scholarly journals Insertional Inactivation of Methylmalonyl Coenzyme A (CoA) Mutase and Isobutyryl-CoA Mutase Genes in Streptomyces cinnamonensis: Influence on Polyketide Antibiotic Biosynthesis

1999 ◽  
Vol 181 (18) ◽  
pp. 5600-5605 ◽  
Author(s):  
Jan W. Vrijbloed ◽  
Katja Zerbe-Burkhardt ◽  
Ananda Ratnatilleke ◽  
Andreas Grubelnik-Leiser ◽  
John A. Robinson
Keyword(s):  
Sole Carbon Source ◽  
Coenzyme A ◽  
Carbon Sources ◽  
Antibiotic Biosynthesis ◽  
Polyketide Biosynthesis ◽  
Streptomyces Cinnamonensis ◽  
Insertional Inactivation ◽  
Hygromycin Resistance Gene ◽  
Monensin A ◽  
Coenzyme B

ABSTRACT The coenzyme B12-dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of n-butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of n- and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer Streptomyces cinnamonensis. Mutants prepared by inserting a hygromycin resistance gene (hygB) into either icmA or mutB, encoding the large subunits of ICM and MCM, respectively, have been characterized. The icmA::hygB mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. ThemutB::hygB mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. 13C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence, n-butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.

scholarly journals MeaA, a Putative Coenzyme B12-Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

2001 ◽  
Vol 183 (6) ◽  
pp. 2071-2080 ◽  
Author(s):  
Weiwen Zhang ◽  
Kevin A. Reynolds
Keyword(s):  
Coenzyme A ◽  
Large Subunit ◽  
Significant Degree ◽  
Structural Features ◽  
Small Subunit ◽  
Erythromycin Resistance ◽  
Partial Restoration ◽  
Streptomyces Cinnamonensis ◽  
Insertional Inactivation ◽  
Monensin A

ABSTRACT The ratio of the major monensin analogs produced byStreptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). ThemeaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B12-dependent mutases. Plasmid-based expression of meaA from the ermE∗ promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (anicm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that themeaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.

Polyketide synthase com plexes: their structure and function in antibiotic biosynthesis

1991 ◽  
Vol 332 (1263) ◽  
pp. 107-114 ◽  
Keyword(s):  
Structural Organization ◽  
Polyketide Synthase ◽  
Structure And Function ◽  
Polyketide Synthases ◽  
Antibiotic Biosynthesis ◽  
Homologous Region ◽  
Polyketide Biosynthesis ◽  
Monensin A ◽  
And Function ◽  
Spore Pigment

This paper gives an overview of existing knowledge concerning the structure and deduced functions of polyketide synthases active in antibiotic-producing streptomycetes. Using monensin A as an example of a structurally complex polyketide metabolite, the problem of understanding how individual strains of microorganism are ‘programmed’ to produce a given polyketide metabolite is first outlined. The question then arises, how is the programming of polyketide assembly related to the structural organization of individual polyketide synthase complexes at the biochemical and genetic levels? Experimental results that help to illuminate these relations are described, in particular, those giving information about the structures and deduced functions of polyketide synthases involved in aromatic polyketide biosynthesis (actinorhodin, granaticin, tetracenomycin, whiE spore pigment and an act homologous region from the monensin-producing organism), as well as the macrolide polyketide synthase active in the biosynthesis of 6-deoxyerythronolide A.

Changes in Size of Intracellular Pools of Coenzyme A and Its Thioesters inEscherichia coliK-12 Cells to Various Carbon Sources and Stresses

1998 ◽  
Vol 62 (6) ◽  
pp. 1122-1128 ◽  
Author(s):  
Shigeru CHOHNAN ◽  
Hiroaki IZAWA ◽  
Hirofumi NISHIHARA ◽  
Yoshichika TAKAMURA
Keyword(s):  
Coenzyme A ◽  
Carbon Sources

Synthesis, characterization, and enzymic conversion of nonhydrolysable analogues of propionylcoenzyme A

1994 ◽  
Vol 72 (1) ◽  
pp. 164-169 ◽  
Author(s):  
Yimin Zhao ◽  
Martina Michenfelder ◽  
János Rétey
Keyword(s):  
Mass Spectrometry ◽  
Nmr Spectroscopy ◽  
Coenzyme A ◽  
The Novel ◽  
Mechanistic Studies ◽  
Preparative Scale ◽  
Propionibacterium Shermanii ◽  
Fab Mass Spectrometry ◽  
Michaelis Constants ◽  
Coenzyme B

