James T. Kealey

Complete Reconstitution of a Highly Reducing Iterative Polyketide Synthase

Dissecting Megaenzyme Mechanisms Filamentous fungi contain a class of multidomain enzymes, the highly-reducing iterative polyketide synthases (HR-IPKSs), which produce important natural products such as the cholesterol-lowering drug lovastatin. To produce their complex products, these megasynthases use multiple catalytic domains repeatedly in different combinations, but mechanistic details remain unclear. Ma et al. (p. 589) now report in vitro reconstitution of the complete catalytic function of lovastatin nonaketide synthase (LovB), the megasynthase that works together with a partner enzyme LovC to complete nearly 40 chemical steps required to construct the core of lovastatin. Analyses of the dependency of enzyme function on cofactors and on the partner enzyme elucidate the programming rules for this system. Reconstitution of catalytic function provides insight into how multifunctional enzymes synthesize important natural products. Highly reducing iterative polyketide synthases are large, multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of the lovastatin nonaketide synthase (LovB) from an engineered strain of Saccharomyces cerevisiae, as well as complete reconstitution of its catalytic function in the presence and absence of cofactors (the reduced form of nicotinamide adenine dinucleotide phosphate and S-adenosylmethionine) and its partner enzyme, the enoyl reductase LovC. Our results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding of the functions and structures of this family of enzymes.

Metabolic engineering of Escherichia coli 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.

Genes for the Biosynthesis of the Fungal Polyketides Hypothemycin from Hypomyces subiculosus and Radicicol from Pochonia chlamydosporia

ABSTRACT Gene clusters for biosynthesis of the fungal polyketides hypothemycin and radicicol from Hypomyces subiculosus and Pochonia chlamydosporia, respectively, were sequenced. Both clusters encode a reducing polyketide synthase (PKS) and a nonreducing PKS like those in the zearalenone cluster of Gibberella zeae, plus enzymes with putative post-PKS functions. Introduction of an O-methyltransferase (OMT) knockout construct into H. subiculosus resulted in a strain with increased production of 4-O-desmethylhypothemycin, but because transformation of H. subiculosus was very difficult, we opted to characterize hypothemycin biosynthesis using heterologous gene expression. In vitro, the OMT could methylate various substrates lacking a 4-O-methyl group, and the flavin-dependent monooxygenase (FMO) could epoxidate substrates with a 1′,2′ double bond. The glutathione S-transferase catalyzed cis-trans isomerization of the 7′,8′ double bond of hypothemycin. Expression of both hypothemycin PKS genes (but neither gene alone) in yeast resulted in production of trans-7′,8′-dehydrozearalenol (DHZ). Adding expression of OMT, expression of FMO, and expression of cytochrome P450 to the strain resulted in methylation, 1′,2′-epoxidation, and hydroxylation of DHZ, respectively. The radicicol gene cluster encodes halogenase and cytochrome P450 homologues that are presumed to catalyze chlorination and epoxidation, respectively. Schemes for biosynthesis of hypothemycin and radicicol are proposed. The PKSs encoded by the two clusters described above and those encoded by the zearalenone cluster all synthesize different products, yet they have significant sequence identity. These PKSs may provide a useful system for probing the mechanisms of fungal PKS programming.

Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster

A generic design of Type I polyketide synthase genes has been reported in which modules, and domains within modules, are flanked by sets of unique restriction sites that are repeated in every module [1]. Using the universal design, we synthesized the six-module DEBS gene cluster optimized for codon usage in E. coli, and cloned the three open reading frames into three compatible expression vectors. With one correctable exception, the amino acid substitutions required for restriction site placements were compatible with polyketide production. When expressed in E. coli the codon-optimized synthetic gene cluster produced significantly more protein than did the wild-type sequence. Indeed, for optimal polyketide production, PKS expression had to be down-regulated by promoter attenuation to achieve balance with expression of the accessory proteins needed to support polyketide biosynthesis.

Determination of the extent of phosphopantetheinylation of polyketide synthases expressed in Escherichia coli and Saccharomyces cerevisiae

A sensitive fluorescent assay was developed to measure the extent of phosphopantetheinylation of polyketide synthase (PKS) acyl carrier protein (ACP) domains in polyketide production strains. The in vitro assay measures PKS fluorescence after transfer of fluorescently labeled phosphopantetheine from coenzyme A to PKS ACP domains in crude protein extracts. The assay was used to determine the extent of phosphopantetheinylation of ACP domains of the erythromycin precursor polyketide synthase, 6-deoxyerythronolide B synthase (DEBS), expressed in a heterologous Escherichia coli polyketide production strain. The data showed that greater than 99.9% of DEBS is phosphopantetheinylated. The assay was also used to interrogate the extent of phosphopantetheinylation of the lovastatin nonaketide synthase (LNKS) heterologously expressed in Saccharomyces cerevisiae. The data showed that LNKS was efficiently phosphopantetheinylated in S. cerevisiae and that lack of production of the lovastatin precursor polyketide was not due to insufficient phosphopantetheinylation of the expressed synthase.

Purification methods for recombinantLactobacillus casei thymidylate synthase and mutants: A general, automated procedure

General procedures for the rapid, large-scale purification of recombinant Lactobacillus casei thymidylate synthase and its mutants have been established. An effective method employs sequential phosphocellulose and hydroxylapatite chromatography. Crude cell extracts are directly applied to phosphocellulose, and the enzyme is obtained in a nearly pure state by stepwise elution with KCl. The eluate is directly applied to hydroxylapatite, and the homogeneous enzyme is eluted with a gradient of potassium phosphate. The method has been successful for the purification of recombinant wild-type enzyme and all mutants thus far examined. The entire purification procedure has been automated using a commonly available FPLC system and can be performed in several hours with minimal operator time.

Chemobiosynthesis of Novel 6-Deoxyerythronolide B Analogues by Mutation of the Loading Module of 6-Deoxyerythronolide B Synthase 1

ABSTRACT Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler “monoketide” acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1o for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.

Macrolide biosynthesis: a single cytochrome P450, PicK, is responsible for the hydroxylations that generate methymycin, neomethymycin, and picromycin in Streptomyces venezuelae.

The final step in the biosynthesis of methymycin, neomethymycin, and picromycin is an hydroxylation, shown to be carried out by the cytochrome P-450 monooxygenase, PicK. Direct comparison of the relative Kcat/K(m) values for the two substrates, YC-17 and narbomycin, showed a threefold rate preference of picK for narbomycin.

The dynamic NMR structure of the TψC-loop: Implications for the specificity of tRNA methylation

AbstracttRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependentmethylation of uridine-54 in the TΨC-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried andthe question arises as to how RUMT gains access to the methylation site. A 17-mer RNAhairpin consisting of nucleotides 49–65 of the TΨ-loop is a substrate for RUMT.Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD)methods were used to determine the solution structure of the 17-mer T-arm fragment. Theloop of the hairpin exhibits enhanced flexibility which renders the conventional NMR averagestructure less useful compared to the more commonly found situation where a molecule existsin predominantly one major conformation. However, when resorting to softer refinementmethods such as MD with time-averaged restraints, the conflicting restraints in the loop canbe satisfied much better. The dynamic structure of the T-arm is represented as an ensembleof 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the TΨ-loop in solution in conjunction with extensive binding studies of RUMT with the TΨC-loop and tRNA suggest that the specificity of the RUMT/tRNA recognition is associated withtRNA tertiary structure elements. For the methylation, RUMT would simply have to breakthe tertiary interactions between the D- and T-loops, leading to a melting of the T-armstructure and making U54 available for methylation.