Exploring the Promiscuous Enzymatic Activation of Unnatural Polyketide Extender Units in Vitro and in Vivo for Monensin Biosynthesis.

The incorporation of new-to-nature extender units into polyketide synthesis is an important source for diversity yet is restricted by limited availability of suitably activated building blocks in vivo. We here describe a straightforward workflow for the biogenic activation of commercially available new-to-nature extender units. Firstly, the substrate scope of a highly flexible malonyl co-enzyme A synthetase from Streptomyces cinnamonensis was characterized. The results were matched by in vivo experiments in which the said extender units were accepted by both the polyketide synthase and the accessory enzymes of the monensin biosynthetic pathway. The experiments gave rise to a series of predictable monensin derivatives by the exploitation of the innate substrate promiscuity of an acyltransferase and downstream enzyme functions.

Flexible enzymatic activation of artificial polyketide extender units by Streptomyces cinnamonensis into the monensin biosynthetic pathway.

Streptomyces cinnamonensis A495 is a variant of the monensin producer which instead of the native polyether antibiotic gives rise to antibiotic and anti-tumour shunt-product premonensin. Through the supplementation of the fermentation medium with suitable precursors, premonensin can be derivatized via the incorporation of new-to-nature extender units into the biosynthetic machinery. Polyketide extender units require activation, typically in form of coenzyme A-thioesters. These are membrane impermeable and thus in the past an artificial mimic was employed. Here, we show the use and preliminary characterization of a highly substrate promiscuous new enzyme for the endogenous thioester formation in a Streptomyces strain. These intracellularly activated alternative extender units are significantly better incorporated into premonensin than the synthetically activated counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyketide natural products are of enormous relevance in medicine. The hit-rate in finding active compounds for the potential treatment of various diseases among this substance family of microbial origin is high. However, most polyketides require derivatization to render them suitable for the application. Of relevance in this field is the incorporation of artificial substances into the biogenesis of polyketides, hampered by both the microbial metabolism and the complexity of the enzymes involved. This manuscript describes the straightforward and selective biosynthetic incorporation of synthetic substances into a reduced polyketide and showcases a promising new enzyme to aid this purpose.

Characterization of three pathway-specific regulators for high production of monensin in Streptomyces cinnamonensis.

Monensin, a polyether ionophore antibiotic, is produced by Streptomyces cinnamonensis and worldwide used as a coccidiostat and growth-promoting agent in the field of animal feeding. The monensin biosynthetic gene cluster (mon) has been reported. In this study, the potential functions of three putatively pathway-specific regulators (MonH, MonRI, and MonRII) were clarified. The results from gene inactivation, complementation, and overexpression showed that MonH, MonRI, and MonRII positively regulate monensin production. Both MonH and MonRI are essential for monensin biosynthesis, while MonRII is non-essential and could be completely replaced by additional expression of monRI. Transcriptional analysis of the mon cluster by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and electrophoresis mobility shift assays (EMSAs) revealed a co-regulatory cascade process. MonH upregulates the transcription of monRII, and MonRII in turn enhances the transcription of monRI. MonRII is an autorepressor, while MonRI is an autoactivator. MonH activates the transcription of monCII-monE, and upregulates the transcription of monT that is repressed by MonRII. monAX and monD are activated by MonRI, and upregulated by MonRII. Co-regulation of those post-polyketide synthase (post-PKS) genes by MonH, MonRI, and MonRII would contribute to high production of monensin. These results shed new light on the transcriptional regulatory cascades of antibiotic biosynthesis in Streptomyces.

DasR positively controls monensin production at two-level regulation in Streptomyces cinnamonensis.

The polyether ionophore antibiotic monensin is produced by Streptomyces cinnamonensis and is used as a coccidiostat for chickens and growth-promoting agent for cattle. Monensin biosynthetic gene cluster has been cloned and partially characterized. The GntR-family transcription factor DasR regulates antibiotic production and morphological development in Streptomyces coelicolor and Saccharopolyspora erythraea. In this study, we identified and characterized the two-level regulatory cascade of DasR to monensin production in S. cinnamonensis. Forward and reverse genetics by overexpression and antisense RNA silence of dasR revealed that DasR positively controls monensin production under nutrient-rich condition. Electrophoresis mobility shift assay (EMSA) showed that DasR protein specifically binds to the promoter regions of both pathway-specific regulatory gene monRII and biosynthetic genes monAIX, monE and monT. Semi-quantitative RT-PCR further confirmed that DasR upregulates the transcriptional levels of these genes during monensin fermentation. Subsequently, co-overexpressed dasR with pathway-specific regulatory genes monRI, monRII or monH greatly improved monensin production.

Production and biological activity of laidlomycin, anti-MRSA/VRE antibiotic from Streptomyces sp. CS684.