We describe the synthesis of three novel analogues of propionyl-coenzyme A, in which the sulfur atom has been replaced by methylene, ethylene, and thiomethylene, respectively. All three analogues, propionyl-dethia(carba)-CoA (1), propionyl-dethia(dicarba)-CoA (2), and S-(2-oxobutanyl)-CoA (3) were characterized by 1H and 31P NMR spectroscopy and FAB mass spectrometry. Propionyl-CoA–oxaloacetate transcarboxylase from Propionibacterium shermanii accepted the novel analogues as substrates and carboxylated them to the corresponding methylmalonyl-CoA analogues (4–6). The latter were further converted into the succinyl-CoA analogues by the coenzyme-B12-dependent methylmalonyl-CoA mutase from the same organism. The succinyl-CoA analogues, succinyl-dethia(carba)-CoA (7), succinyl-dethia(dicarba)-CoA (8), and 4-carboxy(2-oxobutanyl)-CoA (9) were obtained on a preparative scale and their Michaelis constants (Km) with methylmalonyl-CoA mutase were determined to be 0.136, 2.20, and 0.132 mM, respectively (Km for succinyl-CoA is 0.025 mM). The Vmax values for 7, 8, and 9 are 1.1, 0.013, and 0.0047 µmol min−1 U−1, respectively (Vmax for succinyl CoA is 1.0). The utility of the novel coenzyme A analogues in enzyme mechanistic studies is discussed.

Sebatian Metoksiaromatik Sebagai Substrat Pertumbuhan dan Pengaruh Enzim Demetilase dalam Acetobacterium Woodii DSM 1030

2012 ◽  
Author(s):  
Mohd. Sahaid Hj. Kalil ◽  
Muhammad Zaki ◽  
Wan Mohtar Wan Yusoff ◽  
Mohammad Ramlan Mohd. Salleh
Keyword(s):  
Growth Rate ◽  
Steady State ◽  
Sole Carbon Source ◽  
Specific Growth ◽  
Chemostat Culture ◽  
Carbon Sources ◽  
Syringic Acid ◽  
Organic Substrate ◽  
Acetobacterium Woodii ◽  
Vanilic Acid

Penyelidikan ini bertujuan untuk menyaring substrat organik bagi untuk penghasilan sel–sel A. woodii teraruh demetilase. Pertumbuhan A. woodii dilakukan dalam medium “Balch” yang mengandungi sumber karbon berbeza dalam keadaan anaerobik. Sebanyak sebelas substrat telah diuji iaitu anisol, 2– dan 3–metoksifenol, asid vanilik, asid siringik, asid 2,3,4–, 2,4,5– dan 3,4,5–trimetoksi benzoik, 2,3,4–, 2,4,5– dan 3,4,5–trimetoksi benzil alkohol. 2–metoksifenol merupakan substrat terbaik untuk pertumbuhan A. woodii pada kadar pertumbuhan spesifik 0.14 j–1. Penghasilan sel–sel teraruh demetilase dilakukan dalam kultur kemostat pada kadar pencairan (D) 0.0j–1. Sel-sel pada keadaan mantap dituai dalam keadaan anaerobik dan dipekatkan sebelum digunakan. Pertumbuhan A. woodii didapati maksimum dengan menggunakan kepekatan 0.62 g/L 2–metoksifenol sebagai sumber karbon tunggal. Tindak balas penyahmetilan oleh sel–sel A. woodii meningkat sebanyak 78% apabila 2–metoksifenol sebanyak 0.31 g/L ditambah dalam medium yang mengandungi fruktosa (1% w/v) semasa kultur kemostat. Kata kunci: tindak balas penyahmetilan; demetilase; sel-sel tertuai; metosiaromatik, Acetobacteriumwoodii The objective of this project was to screen organic substrate suitable for the growth of A. woodii, and as for the production of demethylase. A. woodii was grown in “Balch” medium containing different carbon sources. Eleven substrates were tested including anisole, 2– and 3–methoxyphenol, vanilic acid, syringic acid, 2,3,4–, 2,4,5– and 3,4,5–trimethoxy benzoic acid and 2,3,4–, 2,4,5– and 3,4,5–trimethoxy benzyl alcohol. It was found that 2–methoxyphenol was the best substrate with a specific growth rate of 0.14 h–1. The production of demethylase induced cells was carried out in a chemostat culture at a dilution rate (D) of 0.08 h–1. Cells were harvested at steady state of growth and concentrated before use. Optimal concentration of 2–methoxvphenol as the sole carbon source was 0.62 g/L. Demethylation reaction of 0.31 g/L 2–methoxyphenol by induced culture increases 78% relative to the chemostat culture containing only fructose. Key words: Demethylation reaction; demethylase; harvested cells; methoxyaromatic; Acetobacteriumwoodii

scholarly journals Comparative metabolomics of Phialemonium curvatum as an omnipotent fungus cultivated on crude palm oil versus glucose

