6-Deoxyerythronolide B analogue production in Escherichia coli through metabolic pathway engineering

The erythromycin precursor polyketide 6-deoxyerythronolide B (6-dEB) is produced from one propionyl-CoA starter unit and six (2S)-methylmalonyl-CoA extender units. In vitro studies have previously demonstrated that the loading module of 6-deoxyerythronolide B synthase (DEBS) exhibits relaxed substrate specificity and is able to accept butyryl-CoA, leading to the production of polyketides with butyrate starter units. We have shown that we can produce butyryl-CoA at levels of up to 50% of the total CoA pool in Escherichia coli cells that overexpress the acetoacetyl-CoA:acetyl-CoA transferase, AtoAD (EC 2.8.3.8), in media supplemented with butyrate. The DEBS polyketide synthase (PKS) used butyryl-CoA and methylmalonyl-CoA supplied in vivo by the AtoAD and methylmalonyl-CoA mutase pathways, respectively, to produce 15-methyl-6-dEB. Priming DEBS with endogenous butyryl-CoA affords an alternative and more direct route to 15-Me-6-dEB than that provided by the chemobiosynthesis method [Jacobsen, J. R., et al. (1997) Science 277, 367-369], which relies on priming a mutant DEBS with an exogenously fed diketide thioester. The approach described here demonstrates the utility of metabolic engineering in E. coli to introduce precursor pathways for the production of novel polyketides.     

Metabolic engineering of<Emphasis Type="Italic"> Escherichia coli</Emphasis> for improved 6-deoxyerythronolide B production

Escherichia coli is an attractive candidate as a host for polyketide production and has been engineered to produce the erythromycin precursor polyketide 6-deoxyerythronolide B (6dEB). In order to identify and optimize parameters that affect polyketide production in engineered E. coli, we first investigated the supply of the extender unit (2S)-methylmalonyl-CoA via three independent pathways. Expression of the Streptomyces coelicolor malonyl/methylmalonyl-CoA ligase (matB) pathway in E. coli together with methylmalonate feeding resulted in the accumulation of intracellular methylmalonyl-CoA to as much as 90% of the acyl-CoA pool. Surprisingly, the methylmalonyl-CoA generated from the matB pathway was not converted into 6dEB. In strains expressing either the S. coelicolor propionyl-CoA carboxylase (PCC) pathway or the Propionibacteria shermanii methylmalonyl-CoA mutase/epimerase pathway, methylmalonyl-CoA accumulated up to 30% of the total acyl-CoA pools, and 6dEB was produced; titers were fivefold higher when strains contained the PCC pathway rather than the mutase pathway. When the PCC and mutase pathways were expressed simultaneously, the PCC pathway predominated, as indicated by greater flux of ¹³C-propionate into 6dEB through the PCC pathway. To further optimize the E. coli production strain, we improved 6dEB titers by integrating the PCC and mutase pathways into the E. coli chromosome and by expressing the 6-deoxyerythronolide B synthase (DEBS) genes from a stable plasmid system.S. Murli and J. Kennedy contributed equally to this work     

Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.     

Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coenzyme B12-dependent acyl-CoA mutases

Coenzyme B(12)-dependent mutases are radical enzymes that catalyze reversible carbon skeleton rearrangement reactions. Here we describe Rhodobacter sphaeroides ethylmalonyl-CoA mutase (Ecm), a novel member of the family of coenzyme B(12)-dependent acyl-CoA mutases, that operates in the recently discovered ethylmalonyl-CoA pathway for acetate assimilation. Ecm is involved in the central reaction sequence of this novel pathway and catalyzes the transformation of ethylmalonyl-CoA to methylsuccinyl-CoA in combination with a second enzyme that was further identified as promiscuous ethylmalonyl-CoA/methylmalonyl-CoA epimerase. In contrast to the epimerase, Ecm is highly specific for its substrate, ethylmalonyl-CoA, and accepts methylmalonyl-CoA only at 0.2% relative activity. Sequence analysis revealed that Ecm is distinct from (2R)-methylmalonyl-CoA mutase as well as isobutyryl-CoA mutase and defines a new subfamily of coenzyme B(12)-dependent acyl-CoA mutases. In combination with molecular modeling, two signature sequences were identified that presumably contribute to the substrate specificity of these enzymes.     

