The allyl moiety of the immunosuppressive agent FK506 is structurally unique amongst polyketides and critical for its potent biological activity. Here, we detail the biosynthetic pathway to allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on a comprehensive chemical, biochemical and genetic interrogation of three FK506 gene clusters. A discrete polyketide synthase (PKS) with noncanonical domain architecture presumably in coordination with the fatty acid synthase pathway of the host catalyzes a multi-step enzymatic reaction to allylmalonyl-CoA via trans-2-pentenyl-acyl carrier protein. Characterization of this discrete pathway facilitated the engineered biosynthesis of novel allyl group-modified FK506 analogs, namely 36-fluoro-FK520 and 36-methyl-FK506, the latter of which exhibits improved * slim@genotech.co.kr .* joonyoon@ewha.ac.kr . 8 These authors contributed equally to this work.

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.

Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and L-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone -rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from -rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-L-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.

The 2-deoxystreptamine-containing aminoglycosides, such as neomycin, kanamycin and gentamicin, are an important class of antibiotics. A detailed understanding of the complete biosynthetic pathway of aminoglycosides and their biosynthetic enzymes will allow us to not only generate more robust antibiotic agents or drugs with other altered biological activities, but also to produce clinically important semi-synthetic antibiotics by direct fermentation. This Highlight focuses on recent advances in the characterization of their biosynthetic enzymes and pathway as well as some chemo-enzymatic and metabolic engineering approaches for the biological production of natural, semi-synthetic, and novel aminoglycosides.

ABSTRACTFK506 is an important 23-member polyketide macrolide with immunosuppressant activity. Its entire biosynthetic gene cluster was previously cloned fromStreptomycessp. strain KCTC 11604BP, and sequence analysis identified three putative regulatory genes,tcs2,tcs7, andfkbN, which encode proteins with high similarity to the AsnC family transcriptional regulators, LysR-type transcriptional regulators, and LAL family transcriptional regulators, respectively. Overexpression and in-frame deletion oftcs2did not affect the production of FK506 or co-occurring FK520 compared to results for the wild-type strain, suggesting thattcs2is not involved in their biosynthesis.fkbNoverexpression improved the levels of FK506 and FK520 production by approximately 2.0-fold, and a deletion offkbNcaused the complete loss of FK506 and FK520 production. Although the overexpression oftcs7decreased the levels of FK506 and FK520 production slightly, a deletion oftcs7caused 1.9-fold and 1.5-fold increases in FK506 and FK520 production, respectively. Finally,fkbNoverexpression in thetcs7deletion strain resulted in a 4.0-fold (21 mg liter−1) increase in FK506 production compared to that by the wild-type strain. This suggests thatfkbNencodes a positive regulatory protein essential for FK506/FK520 biosynthesis and that the gene product oftcs7negatively regulates their biosynthesis, demonstrating the potential of exploiting this information for strain improvement. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of the transcription levels of the FK506 biosynthetic genes in the wild-type and mutant strains proved that most of the FK506 biosynthetic genes are regulated byfkbNin a positive manner and negatively bytcs7.