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Arief Izzairy Zamani ◽  
Susann Barig ◽  
Sarah Ibrahim ◽  
Hirzun Mohd. Yusof ◽  
Julia Ibrahim ◽  
...  
Keyword(s):  
Carbon Source ◽  
Organic Acids ◽  
Vegetable Oil ◽  
Carbon Metabolism ◽  
Sole Carbon Source ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Targeted Metabolomics ◽  
Central Carbon

Abstract Background Sugars and triglycerides are common carbon sources for microorganisms. Nonetheless, a systematic comparative interpretation of metabolic changes upon vegetable oil or glucose as sole carbon source is still lacking. Selected fungi that can grow in acidic mineral salt media (MSM) with vegetable oil had been identified recently. Hence, this study aimed to investigate the overall metabolite changes of an omnipotent fungus and to reveal changes at central carbon metabolism corresponding to both carbon sources. Results Targeted and non-targeted metabolomics for both polar and semi-polar metabolites of Phialemonium curvatum AWO2 (DSM 23903) cultivated in MSM with palm oil (MSM-P) or glucose (MSM-G) as carbon sources were obtained. Targeted metabolomics on central carbon metabolism of tricarboxylic acid (TCA) cycle and glyoxylate cycle were analysed using LC–MS/MS-TripleQ and GC–MS, while untargeted metabolite profiling was performed using LC–MS/MS-QTOF followed by multivariate analysis. Targeted metabolomics analysis showed that glyoxylate pathway and TCA cycle were recruited at central carbon metabolism for triglyceride and glucose catabolism, respectively. Significant differences in organic acids concentration of about 4- to 8-fold were observed for citric acid, succinic acid, malic acid, and oxaloacetic acid. Correlation of organic acids concentration and key enzymes involved in the central carbon metabolism was further determined by enzymatic assays. On the other hand, the untargeted profiling revealed seven metabolites undergoing significant changes between MSM-P and MSM-G cultures. Conclusions Overall, this study has provided insights on the understanding on the effect of triglycerides and sugar as carbon source in fungi global metabolic pathway, which might become important for future optimization of carbon flux engineering in fungi to improve organic acids production when vegetable oil is applied as the sole carbon source.

A Method for Trapping Intermediates of Polyketide Biosynthesis with a Nonhydrolyzable Malonyl-Coenzyme A Analogue

2005 ◽  
Vol 44 (43) ◽  
pp. 7079-7082 ◽  
Author(s):  
Dieter Spiteller ◽  
Claire L. Waterman ◽  
Jonathan B. Spencer
Keyword(s):  
Coenzyme A ◽  
Polyketide Biosynthesis

Control of growth, secondary metabolism and sporulation in Streptomyces venezuelae ISP5230 by jadW 1, a member of the afsA family of γ-butyrolactone regulatory genes

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 1991-2004 ◽  
Author(s):  
Liru Wang ◽  
Leo C. Vining
Keyword(s):  
Growth Rate ◽  
Secondary Metabolism ◽  
Morphological Differentiation ◽  
Nitrogen Sources ◽  
Repeat Sequence ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
Streptomyces Venezuelae ◽  
New Genes ◽  
Insertional Inactivation