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

When (methyl-2H3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 1H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion. When [methyl-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA). By addition of [14C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)- and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using (2H4)succinyl-CoA. When (1-13C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (less than 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place. (1,4-13C2)Succinic acid and (1,4-13C2)succinyl-CoA were synthesised and their 13C-NMR chemical shifts were exactly determined. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonyl-CoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3H to water and slow washing-in from the medium. Whenever migration of protium from the sterically less preferred 3-pro(S)- position of succinyl-CoA occurs and simultaneously a heavy isotope is maneuvered from the migratable 3-pro(R)- position into the labile alpha-position of methylmalonyl-CoA, the substitution by the COSCoA group takes place with inversion of configuration. When the sterically preferred 3-pro(R)-hydrogen atom migrates, the previously reported stereochemical retention occurs. A mechanistic and stereochemical scheme is discussed that fully accounts for all observations.     

A new substrate specificity for acyl transferase domains of the ascomycin polyketide synthase in Streptomyces hygroscopicus.

Ascomycin (FK520) is a structurally complex macrolide with immunosuppressant activity produced by Streptomyces hygroscopicus. The biosynthetic origin of C12-C15 and the two methoxy groups at C13 and C15 has been unclear. It was previously shown that acetate is not incorporated into C12-C15 of the macrolactone ring. Here, the acyl transferase (AT) of domain 8 in the ascomycin polyketide synthase was replaced with heterologous ATs by double homologous recombination. When AT8 was replaced with methylmalonyl-CoA-specific AT domains, the strains produced 13-methyl-13-desmethoxyascomycin, whereas when AT8 was replaced with a malonyl-specific domain, the strains produced 13-desmethoxyascomycin. These data show that ascomycin AT8 does not use malonyl- or methylmalonyl-CoA as a substrate in its native context. Therefore, AT8 must be specific for a substrate bearing oxygen on the alpha carbon. Feeding experiments showed that [(13)C]glycerol is incorporated into C12-C15 of ascomycin, indicating that both modules 7 and 8 of the polyketide synthase use an extender unit that can be derived from glycerol. When AT6 of the 6-deoxyerythronolide B synthase gene was replaced with ascomycin AT8 and the engineered gene was expressed in Streptomyces lividans, the strain produced 6-deoxyerythronolide B and 2-demethyl-6-deoxyerythronolide B. Therefore, although neither malonyl-CoA nor methylmalonyl-CoA is a substrate for ascomycin AT8 in its native context, both are substrates in the foreign context of the 6-deoxyerythronolide B synthase. Thus, we have demonstrated a new specificity for an AT domain in the ascomycin polyketide synthase and present evidence that specificity can be affected by context.     

Gluconeogenesis from propionate in kidney and liver of the vitamin B12-deficient rat

1. Kidney-cortex slices and the perfused livers of vitamin B(12)-deficient rats removed propionate from the incubation and perfusion media at 33 and 17% respectively of the rates found with tissues from rats receiving either a normal or a vitamin B(12)-supplemented diet. There was a corresponding fall in the rates of glucose synthesis from propionate in both tissues. 2. The addition of hydroxocobalamin or dimethylbenzimidazolylcobamide coenzyme to kidney-cortex slices from vitamin B(12)-deficient rats in vitro failed to restore the normal capacity for propionate metabolism. 3. Although the vitamin B(12)-deficient rat excretes measurable amounts of methylmalonate, no methylmalonate production could be detected (probably because of the low sensitivity of the method) when kidney-cortex slices or livers from deficient rats were incubated or perfused with propionate. 4. The addition of methylmalonate (5mm) to kidney-cortex slices from rats fed on a normal diet inhibited gluconeogenesis from propionate by 25%. 5. Methylmalonate formation is normally only a small fraction of the flux through methylmalonyl-CoA. This fraction increases in vitamin B(12)-deficient tissues (as shown by the urinary excretion of methylmalonate) presumably because the concentration of methylmalonyl-CoA rises as a result of low activity of methylmalonyl-CoA mutase (EC 5.4.99.2). Slow removal of methylmalonyl-CoA might depress propionate uptake owing to the reversibility of the steps leading to methylmalonyl-CoA formation.     

Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are giant multienzymes that biosynthesize a number of clinically important natural products. The modular nature of PKSs suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides, but the efficacy of such a strategy depends critically on gaining fundamental insight into PKS structure and function, most directly through experiments with purified PKS proteins. Several recent investigations into important aspects of the activity of these enzymes have used only partially purified proteins (often 3-4% of total protein), reflecting how difficult it is to purify these multienzymes in amounts adequate for kinetic and structural analysis. We report here the steady-state kinetic analysis of a typical bimodular PKS, 6-deoxyerythronolide B synthase 1-thioesterase (DEBS 1-TE), purified from recombinant Saccharopolyspora erythraea JCB101 by a new, high-yielding procedure consisting of three steps: ammonium sulfate precipitation, hydrophobic interaction chromatography and size-exclusion chromatography. The method provides 13-fold purification with a recovery of 11% of the applied PKS activity. The essentially homogeneous synthase exhibits an intrinsic methylmalonyl-CoA hydrolase activity, which competes with polyketide chain extension. The most reliable value for the kcat for synthesis of (3S,5R)-dihydroxy-(2R,4R)-dimethyl-n-heptanoic acid-delta-lactone is 0.84 min-1, and the apparent Km for (2RS)-methylmalonyl-CoA is 17 microM. This kcat is approximately 10-fold lower than the value reported previously for a differently engineered version of the truncated PKS, DEBS 1+TE. The difference likely reflects the fact that the DEBS 1-TE contains a hybrid acyl carrier protein (ACP) domain in its second module, which lowers its catalytic efficiency.     

Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold

BACKGROUND: Methylmalonyl-CoA epimerase (MMCE) is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. Present in many bacteria and in animals, it catalyzes the conversion of (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the substrate for the B12-dependent enzyme, methylmalonyl-CoA mutase. Defects in this pathway can result in severe acidosis and cause damage to the central nervous system in humans. RESULTS: The crystal structure of MMCE from Propionibacterium shermanii has been determined at 2.0 A resolution. The MMCE monomer is folded into two tandem betaalphabetabetabeta modules that pack edge-to-edge to generate an 8-stranded beta sheet. Two monomers then pack back-to-back to create a tightly associated dimer. In each monomer, the beta sheet curves around to create a deep cleft, in the floor of which His12, Gln65, His91, and Glu141 provide a binding site for a divalent metal ion, as shown by the binding of Co2+. Modeling 2-methylmalonate into the active site identifies two glutamate residues as the likely essential bases for the epimerization reaction. CONCLUSIONS: The betaalphabetabetabeta modules of MMCE correspond with those found in several other proteins, including bleomycin resistance protein, glyoxalase I, and a family of extradiol dioxygenases. Differences in connectivity are consistent with the evolution of these very different proteins from a common precursor by mechanisms of gene duplication and domain swapping. The metal binding residues also align precisely, and striking structural similarities between MMCE and glyoxalase I suggest common mechanisms in their respective epimerization and isomerization reactions.     

Purification and properties of the Streptomyces peucetius DpsC beta-ketoacyl:acyl carrier protein synthase III that specifies the propionate-starter unit for type II polyketide biosynthesis.

Biosynthesis of the polyketide-derived carbon skeleton of daunorubicin (DNR) begins with propionate rather than acetate, which is the starter unit for most other aromatic polyketides. The dpsCgene has been implicated in specifying the unique propionate-starter unit, and it encodes a protein that is very similar to the Escherichia coli beta-ketoacyl:acyl carrier protein (ACP) synthase III (FabH or KS III) enzyme of fatty acid biosynthesis. Purified DpsC was found to use propionyl-coenzyme A as substrate and to be acylated by propionate at the Ser-118 residue. DpsC exhibits KS III activity in catalyzing the condensation of propionyl-CoA and malonyl-ACP, and also functions as an acyltransferase in the transfer of propionate to an ACP. The DpsC enzyme has a high-substrate specificity, utilizing only propionyl-CoA, and not malonyl-CoA, 2-methylmalonyl-CoA or acetyl-CoA, as the starter unit of DNR biosynthesis.     

Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l(-1) (OD600 approximately 60) and the production of 700 mg l(-1) of 6-dEB. Retention of the two plasmids harboring the heterologous genes was maintained between 90 and 100% even in the absence of antibiotic selection. However, the accumulation of excess ammonia in the culture medium was found to significantly decrease the productivity of the cells. Through optimization of the medium composition and fermentation conditions, the maximum cell density was increased by two-fold, and a final titer of 1.1 g l(-1) of 6-dEB was achieved. This represents an 11-fold improvement compared to the highest reported titer of 100 mg l(-1) with E. coli as the production host.     

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. The reaction of (2R)-methylmalonyl-CoA in tritiated water.

The reaction catalysed by methylmalonyl-CoA epimerase from Propionibacterium shermanii was studied in tritiated water, in the direction with (2R)-methylmalonyl-CoA as substrate, under 'irreversible' conditions. After partial reaction, even when most of the substrate had been converted into product (isolated as propionyl-CoA) essentially no solvent tritium appeared in residual (2R)-methylmalonyl-CoA. The product, however, did contain tritium, and the specific radioactivity of the (2S)-epimer was deduced to be 0.33 times that of the solvent. These results provide further support for the mechanism proposed for the epimerase-catalysed reaction in the accompanying paper [Leadlay & Fuller (1983) Biochem. J. 213, 635-642], in which two enzyme bases act respectively as proton donor and acceptor. The observed low discrimination against solvent tritium entering the product can be accounted for by a mechanism in which the release of product is slow, and the re-protonation step on the enzyme is reversible, without leading to isotopic exchange with the solvent.     

Quantitative analysis of loading and extender acyltransferases of modular polyketide synthases

The acyltransferase (AT) domains of modular polyketide synthases (PKSs) are the primary determinants of building block specificity in polyketide biosynthesis and are therefore attractive targets for protein engineering. Thus far, investigations into the fundamental biochemical properties of AT domains have been hampered by the inability to produce these enzymes as self-standing polypeptides. Here we describe an alternative, generally applicable strategy for overexpression and analysis of AT domains from modular PKSs as truncated didomain proteins (approximately 60 kDa). Recently, we reported the expression and reconstitution of the loading didomain of 6-deoxyerythronolide B synthase (Lau, J., Cane, D. E., and Khosla, C. (2000) Biochemistry 39, 10514-20). By replacing the AT domain of this protein with a methylmalonyl-CoA specific AT domain from module 6 of the 6-deoxyerythronolide B synthase, or alternatively a malonyl-CoA specific AT domain from module 2 of the rapamycin synthase, each of these extender unit AT domains could be overproduced and purified to homogeneity. Using acyl-CoA substrates as acyl group donors and N-acetylcysteamine as the thiol acceptor, we devised a steady-state kinetic assay to probe the properties of these three didomain proteins and selected mutants. Propionyl-CoA was the preferred substrate of the loading didomain, although acetyl- and butyryl-CoA were also accepted with approximately 40-fold-lower specificity. In contrast to the relatively relaxed specificity of the loading AT domain, the methylmalonyl- and malonyl-specific AT domains had high specificity (>1000-fold) toward their natural substrates. The acyl transfer reaction was inhibited by coenzyme A (CoASH) with both a competitive and a noncompetitive component. Use of an exogenous holo-acyl carrier protein (ACP) as an acceptor thiol did not increase the rate of acyl transfer relative to the reaction involving N-acetylcysteamine, suggesting that either the on-rate of the acyl group is rate-limiting or that the apo-ACP component of the didomain protein precludes effective docking of a second ACP onto the AT active site. Mutation of Trp-222 in the loading AT domain to an Arg residue that is universally conserved in all extender unit AT domains failed to enable the loading AT domain to accept methylmalonyl-CoA as an alternative substrate. In contrast, mutation of the equivalent Arg residue in an extender AT domain resulted in a protein with no activity. Together, these results provide a foundation for future structural and mechanistic investigations into the properties of AT domains of modular PKSs.     

