Institute for Structural Biology and Drug Discovery, Suite 212B, Biotechnology Park, Virginia Commonwealth University, 800 East Leigh Street, Richmond, VA 23219, USA
Correspondence
Kevin A. Reynolds
kareynol{at}hsc.vcu.edu
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ABSTRACT |
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INTRODUCTION |
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Methylmalonyl-CoA is one of the most common precursors used by modular type I PKSs. All of the six polyketide extension steps in erythromycin biosynthesis utilize this precursor (Cortes et al., 1990; Donadio et al., 1991
), while seven of the steps in monensin B biosynthesis utilize this precursor (Liu & Reynolds, 1999
). The availability of sufficient intracellular methylmalonyl-CoA represents a limiting factor for production of significant quantities of polyketide products in fermentation processes, both in natural hosts (Zhang & Reynolds, 2001
; Zhang et al., 1999a
) and engineered hosts (Dayem et al., 2002
). A thorough understanding of the relative roles of pathways which contribute to generation of methylmalonyl-CoA is required if genetic manipulation of precursor pathways to improve polyketide titres in fermentation processes is to become a reality.
Currently, there are three recognized pathways which could contribute to generation of methylmalonyl-CoA (Fig. 1). (i) Isomerization of succinyl-CoA, catalysed by methylmalonyl-CoA mutase (MCM) (Hunaiti & Kolattukudy, 1984
; Marsh, 1999
; Thomä & Leadlay, 1998
); (ii) Carboxylation of propionyl-CoA, catalysed by propionyl-CoA carboxylase (Bramwell et al., 1996
; Rodríguez & Gramajo, 1999
) or methylmalonyl-CoA transcarboxylase (Hunaiti & Kolattukudy, 1982
). The propionyl-CoA is either from the valine catabolism pathway of pseudomonads and mammals, degradation of odd chain fatty acids or other sources. (iii) A multistep oxidation of isobutyryl-CoA, via methacrylyl-CoA,
-hydroxyisobutyryl-CoA and methylmalonyl-CoA semialdehyde (Reynolds et al., 1988
). The isobutyryl-CoA is generated either from isomerization of butyryl-CoA via isobutyryl-CoA mutase (ICM), or valine catabolism. Sources of butyryl-CoA include metabolism of even-numbered fatty acids, and reduction of crotonyl-CoA generated from the condensation of two acetyl-CoA molecules. More recently a fourth and as yet undetermined pathway utilizing MeaA has emerged. The gene encoding the MeaA protein was found in Streptomyces collinus, Streptomyces cinnamonensis and Methylobacterium extorquens. It encodes a MCM-like protein and appears to be involved in methylmalonyl-CoA formation (Smith et al., 1996
; Zhang & Reynolds, 2001
). Finally, a monensin A labelling study with labelled acetoacetate in meaA and meaA icm mutants of S. cinnamonensis C730.1 (L1) has indicated the presence of potentially another pathway which links acetoacetyl-CoA with methylmalonyl-CoA, but which does not use MeaA or proceed through a butyryl-CoA intermediate (Zhang & Reynolds, 2001
).
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Similar types of study in an early industrial strain of S. cinnamonensis C730.1 have indicated that crotonyl-CoA reductase (CCR), which catalyses the conversion of crotonyl-CoA to butyryl-CoA, plays an important role in providing ethylmalonyl-CoA for monensin biosynthesis, but not methylmalonyl-CoA (Liu & Reynolds, 1999). Two major monensin products from fermentations of S. cinnamonensis are monensin A and monensin B (Fig. 2a
). Monensin A is made using an ethylmalonyl-CoA instead of methylmalonyl-CoA during the fifth elongation step (Liu & Reynolds, 1999
). We have previously cloned and sequenced S. cinnamonensis ccr and showed that insertional inactivation of this gene in the C730.1 strain generates an L1 mutant which produces a significantly higher monensin B to monensin A ratio. As there was no impact on overall monensin titres, this study suggested that the butyryl-CoA product of CCR was important for producing ethylmalonyl-CoA but not generating methylmalonyl-CoA (Liu & Reynolds, 1999
). This conclusion was supported by labelling studies with [1,2-13C2]acetate which led to significantly higher intact labelling of the ethylmalonyl-CoA-derived than the methylmalonyl-CoA-derived positions of monensin (Liu & Reynolds, 1999
).
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METHODS |
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Fermentation of S. cinnamonensis in an oil-based medium.
