Leu DLeu

Fig. 35.10. (A) Surfactin A, which is produced by the seven modules. Two new analogs were produced by replacing the d-Leu module with phenylalanine and ornithine incorporation modules. (B) Placing the thioesterase domain immediately downstream of the srfA-A gene (which encodes the first three amino acid incorporation modules) results in the production of the expected truncated peptide.

Combinatorial Biosynthesis of Carbohydrates

Deoxysugars are essential appendages for many secondary metabolites. Their biosynthesis has been reviewed extensively elsewhere [9-11, 82, 83]. In natural products they often act as guidance systems, determining the specificity and biological activity toward a target [84, 85]. The ability to create diverse deoxysugars and incorporate them into larger structures provides an opportunity to devise natural product analogs with altered targets, specificity, activity, and physical properties.

As indicated in Fig. 35.2C, three types of enzymes are necessary for the incorporation of deoxysugar moieties into larger natural products: (1) nucleotidyl-transferases are responsible for adding nucleotide diphosphate appendages to carbohydrates; (2) modifying enzymes, which, for example, alter the oxidation state of the molecule or replace hydroxyls with amines; and (3) glycosyltransferases, which couple the fully derivatized sugar with its intended aglycon partners. To produce novel deoxysugars, one can alter or relax the specificity of enzymes through the introduction of mutations, replace a ''natural'' enzyme in one pathway with those from other pathways, or take advantage of the relaxed specificity some of these enzymes already demonstrated to incorporate non-natural substrates.

In one example, non-natural analogs of desosamine, the sugar moiety found in methymycin and neomethymycin, were produced (Fig. 35.12). The desosamine biosynthetic cluster was altered by deletion of the desV (reductive amination) and desVI (dimethylation) genes [86, 87]. Several new macrolides were produced from the deletion mutants, including some that were quite unexpected. In addition to

Ck Me

DesVIII

OH OH

HO 1

O' H0 OTDP disrupt / DesV

HO 1

O' H0 OTDP disrupt / DesV

Fig. 35.12. Deletion of DesV or DesVI results in the production of two new methamycin analogs.

Fig. 35.12. Deletion of DesV or DesVI results in the production of two new methamycin analogs.

producing potentially useful new macrolides, this work also demonstrates that the glycosyltransferase involved has a relaxed substrate specificity, in that it could incorporate a variety of deoxysugars.

The ability to swap genes between pathways is another important demonstration of the potential for deoxysugar combinatorial biosynthesis. In the methymycin pathway, the desVI gene can be replaced without difficulty by an analogous gene, tylMl, that is involved in the biosynthesis of mycaminose in the tylosin pathway (Fig. 35.13) [88]. Similarly, the tylB gene can replace desV [87]. The ease of such replacements might suggest that very few protein-protein interactions are essential in the deoxysugar biosynthetic pathway, thus reducing the chances that communication problems may be stumbling blocks when mixing and matching deoxysugar biosynthetic proteins in combinatorial biosynthesis.

The appending of alternative sugar moieties to aglycons for combinatorial biosynthesis also rests upon our ability to reengineer or utilize the existing reduced specificity of the nucleotidyltransferases and glycosyltransferases involved. For instance, the wild-type a-D-glucopyranosyl phosphate thymidylyltransferase (Ep) shows very broad acceptance of sugar substrates for coupling to thymidylate (Fig. 35.14) [89, 90]. Structural analysis suggested that introducing specific point mutations should further relax the specificity and better allow incorporation of deoxy-sugars modified at C2, C3 or C6 [91]. The pooled Ep mutants were capable of incorporating non-natural deoxysugars, including some that are unable to be in-

35.2 Combinatorial Biosynthesis: Creation of Novel Small-molecule Natural Products | 1079 oh oh ho ho sfc^ -nafcA an

Fig. 35.13. DesVand DesVI can be replaced by analogous or similar genes from the tylosin pathway.

Desl rDesll

o otdp otdp o otdp

TylM1 TylB

corporated by the wild-type Ep. These results demonstrate the feasibility of a rational redesign approach.

Another strategy for combinatorial biosynthesis using deoxysugars takes advantage of the broad specificity of some glycosyltransferases [92]. In one striking example, a cosmid (essentially, a very large piece of circular DNA) that contained 25 kb of the elloramycin biosynthetic pathway from Streptomyces olivaceus was transformed into mutants of Streptomyces fradiae and Streptomyces argillaceus (Fig. 35.15) [93]. The wild-type versions of these strains normally produce urdamycin A or mi-thramycin, respectively, but the PKS genes for these natural products were deleted in the mutant strains. The glycosyltransferase gene from the elloramycin pathway was capable of accepting the alternate deoxysugars produced by the mutants, resulting in the production of new glycosylated tetracenomycins. Further investigation revealed that this glycosyltransferase was capable of glycosylating tetraceno-mycins with several non-native deoxysugars, including a disaccharide. This glyco-syltransferase element has been recently identified [94]. A related approach has also been used with the erythromycin-producing strain Saccharopolyspora erythraea [95]. The inverse approach - instead of employing different deoxysugars, using several different macrolides as substrates for a single glycosyltransferase - has also been successful with the glycosyltransferase from the picromycin biosynthetic pathway in Streptomyces lividans [96].

The ability to reengineer enzymes or to introduce wild-type enzymes into new deoxysugar biosynthetic pathways holds great promise for the combinatorial biosynthesis of a highly diverse array of deoxysugar-containing macrolides. While a substantial amount of work has already been devoted to understanding the genetics and enzymology of deoxysugar biosynthesis, further insight can only assist efforts to produce combinatorial deoxysugar-containing biosynthetic libraries. Recent identification of the elements in a glycosyltransferase responsible for substrate specificity and catalytic activity is an important advance along these lines [97].

OH OH

HO HO

OH HO'VvO

0P032"

HO HO

HO I

OPOa^

HO HO

OH OH

oXJvi,

0P032

NH2 HO 1

opo3^

0P03Z

OPO32" OPO32"

0P032" 0P032" 0P032" 0P032"

HO HO

C02" NH

OPO32" OPO32

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