Homology modeling of human methylmalonyl-CoA mutase: a structural basis for point mutations causing methylmalonic aciduria.

Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent methylmalonyl-CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric methylmalonyl-CoA mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii methylmalonyl-CoA mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of methylmalonyl-CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.     

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.     

Methylmalonyl coenzyme A selectivity of cloned and expressed acyltransferase and beta-ketoacyl synthase domains of mycocerosic acid synthase from Mycobacterium bovis BCG.

Methyl-branched fatty acids and polyketides occur in a variety of living organisms. Previous studies have established that multifunctional enzymes use methylmalonyl coenzyme A (CoA) as the substrate to generate methyl-branched products such as mycocerosic acids and polyketides. However, we do not know which of the component activities show selectivity for methylmalonyl-CoA in any biological system. A comparison of homologies of the domains of the multifunctional synthases that selectively use malonyl-CoA or methylmalonyl-CoA suggested that the acyltransferase (AT) and beta-ketoacyl synthase (KS) domains might be responsible for the substrate selectivity. To test this hypothesis, we expressed the AT and KS domains of the mycocerosic acid synthase (MAS) gene from Mycobacterium bovis BCG in Escherichia coli and examined whether they confer to synthases that normally do not use methylmalonyl-CoA the ability to incorporate methylmalonyl-CoA into fatty acids. Both the AT and the KS domains of MAS showed selectivity for methylmalonyl-CoA over malonyl-CoA. Acyl carrier protein (ACP)-dependent elongation of the n-C12 acyl primer mainly by one methylmalonyl-CoA unit was catalyzed by an E. coli fatty acid synthase preparation only in the presence of the expressed MAS domains. An ACP-dependent elongation of the n-C20 acyl primer by one methylmalonyl-CoA extender unit was catalyzed by fatty acid synthase from Mycobacterium smegmatis only in the presence of the expressed MAS domains. These results show methylmalonyl-CoA selectivity for the AT and KS domains of MAS. These domains may be useful in producing novel polyketides by genetic engineering.     

Purification and characterization of methylmalonyl-CoA epimerase from Propionibacterium shermanii.

Methylmalonyl-CoA epimerase, which specifically interconverts the (2R)- and (2S)- epimers of methylmalonyl-CoA, was purified 95-fold from Propionibacterium shermanii by a new method that affords apparently homogeneous enzyme, in 80-100mg quantities, in yields representing about 40% of the activity in cell-free extracts. The specific activity of the purified enzyme, 10.1 mukat/mg, is much greater than previously reported. Native methylmalonyl-CoA epimerase has Mr about 33000, and apparently consists of two identical subunits. The purified enzyme is stable indefinitely when stored at -20 degrees C and pH 8.5, but contrary to previous reports it is not unusually acid-stable. The activity of methylmalonyl-CoA epimerase is increased by Co2+, and to a smaller extent by Ni2+, Mn2+ and Zn2+.     

Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase

BACKGROUND: 6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system. RESULTS: A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E. coli. As reported earlier for the enzyme isolated from P. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate. CONCLUSIONS: NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E. coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.     

Crystal structure of substrate complexes of methylmalonyl-CoA mutase.

X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.     

异源生物合成聚酮化合物的研究进展

聚酮是一大类具有重要生物活性的天然产物,其生物合成途径复杂多样。利用异源宿主合成聚酮化合物要比使用天然生产菌有很多优点。异源宿主的选择是异源生物合成聚酮的关键。这种宿主必须能够大量表达大分子聚酮合成酶(300 kDa或更大)且能够大规模的转译后修饰这些蛋白;还要能够形成大量的像丙二酰CoA、甲基丙二酰CoA等细胞内起始单元。随着各种技术的不断进步,异源宿主很可能成为大规模生产聚酮化合物的一个强有力平台。本文对聚酮合成酶,异源生产聚酮的优点、条件和应用都有所阐述。