The fermentation was carried out in three stages. First, 50 ml of MOV medium in a 300-ml flask was inoculated with a fresh R2YE agar plate culture and incubated at 32 °C, 300 r.p.m. for 18 h. Then 0·5 ml of this MOV medium culture was transferred into a 300-ml flask containing 50 ml MOB medium and was incubated at 32 °C, 300 r.p.m. for 24 h. Finally 0·5 ml of MOB medium culture was used to inoculate 5 ml MOF medium in a 50-ml flask and incubated for 10 days at 34 °C at 260 r.p.m. On the fifth day, the MOF culture was supplemented with 1·2 ml natural oils. Purification of monensin A and quantification of monensins from the fermentation broth were performed as described previously (Reynolds et al., 1988).
Western blotting analyses.
The polyclonal antibodies against CCR were raised using recombinant CCR expressed in E. coli and purified by metal-chelate affinity chromatography. Samples at the different time-points of fermentations were washed with water and diluted in a SDS-loading buffer. Subsequently, the samples were heated to 100 °C for 10 min and cooled on ice, before loading onto NuPAGE 10 % Bis-Tris gel (Cat. No. NP0301, Invitrogen) with NuPAGE MOPS SDS running buffer (Cat. No. NP0001, Invitrogen). Proteins were then electroblotted on microporous polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h, washed, and incubated with primary antibodies (1 : 750) overnight at 4 °C. After a wash step, immunocomplexes were detected using an anti-mouse IgG (Sigma). The membranes were developed with the NBT/BCIP (Roche) according to the manufacturer's protocol.
Transcript analysis.
Total RNA was isolated from S. cinnamonensis grown in oil-based medium at different times of fermentation. In order to prevent mRNA degradation, two volumes of RNAprotect reagent (Qiagen) were added to one volume of the broth. The RNeasy Midi kit (Qiagen) was used for total RNA preparation according to the manufacturer's instructions. Nucleic acid preparations were treated with DNase I (DNA-free kit; Ambion) as recommended by the manufacturer. Two primer sets were used to detect ccr and mcm transcripts using a One-Step RT-PCR kit (Qiagen) following methods recommended by the manufacturer: ccr forward, 5'-CAAGGACGAGACGGAGATGTT-3'; ccr reverse, 5'-CGACACAGATGGGGTTGGC-3' and mcm forward, 5'-TGCCGCGCTACAACTCCATCTCG-3'; mcm reverse, 5'-CTCGTCCAGGGCGTTCGTGTGC-3'. Dimethyl sulfoxide (5 %, v/v final) was added to the RT-PCR mixture. For RT-PCR, conditions were as follows: an initial DNA strand synthesis with reverse transcriptase, 52 °C for 30 min, followed by 95 °C for 15 min to activate the DNA polymerase, and then 35 cycles of 94 °C for 10 s, 55 °C for 20 s, 72 °C for 45 s. The negative controls were carried out with each experimental reaction using same enzyme mix but without the initial reverse transcription step.
Incorporation of ethyl [3,4-13C2]acetoacetate and [2,4-13C2]butyrate into monensin A.
Ethyl [3,4-13C2]acetoacetate (22·5 mM final concentration) and 30 mM (final concentration) of [2,4-13C2]butyrate were added into two separate fermentation broths in three equal portions on days 3, 4 and 5. After 10 days of fermentation monensin A (1625 mg) was purified and used for NMR analysis.
HPLC analysis of acyl-CoAs.
HPLC analyses of acyl-CoAs was performed with a 250x4·6 mm 5 µm Luna C18 column (Phenomenex), with only minor modifications to a recently reported method (Dayem et al., 2002). For non-radiolabelled samples, the eluant was monitored at 260 nm with a UV detector. Radiolabelled samples were analysed with
-RAM model 2 Radio flow-through detector (IN/US Systems). The ratio of scintillation fluid : eluant was 1·5 : 1. Peaks were assigned by comparing elution time with CoA, malonyl-CoA and methylmalonyl-CoA standards.
Analysis of intracellular acyl-CoA pools.
[3H]--Alanine (3·7 MBq total) was added into the MOF fermentation broth in two equal portions on day 1 and day 5. On days 3, 7 and 10 of fermentation the cells were collected by centrifugation and washed with sterilized deionized water several times until the supernatant was colourless. The cell pellet was resuspended with 4 ml ice-cold 10 % TCA, and then sonicated on ice 5x30 s at 5 W. Precipitants were removed by centrifugation and supernatants were filtered with a 0·2 µm syringe filter and then used for HPLC analysis (Dayem et al., 2002
).