Three new genes (jadW 1, jadW 2 and jadW 3) were isolated from a region of the Streptomyces venezuelae ISP5230 chromosome at the left-hand end of the jad cluster for jadomycin B (JdB) biosynthesis. The deduced amino acid sequence of jadW 1 showed strong similarity to gene products associated in several streptomycetes with γ-butyrolactone autoregulators controlling morphological differentiation and secondary metabolism. Examination of JadW1 for conserved domains detected a repeat sequence characteristic of proteins in the AfsA regulatory family. Insertional inactivation of jadW 1 reduced the growth rate of S. venezuelae cultures in aerated liquid media containing complex nitrogen sources and altered growth morphology in minimal medium. It also affected sporulation on agar media. Cultures of jadW 1-disrupted mutants grown under conditions supporting biosynthesis of JdB or chloramphenicol by the wild-type strain failed to produce either of the antibiotics. Complementing the disrupted strain by transformation with pJV435, containing a cloned copy of the gene, improved sporulation and restored antibiotic biosynthesis in transformants to titres close to those of the wild-type similarly transformed with pJV435 as a control. The results are consistent with a role for jadW 1 in regulating morphological and metabolic differentiation. Further sequence analysis of jadR 2, which functions with jadR 1 in stress-induced activation of JdB biosynthesis, indicated that this gene encodes a γ-butyrolactone receptor homologue. The growth-rate-sensitive phenotype of the jadW 1-disrupted mutant, and the proximity of jadW 1 to jadR 2 indicate that this region of the jad gene cluster contains a regulatory mechanism incorporating γ-butyrolactone signalling and sensitivity to environmental stress.

scholarly journals Cloning and Characterization of Sialidases with 2-6′ and 2-3′ Sialyl Lactose Specificity from Pasteurella multocida

2000 ◽  
Vol 182 (24) ◽  
pp. 6874-6883 ◽  
Author(s):  
Shaikh Mizan ◽  
Adam Henk ◽  
Amy Stallings ◽  
Marie Maier ◽  
Margie D. Lee
Keyword(s):  
Pasteurella Multocida ◽  
Carbon Sources ◽  
Sialidase Activity ◽  
Fowl Cholera ◽  
E Coli ◽  
Colominic Acid ◽  
Mucosal Surfaces ◽  
Insertional Inactivation ◽  
Significant Amino Acid Sequence

ABSTRACT Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates ofP. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene, nanH, from a fowl cholera isolate ofP. multocida. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3′ sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3′ and 2-6′ sialyl lactose, while NanH demonstrated activity only on 2-3′ sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8′ sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces.

scholarly journals Role of Crotonyl Coenzyme A Reductase in Determining the Ratio of Polyketides Monensin A and Monensin B Produced byStreptomyces cinnamonensis

1999 ◽  
Vol 181 (21) ◽  
pp. 6806-6813 ◽  
Author(s):  
Haibin Liu ◽  
Kevin A. Reynolds
Keyword(s):  
Coenzyme A ◽  
Streptomyces Coelicolor ◽  
Fermentation Medium ◽  
Malt Extract ◽  
Moderate Increase ◽  
Wild Type ◽  
Coding Region ◽  
Gene Encoding ◽  
Monensin A ◽  
Coa Reductase

ABSTRACT The ccr gene, encoding crotonyl coenzyme A (CoA) reductase (CCR), was cloned from Streptomyces cinnamonensisC730.1 and shown to encode a protein with 90% amino acid sequence identity to the CCRs of Streptomyces collinus andStreptomyces coelicolor. A ccr-disrupted mutant, S. cinnamonensis L1, was constructed by inserting the hyg resistance gene into a unique BglII site within the ccr coding region. By use of theermE* promoter, the S. collinus ccr gene was expressed from plasmids in S. cinnamonensis C730.1/pHL18 and L1/pHL18. CCR activity in mutant L1 was shown to decrease by more than 90% in both yeast extract-malt extract (YEME) medium and a complex fermentation medium, compared to that in wild-type C730.1. Compared to C730.1, mutants C730.1/pHL18 and L1/pHL18 exhibited a huge increase in CCR activity (14- and 13-fold, respectively) in YEME medium and a moderate increase (3.7- and 2.7-fold, respectively) in the complex fermentation medium. In the complex fermentation medium,S. cinnamonensis L1 produced monensins A and B in a ratio of 12:88, dramatically lower than the 50:50 ratio observed for both C730.1 and C730.1/pHL18. Plasmid (pHL18)-based expression of theS. collinus ccr gene in mutant L1 increased the monensin A/monensin B ratio to 42:58. Labeling experiments with [1,2-13C2]acetate demonstrated the same levels of intact incorporation of this material into the butyrate-derived portion of monensin A in both C730.1 and mutant C730.1/pLH18 but a markedly decreased level of such incorporation in mutant L1. The addition of crotonic acid at 15 mM led to significant increases in the monensin A/monensin B ratio in C730.1 and C730.1/pHL18 but had no effect in S. cinnamonensis L1. These results demonstrate that CCR plays a significant role in providing butyryl-CoA for monensin A biosynthesis and is present in wild-type S. cinnamonensis C730.1 at a level sufficient that the availability of the appropriate substrate (crotonyl-CoA) is limiting.

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