA, block both ST production and a sexual sporulation, consistent with a model in which both asexual sporulation and ST production require inactivation of proliferative growth through inhibition of FadA‐dependent signaling.
Abstract: The filamentous fungus Aspergillus nidulans contains a cluster of 25 genes that encode enzymes required to synthesize a toxic and carcinogenic secondary metabolite called sterigmatocystin (ST), a precursor of the better known fungal toxin aflatoxin (AF). One ST Cluster (stc) gene, aflR, functions as a pathway-specific transcriptional regulator for activation of other genes in the ST pathway. However, the mechanisms controlling activation of aflR and synthesis of ST and AF are not understood. Here we show that one important level for control of stc gene expression requires genes that were first identified as early acting regulators of asexual sporulation. Specifically, we found that loss-of-function mutations in flbA, which encodes a RGS domain protein, or dominant activating mutations in fadA, which encodes the alpha subunit of a heterotrimeric G protein, block both ST production and asexual sporulation. Moreover, overexpression of flbA or dominant interfering fadA mutations cause precocious stc gene expression and ST accumulation, as well as unscheduled sporulation. The requirement for flbA in sporulation and ST production could be suppressed by loss-of-function fadA mutations. The ability of flbA to activate stc gene expression was dependent upon another early acting developmental regulator, fluG, and AflR, the stc gene-specific transcription factor. These results are consistent with a model in which both asexual sporulation and ST production require inactivation of proliferative growth through inhibition of FadA-dependent signaling. This regulatory mechanism is conserved in AF-producing fungi and could therefore provide a means of controlling AF contamination.
324 citations
"Lovastatin Biosynthesis by Aspergil..." refers background in this paper
...An implication of starvation as an eliciting factor has been demonstrated for the induction of sterigmatocystin in A. nidulans ( 17 , 34)....
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...The transcriptional regulator aflR is regulated by flbA, fluG, and aflR, showing that both asexual sporulation and sterigmatocystin require inactivation of proliferative growth through inhibition of fadA-dependent signaling ( 17 )....
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TL;DR: The data suggest that CREA represses the ethanol regulon by a double lock mechanism repressing both the trans‐acting gene, alcR, and the structural gene,Alc A, encoding the alcohol dehydrogenase I.
Abstract: Summary
The CREA repressor responsible for carbon catabolite repression in Aspergillus nidulans represses the transcription of the ethanol regulon. The N-terminal part of the CREA protein encompassing the two zinc fingers (C2H2 class family) and an alanine-rich region was expressed in Escherichia colias a fusion protein with giutathione-S-transferase. Our results show that CREA is a DNA-binding protein able to bind to the promoters of both the specific trans-acting gene, alcR, and of the structural gene, alcA, encoding the alcohol dehydrogenase I. DNase I protection foot-printing experiments revealed several specific binding sites in the alcR and in the alcA promoters having the consensus sequence 5′-G/CPyGGGG-3′. The disruption of one of these CREA-binding sites in the alcR promoter overlapping the induction target for the trans-activator ALCR results in a partially derepressed alc phenotype and derepressed alcR transcription, showing that this binding site is functional in vivo. Our data suggest that CREA represses the ethanol regulon by a double lock mechanism repressing both the trans-acting gene, alcR, and the structural gene, alc A.
321 citations
"Lovastatin Biosynthesis by Aspergil..." refers background in this paper
...The utilization of ethanol in A. nidulans is repressed by double lock control by CreAp of activator alcR and structural gene alcA, encoding alcohol dehydrogenase I ( 19 )....
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