Expression of S. collinus ccr in S. cinnamonensis C730.1 L1.
S. collinus ccr was PCR amplified from the expression plasmid pHL18 (Liu & Reynolds, 1999). The DNA primers were 5'-GAATTCGAGCTCGGTACCAGC-3' (rightward primer mapping onto upstream sequence of PermE) and 5'-ATCACAGTTGAATTCCTAAGCCAGG-3' (leftward primer mapping onto sequence downstream of ccr). The PCR primers both contained an EcoRI site (italicized) to facilitate cloning. The EcoRI-digested PCR product was cloned into the integrative shuttle vector pSET152 to give pGF200. pGF200 was introduced by conjugation (Hopwood et al., 1985
) into the S. cinnamonensis L1 strain and exconjugants were selected in the presence of 50 mg apramycin ml1, following standard protocols. Monensin production by the strain L1/pGF200 was determined in a standard fermentation procedure (as described above). Monensin production by C730.1/pSET152 was also evaluated.
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RESULTS |
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We confirmed that the decrease in monensin titre in the L1 mutant was due specifically to loss of CCR by carrying out a complementation experiment. In this work a pSET152 derivative (pGF200) carrying the S. collinus ccr gene under the control of the ermE* promoter was conjugated into the L1 strain. The resulting L1/pGF200 strain generated comparable levels of monensin compared to both the C730.1 and C730.1/pSET152 strains. This set of experiments demonstrates that a single copy of a heterologous ccr gene expressed from ermE* promoter can fully restore monensin titres to the L1 strain. The work also demonstrates that pSET152 and derivatives thereof can be integrated into the C31-attB site in S. cinnamonensis C730.1 without impacting fermentation titres. It has been shown that in some strains such site-specific integration can lead to decreased fermentation titres (Baltz, 1998
).
The incorporation of ethyl [3,4-13C2]acetoacetate and [2,4-13C2]butyrate into monensin A
It has long been established that dual-labelled acetoacetyl-CoA can be incorporated intact into methylmalonyl-CoA-derived positions of monensin (Reynolds et al., 1988). The intact incorporation is inconsistent with a pathway in which acetyl-CoA generated from acetoacetyl-CoA is converted via the citric acid cycle and succinyl-CoA to methylmalonyl-CoA. Rather, the intact labelling is consistent with a more direct pathway which may involve a butyryl-CoA intermediate (Zhang & Reynolds, 2001
). In all experiments reported to date there is incorporation into the acetyl-CoA (malonyl-CoA) methylmalonyl-CoA and butyryl-CoA (ethylmalonyl-CoA)-derived positions suggesting no overwhelming flux in pathways originating from acetoacetyl-CoA (Zhang & Reynolds, 2001
). Also, the level of incorporation into the butyryl-CoA (ethylmalonyl-CoA)-derived positions is typically 10-fold higher than either the acetyl-CoA (malonyl-CoA) or methylmalonyl-CoA, suggesting significant dilution of the labelled material by other pathways (Zhang & Reynolds, 2001
). It has thus been argued that a pathway passing though acetoacetyl-CoA and potentially butyryl-CoA as an intermediate is not the primary source of methylmalonyl-CoA for monensin production (Zhang & Reynolds, 2001
).