Culture broth of a streptomycete isolate, Streptomyces sp. CS684 showed antibacterial activity on methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Among purified substances from the organism, CSU-1, which is active against MRSA and VRE, is a C37H62O12Na (M+, 721.3875), and identified as laidlomycin. The anti-MRSA and anti-VRE activity of CSU-1 was stronger than oxacillin and vancomycin. Phylogenetic analysis showed that strain CS684 is very similar to Streptomyces ardus NRRL 2817T, whereas the ability of Streptomyces sp. CS684 to produce laidlomycin was shown to be unique.

Evidence that a novel thioesterase is responsible for polyketide chain release during biosynthesis of the polyether ionophore monensin.

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the alpha/beta-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.

Evidence for the role of the monB genes in polyether ring formation during monensin biosynthesis.

Ionophoric polyethers are produced by the exquisitely stereoselective oxidative cyclization of a linear polyketide, probably via a triepoxide intermediate. We report here that deletion of either or both of the monBI and monBII genes from the monensin biosynthetic gene cluster gave strains that produced, in place of monensins A and B, a mixture of C-3-demethylmonensins and a number of minor components, including C-9-epi-monensin A. All the minor components were efficiently converted into monensins by subsequent acid treatment. These data strongly suggest that epoxide ring opening and concomitant polyether ring formation are catalyzed by the MonB enzymes, rather than by the enzyme MonCII as previously thought. Consistent with this, homology modeling shows that the structure of MonB-type enzymes closely resembles the recently determined structure of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis.

Identification and disruptional analysis of the Streptomyces cinnamonensis msdA gene, encoding methylmalonic acid semialdehyde dehydrogenase.

The msdA gene encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is known to be involved in valine catabolism in Streptomyces coelicolor. Using degenerative primers, a homolog of msdA gene was cloned and sequenced from the monensin producer, Streptomyces cinnamonensis. RT-PCR results showed msdA was expressed in a vegetative culture, bump-seed culture and the early stages of oil-based monensin fermentation. However, isotopic labeling of monensin A by [2, 4-(13)C(2)]butyrate revealed that this MSDH does not play a role in providing precursors such as methylmalonyl-CoA for the monensin biosynthesis under these fermentation conditions. Using a PCR-targeting method, msdA was disrupted by insertion of an apramycin resistance gene in S. cinnamonensis C730.1. Fermentation results revealed that the resulting DeltamsdA mutant (CXL1.1) produced comparable levels of monensin to that observed for C730.1. This result is consistent with the hypothesis that butyrate metabolism in S. cinnamonensis in the oil-based fermentation is not mediated by msdA, and that methylmalonyl-CoA is probably produced through direct oxidation of the pro-S methyl group of isobutyryl-CoA. The CXL1.1 mutant and C730.1 were both able to grow in minimal medium with valine or butyrate as the sole carbon source, contrasting previous observations for S. coelicolor which demonstrated msdA is required for growth on valine. In conclusion, loss of the S. cinnamonensis msdA neither affects valine catabolism in a minimal medium, nor butyrate metabolism in an oil-based medium, and its role remains an enigma.

Crotonyl-coenzyme A reductase provides methylmalonyl-CoA precursors for monensin biosynthesis by Streptomyces cinnamonensis in an oil-based extended fermentation.

It is demonstrated that crotonyl-CoA reductase (CCR) plays a significant role in providing methylmalonyl-CoA for monensin biosynthesis in oil-based 10-day fermentations of Streptomyces cinnamonensis. Under these conditions S. cinnamonensis L1, a derivative of a high-titre producing industrial strain C730.1 in which ccr has been insertionally inactivated, produces only 15 % of the monensin yield. Labelling of the coenzyme A pools using [3H]-beta-alanine and analysis of intracellular acyl-CoAs in the L1 and C730.1 strains demonstrated that loss of ccr led to lower levels of the monensin precursor methymalonyl-CoA, relative to coenzyme A. Expression of a heterologous ccr gene from Streptomyces collinus fully restored monensin production to the L1 mutant. Using C730.1 and an oil-based extended fermentation an exceptionally efficient and comparably intact incorporation of ethyl [3,4-13C2]acetoacetate into both the ethylmalonyl-CoA- and methylmalonyl-CoA-derived positions of monensin was observed. No labelling of the malonyl-CoA-derived positions was observed. The opposite result was observed when the incorporation study was carried out with the L1 strain, demonstrating that ccr insertional inactivation has led to a reversal of carbon flux from an acetoacetyl-CoA intermediate. These results dramatically contrast similar analyses of the L1 mutant in glucose-soybean medium which indicate a role in providing ethylmalonyl-CoA but not methylmalonyl-CoA, thus causing a change in the ratio of monensin A and monensin B analogues, but not the overall monensin titre. These results demonstrate that the relative contributions of different pathways and enzymes to providing polyketide precursors are thus dependent upon the fermentation conditions. Furthermore, the generally accepted pathways for providing methylmalonyl-CoA for polyketide production may not be significant for the S. cinnamonensis high-titre monensin producer in oil-based extended fermentations. An alternative pathway, leading from the fatty acid catabolite acetyl-CoA, via the CCR-catalysed reaction is proposed.

Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization.

The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.

Measurement of potency and lipids in monensin fermentation broth by near-infrared spectroscopy.

A transmission near-infrared (NIR) spectroscopic method for quantification of potency and lipids in monensin fermentation broth was developed and validated. Two multiple linear regression calibration curves were established for a set of 100 fermentation samples, correlating the appropriate absorption bands in the NIR spectrum to the laboratory reference methods; high-performance liquid chromatography for potency, and chloroform extraction for lipids. During method development, potency was found to be well correlated to NIR absorbances specific for monensin. While acceptable, correlation of NIR absorbances characteristic of oil to the chloroform lipid method was weaker due to a greater amount of relative variation in the lipid measurements. Following establishment of the optimal calibration curves, the NIR method for potency and lipids was validated for selectivity, accuracy, precision, and robustness. In order to investigate long-term drift in the measurement system, samples were tested both by the NIR and the reference methods over a 7-month period. The differences between results from the two measurements were calculated and statistically analyzed.

MeaA, a putative coenzyme B12-dependent mutase, provides methylmalonyl coenzyme A for monensin biosynthesis in Streptomyces cinnamonensis.

The ratio of the major monensin analogs produced by Streptomyces 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). The meaA 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 (an icm 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 the meaA 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.

Precursor supply for polyketide biosynthesis: the role of crotonyl-CoA reductase.

Crotonyl-CoA reductase (CCR), which catalyzes the reduction of crotonyl-CoA to butyryl-CoA, is common to most streptomycetes and appears to be inducible by either lysine or its catabolites in Streptomyces cinnamonensis grown in chemically defined medium. A major role of CCR in providing butyryl-CoA from acetate for monensin A biosynthesis has been demonstrated by the observation of a change in the monensin A/monensin B ratio in the parent C730.1 strain (50/50) and a ccr (encoding CCR) disruptant (12:88) of S. cinnamonensis in a complex medium. Both strains produce significantly higher monensin A/monensin B ratios in a chemically defined medium containing valine as a major carbon source than in either complex medium or chemically defined medium containing alternate amino acids. This observation demonstrates that under certain growth conditions valine catabolism may have a more significant role than CCR in providing butyryl-CoA. Such a process most likely involves an isomerization of the valine catabolite isobutyryl-CoA, catalyzed by the coenzyme B(12)-dependent isobutyryl-CoA mutase. Monensin labeling experiments using dual (13)C-labeled acetate in the ccr-disrupted S. cinnamonensis indicate the presence of an additional coenzyme B(12)-dependent mutase linking branched and straight-chain C(4) compounds by a new pathway.

Engineering of complex polyketide biosynthesis--insights from sequencing of the monensin biosynthetic gene cluster.

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation.

Genetic approaches for controlling ratios of related polyketide products in fermentation processes.

Simple acyl thioesters are used as precursors for both the initiation and elongation steps in polyketide biosynthetic processes. Several structurally related polyketide products are sometimes made in these processes. These analogs are typically generated by a combination of two factors: availability of structurally similar biosynthetic precursors, and biosynthetic enzymes unable to effectively discriminate between them. Often, only one polyketide product is desired from a fermentation process, requiring a method to control the ratio of these different analogs. Preferential production of one desired analog is accomplished using random mutagenesis and manipulation of fermentation conditions. A genetic enzymatic understanding of polyketide biosynthesis, as well as the pathways that provide the relevant precursors, allows for a rational and more contemporary approach for control of analogs produced in fermentation processes. This approach involves genetic manipulation of either the pathways that provide pools of the acyl CoA thioester precursors, or the function/specificity of the appropriate biosynthetic enzymes. Reviewed herein are three such examples where these approaches have been carried out successfully with polyketide biosynthetic processes.

Role of crotonyl coenzyme A reductase in determining the ratio of polyketides monensin A and monensin B produced by Streptomyces cinnamonensis.

The ccr gene, encoding crotonyl coenzyme A (CoA) reductase (CCR), was cloned from Streptomyces cinnamonensis C730.1 and shown to encode a protein with 90% amino acid sequence identity to the CCRs of Streptomyces collinus and Streptomyces 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 the ermE* 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 the S. collinus ccr gene in mutant L1 increased the monensin A/monensin B ratio to 42:58. Labeling experiments with [1, 2-(13)C(2)]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.

Insertional inactivation of methylmalonyl coenzyme A (CoA) mutase and isobutyryl-CoA mutase genes in Streptomyces cinnamonensis: influence on polyketide antibiotic biosynthesis.

The coenzyme B(12)-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. The mutB::hygB mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. (13)C-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.