Application of a Newly Identified and Characterized 18-O-Acyltransferase in Chemoenzymatic Synthesis of Selected Natural and Nonnatural Bioactive Derivatives of Phoslactomycins

Attachment of either short (C2 to C6) or medium (C8 to C12) acyl chains to both amine and alcohol moieties on polyketide and polypeptide natural products can represent a key step in generating the final biologically active molecule. This step is often, but not always, one of the later biosynthetic steps and is catalyzed by an acyltransferase. The corresponding gene is typically associated with the polyketide or polypeptide biosynthetic gene cluster. Despite the importance of this step, a relatively small number of these acyltransferases from actinomycetes have been identified, and very few have been fully characterized (2, 10, 15, 18). Studies of biosynthetic processes where there is an acylation of a polyketide chain, have indicated that the enzymes have various degrees of promiscuity for the carboxylic acid substrates. For instance, genetic evidence has shown that mdmB and acyA encode 3-O-acyltransferases which transfer either acetyl or propionyl groups to position 3 in 16-membered macrolides such as midecamycin, spiramycin, carbomycin, and tylosin (3, 10). The asm19 gene product has been identified as the 3-O-acyltransferase which catalyzes the attachment of the biologically essential acyl group in the macrocyclic ansamitocins (18). The asm19 mutant accumulates an N-demethyl-4,5-desepoxymytansinol, indicating that acylation of the macrocycle precedes N methylation and epoxidation. Escherichia coli cell extracts containing a recombinant Asm19 protein have been shown to catalyze acylation of this mytansinol intermediate using a range of short straight- and branched-chain acyl coenzyme A (CoA) thioesters (C2 to C5). Finally, LovD, which catalyzes the acylation of the C-8 hydroxyl group of monacolin J to yield the natural product lovastatin, a pharmaceutically important fungal polyketide product produced by Aspergillus nidulans, has been characterized (30). This enzyme is able to utilize a wide range of different acyl donors activated as CoA, N-acetylcysteamine (NAC), or methyl thioglycolate esters and thus offers an economically attractive route for generating novel lovasotatin analogs for treatment of hypercholesterolemia. One or multiple O-acyltransferases have been implied to be involved in the post-polyketide synthase tailoring steps leading to a series of natural products known as the phoslactomycins (PLMs) (Fig. ​(Fig.1)1) (6). These compounds (also known as leustroducsins [LSNs], phospholines, and phosphazomycins) have been isolated from various actinomycetes, and their structures are all identical with the exception of the acyl substituent at C-18 (4, 5, 12, 13, 20, 27, 28). Streptomyces sp. strain HK 803 produces at least five such acylated analogs (PLM A and PLMs C to F) (Fig. ​(Fig.1)1) as well as PLM B, in which the C-18 hydroxyl substituent is absent (4, 5, 27). FIG. 1. Proposed biosynthetic relationship between PLM products made by Streptomyces sp. strain HK 803. A cytochrome P450 monooxygenase (PlmS2) catalyzes C-18 hydroxylation of PLM B to generate PLM G, which is subsequently 18-O acylated by PlmS3. These natural products have been isolated based on their potent activity (as low as 0.008 μg/ml) against some phytopathogenic fungi (27, 28). The compounds also have relatively weak antitumor activity (50% inhibitory concentration of 2 to 3 μg/ml against L1210, P38,8, and El-4 cell lines) (19) which may arise from their activity as selective inhibitors of protein phosphatase 2A. (26). These natural products also show induction of a colony-stimulating factor (12) via NF-κB activation and thrombopoiesis (14). This array of promising biological activities has stimulated research into the field of PLMs for treatments of various diseases. Low yields and the presence of multiple acylated products from fermentations have provided a barrier to this work, and circuitous routes to obtaining individual compounds have been described. For instance, there have been no reports of any actinomycetes which produce PLM G (LSN H) (Fig. ​(Fig.1),1), in which the C-18 hydroxyl residue is present but not acylated, and this compound has been reported to be obtained only by cleavage of the acyl groups of a mixture of other PLMs and LSNs using porcine liver esterase (24). A multistep synthetic route to selectively acylate PLM G with 6-methyloctanoic acid (producing LSN B) has also been described (17). Recently the entire 75-kbp Plm biosynthetic gene cluster has been cloned, sequenced, and analyzed (21) and has provided an opportunity to study the enzymatic hydroxylation and acylation processes which give rise to the range of PLM products. Deletion of the plmS2 open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases, has resulted in an NP1 mutant producing only PLM B (Fig. ​(Fig.1)1) (21). The plmS2 ORF has been expressed as an N-terminally polyhistidine-tagged protein in Streptomyces coelicolor, and the purified protein has been shown to catalyze conversion of PLM B to PLM G (7). This work in conjunction with other studies (1, 23) has led to a proposal that the final two biosynthetic steps involve hydroxylation of PLM B (to give PLM G) and subsequent acylation with a broad range of acyl-CoA substrates (to give PLM A and PLMs C to F). The acylation step is required for potent antifungal activity of the PLMs. Initial analysis of the PLM biosynthetic gene cluster (21) did not reveal a candidate gene or genes whose products might be responsible for this acylation. Here we identify the plmS3 gene product as the singular acyltransferase in Streptomyces sp. strain HK 803 responsible for C-18 acylation of PLM G. Generation of a plmS3 deletion mutant results in selective production of PLM G, supporting the proposed role of this gene product and providing the first direct fermentation method to access this intermediate. The 921-bp plmS3 ORF was cloned and expressed as an N-terminally polyhistidine-tagged protein in E. coli, and the recombinant purified protein was shown to catalyze acylation of PLM G with isobutyryl-CoA, 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA to give PLM A, PLM C, and PLM E, respectively. This efficient one-step enzymatic process offers an attractive alternative to the multistep synthetic process for selective acylation of PLM G. PlmS3 was also shown to catalyze esterification of PLM G with CoA and NAC thioesters with a remarkably wide range of various saturated, unsaturated, and aromatic carboxylic acids and thus provides an efficient chemoenzymatic route to new PLM analogs.