A dramatically different series of results were obtained from incorporation studies using the C730.1 S. cinnamonensis strain in the oil-based extended fermentation. As shown in Fig. 2(b) comparable intact labelling into both the butyryl-CoA- (ethylmalonyl-CoA) and methylmalonyl-CoA-derived positions was observed. Enriched doublets surrounding the natural abundance signals for C-2, C-4, C-6, C-12, C-18, C-22 and C-24 and the corresponding methyl substituents were observed, consistent with incorporation of [2,3-13C2]methylmalonyl-CoA. The level of labelling was approximately 1·1±0·12 %. A similar set of enriched-doublets was observed for C-32 and C-33, consistent with incorporation of [3,4-13C2]ethylmalonyl-CoA. In this case the level of enrichment was 1·35±0·1 %. These data suggest that the butyryl-CoA pool used to generate ethylmalonyl-CoA precursor also generates more than 80 % of the methylmalonyl-CoA precursor pool. An alternative interpretation is that the labelled acetoacetyl-CoA is converted to methylmalonyl-CoA primarily via a pathway not using a butyryl-CoA intermediate, and that labelled butyryl-CoA generated from this acetoacetyl-CoA is diluted by a large pool of butyryl-CoA generated from the degradation of long chain fatty acids. This possibility was discounted by carrying out additional incorporation studies with ethyl [3,4-13C2]acetoacetate (the L1 mutant, described below) and [2,4-13C2]butyrate (C730.1 strain). In the latter case, a threefold enrichment of both C16 and C33 (derived from C2 and C4 of ethylmalonyl-CoA) and the methyl substituents of monensin (derived from C3 of methylmalonyl-CoA) were observed (data not shown). These observations clearly support the hypothesis that the major pathway providing methylmalonyl-CoA under these conditions passes through a butyryl-CoA intermediate. The final observation from these incorporation studies was that there was no detectable level of labelling of the malonyl-CoA-derived positions of monensin (see C10 in Fig. 2b
), from either labelled ethyl acetoacetate or butyrate. Thus the pathway flux from acetoacetyl-CoA, the last intermediate in the degradation of straight chain fatty acids, is not towards acetyl-CoA, a surprising observation given that fatty acid degradation provides the primary carbon source in the fermentation. The observation indicates that instead the carbon flux is from acetoacetyl-CoA towards butyryl-CoA (which is subsequently efficiently converted to both ethylmalonyl-CoA and methylmalonyl-CoA).
A labelling study with ethyl [3,4-13C2]acetoacetate was also carried out with the S. cinnamonensis L1 mutant. Intact labelling of the methylmalonyl-CoA-derived positions decreased to 0·16 % (a 95 % decrease from that observed in the C730.1 strain). A significant drop in intact labelling of the positions derived from C3 and C4 of butyryl-CoA (ethylmalonyl-CoA) to 0·4 % (a 70 % decrease) was also observed. Previous analyses of the L1 mutant have shown that labelling of these units by [1,2-13C2]acetate (which would generate [3,4-13C2]acetoacetyl-CoA in vivo) also decreases by more than 75 % relative to the C730.1, but that intact low-level labelling of the methylmalonyl-CoA-derived positions is unaffected (Liu & Reynolds, 1999). The observations of loss of labelling of ethylmalonyl-CoA- and methylmalonyl-CoA-derived positions in the oil-based extended fermentation is again consistent with the hypothesis that these precursors are obtained from a CCR-mediated pathway in which acetoacetyl-CoA is channelled through a common butyryl-CoA intermediate. Loss of CCR blocks this pathway, leading to the observed decreased monensin titres. The acetoacetyl-CoA intermediate from straight chain fatty acid degradation, unable to generate butyryl-CoA, is thus converted to acetyl-CoA (malonyl-CoA), representing a switch in flux from this pathway intermediate. Consistent with this hypothesis, the decreased levels of labelling of the methylmalonyl-CoA and ethylmalonyl-CoA-derived positions of monensin were matched by a concomitant increase in labelling of the acetyl-CoA (malonyl-CoA)-derived positions of monensin (see Fig. 2c
). The level of labelling in the product of the L1 strain was 0·4 % (Fig. 2c
), in contrast to the lack of any detectable labelling in that of C730.1 strain (Fig. 2b
) grown under similar conditions.
Direct analysis of intracellular acyl-CoA pools
The loss of overall monensin titres and labelling studies with the L1 mutant were all consistent with an inability to convert the primary carbon source in the fermentation into methylmalonyl-CoA. Direct evidence for this hypothesis was sought by carrying out fermentations in the presence of [3H]--alanine. The radiolabelled
-alanine is a biosynthetic precursor of coenzyme A and thus serves to radiolabel all acyl-CoA pools within a cell (Dayem et al., 2002
). The 3H-labelled acyl-CoAs in the crude lysates are separated by HPLC and detected by radioactive detector. Analysis of radiolabelled pools in this way has been described previously for E. coli panD strains and has led to very efficient labelling of the coenzyme A pools (Cronan, 1980
; Dayem et al., 2002
; Jackowski & Rock, 1984
). Creation of a panD equivalent mutant in S. cinnamonensis strain L1 would introduce a second mutation and thus cloud any interpretation of the effect of the ccr loss to acyl-CoA pools in the C730.1 strain. Despite the associated loss in sensitivity, we were able to compare the relative levels of malonyl-CoA, CoASH and methylmalonyl-CoA (Fig. 5
), and observed clear and reproducible differences in the relative ratio of intracellular acyl-CoAs between S. cinnamonensis C730.1 and the ccr mutant C730.1 L1 on each of days 3, 7 and 10. In both strains, we observed relatively small levels of malonyl-CoA which did not differ significantly either between the two strains, or over the cause of the fermentation. The loss of ccr, however, causes a decrease in the level of methylmalonyl-CoA relative to CoASH during the time of monensin production (day 3 and 7). In S. cinnamonensis C730.1, the amount of methylmalonyl-CoA is significantly larger than the CoA pool (comprising about 7482 % of combined pools). The situation is reversed in the L1 mutant, where methylmalonyl-CoA pools represent only 1826 % of this combined CoA pool. These observations support the role of CCR in providing the monensin biosynthetic pathway with methylmalonyl-CoA. One additional interesting observation is that on day 10 of the fermentation the percentage of CoASH decreased, while methylmalonyl-CoA increased, in both strains. In the C730.1 strain only malonyl-CoA and methylmalonyl-CoA were detectable. In the L1 strain the levels of methylmalonyl-CoA exceeded the CoA levels. While all interpretations of relative levels of acyl-CoA pools should be made cautiously, these observations suggest that the loss of monensin production is not lack of precursor availability. Rather, lower monensin production causes a build up of these precursors.
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DISCUSSION |
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Sources of methylmalonyl-CoA
The source of methylmalonyl-CoA production in most fermentation processes has been a source of some controversy. Clearly the predominant carbon source in the fermentation will determine which pathway or pathways play significant roles. Oxidation of the valine catabolite isobutyryl-CoA has been well studied in many streptomycete strains. However, studies have clearly shown that this pathway does not contribute significantly to precursor supply for either doramectin biosynthesis in a Streptomyces avermitilis bkd mutant, or monensin biosynthesis in S. cinnamonensis C730.1 grown in glucose-soybean meal (Cropp et al., 2000; Zhang & Reynolds, 2001
). Valine is also not the predominant carbon source in the complex media used in industrial fermentations for polyketide products, and is thus unlikely to provide the majority of methylmalonyl-CoA precursors. The same is likely to be true for a pathway which involves direct carboxylation of propionyl-CoA, as only a small proportion of the carbon sources present in most industrial fermentations will be catabolized to propionyl-CoA. An isomerization of the citric acid cycle intermediate succinyl-CoA to generate (2R)-methylmalonyl-CoA and a subsequent epimerization step is likely the major source of (2S)-methylmalonyl-CoA in fermentation processes grown in carbohydrate and/or protein-based media. Indeed, it has been shown that MCM can play a significant role in providing methylmalonyl-CoA precursors for monensin biosynthesis for S. cinnamonensis C730.1 grown in a glucose-soybean medium (Zhang & Reynolds, 2001
; Zhang et al., 1999a
). This pathway has been introduced into E. coli strains and shown to be essential for production of polyketide products such as 6-deoxyerythonolide B (Dayem et al., 2002
).
Fermentations in which the carbon source is primarily fats and oils will generate significant quantities of acetyl-CoA, which needs to be converted to methylmalonyl-CoA in order to generate high titres of the polyketide product. In most bacteria, acetate assimilation is accomplished via the glyoxylate cycle, which catalyses the net synthesis of succinyl-CoA from two molecules of acetyl-CoA. Analysis of the Streptomyces coelicolor genome sequence reveals the presence of genes putatively encoding isocitrate lyase and malate synthase, the two essential enzymes for this process. Malate synthase and isocitrate lyase have recently been studied from several streptomyces (Chan & Sim, 1998; Goh et al., 2003
; Huttner et al., 1997
; Loke & Sim, 2000
; Loke et al., 2002
; Soh et al., 2001
). We have cloned and sequenced homologues of these genes from S. cinnamonensis. While glyxoylate enzymes appear to be present in a variety of streptomycetes, their role in acetate assimilation or providing precursors such as methylmalonyl-CoA for polyketide biosynthesis remains undetermined. An alternative pathway for acetate assimilation involving CCR has been proposed for streptomyces (Han & Reynolds, 1997
). This pathway, which leads directly to methylmalonyl-CoA, bypassing succinyl-CoA, was found over 15 years ago with the discovery of the coenzyme B12-dependent rearrangement which catalyses the interconversion of butyryl-CoA and isobutyryl-CoA (Reynolds & Robinson, 1985
; Reynolds et al., 1986
, 1988
). To date, the evidence from isotopic labelling studies and gene deletion experiments have indicated that CCR and this butyryl-CoA pathway play a minor role in providing methylmalonyl-CoA for polyketide biosynthesis. However, these analyses were not conducted under conditions where the fermentation titres were optimized using an oil-based extended fermentation process. Under these conditions the labelling studies, the dependence of monensin titre on CCR, and the analysis of the acyl-CoA pools, together indicate that the butyryl-CoA pathway may indeed provide the majority of the methylmalonyl-CoA precursor, despite the observation that the MCM genes are clearly expressed and that genes encoding glyoxylate cycle enzymes are present.
The low-level monensin production and acyl-CoA pool analysis of the L1 strain together indicate that CCR is not essential for generation of all of the methylmalonyl-CoA. Clearly, the butyryl-CoA-derived intermediate from straight chain fatty acid degradation can be converted into methylmalonyl-CoA in the L1 mutant. Presumably other pathways, including MCM, also can contribute. The incorporation of dual-labelled ethyl acetoacetate into the butyryl-CoA (ethylmalonyl-CoA)-derived unit of monensin A in the L1 mutant (albeit at a much lower level than the C730.1 strain) also indicates that the butyryl-CoA pathway may still function. The conversion of acetoacetyl-CoA to butyryl-CoA in the L1 strain has previously been observed in glucose-soybean meal fermentations (Liu & Reynolds, 1999) and may reflect the presence of another enoyl thioester reductase able to catalyse this reaction.
Several studies have indicated the presence of an additional pathway or pathways which link acetoacetyl-CoA with methylmalonyl-CoA, but which do not proceed through a butyryl-CoA intermediate. Labelling studies with [1,2-13C2]acetate in the L1 mutant and C730.1 strain in a glucose-soybean medium have demonstrated comparable low-level intact incorporation into the monensin positions derived from [2,3-13C2] of methylmalonyl-CoA (Liu & Reynolds, 1999, 2001
). In a separate set of studies [1-13C]-butyrate and ethyl [1,3-13C2]acetoacetate were incorporated into the methylmalonyl-CoA derived positions of monensin A in both an icmA : : hyg mutant and a mutB : : hyg mutant of S. cinnamonensis (Vrijbloed et al., 1999
). Thus it has been argued that in addition to the isomerization of butyryl-CoA (by ICM) and succinyl-CoA (by MCM) another pathway can generate methylmalonyl-CoA from acetoacetyl-CoA (Liu & Reynolds, 2001
; Vrijbloed et al., 1999
; Zhang & Reynolds, 2001
). The current studies show that such a pathway or pathways cannot be important for S. cinnamonensis grown in the oil-based extended fermentation as both the monensin titres decreased, and there was almost a complete loss of labelling of methylmalonyl-CoA derived positions by acetoacetyl-CoA in the L1 mutant (in fact, the flux from this intermediate switches towards the formation of acetyl-CoA).
Implications for strain improvement
Until now the butyryl-CoA pathway has not been considered as important in providing methylmalonyl-CoA for polyketide biosynthetic processes. Understanding changes that have led to strain improvement and attempts to rationally improve fermentation titres have often focused on the pathway from succinyl-CoA. Carboxylation of propionate and degradation of valine have also been considered as alternative potentially important sources. The present work demonstrates that in a S. cinnamonensis fermentation where the carbon source is primarily lipids, the butyryl-CoA pathway produces the majority of the methylmalonyl-CoA. It remains to be determined if this is the case for other oil-based fermentation processes. Nonetheless, it is clear that alternative pathways must be considered to have important roles and that the role of the fermentation medium is critical. Studies that aim to understand the role of precursor pathways involved in high titre production strains cannot be done under alternative fermentation conditions that provide lower titres.
In S. cinnamonensis we have demonstrated that the flux of the primary lipid metabolite acetyl-CoA (a precursor to malonyl-CoA) passes through butyryl-CoA (a precursor to ethylmalonyl-CoA) to methylmalonyl-CoA. We have cloned and sequenced many of the genes in this process (including four different acyl-CoA carboxylases). It may therefore be possible to make rational changes in the relative levels of the common polyketide precursors, malonyl-CoA, ethylmalonyl-CoA and methylmalonyl-CoA. Thus it might be possible to alter C730.1 or other monensin-producing strains to be a host for production of a range of different polyketide aglycone structures, regardless of which of these precursors they require/utilize.
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ACKNOWLEDGEMENTS |
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Received 16 April 2004;
revised 15 June 2004;
accepted 17 June 2004.
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