Rhamnose Biosynthesis Pathway Supplies Precursors for Primary and Secondary Metabolism in Saccharopolyspora spinosa

Abstract

Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in Saccharopolyspora spinosa are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products.Spinosyns, the active ingredients in Dow AgroSciences' new Naturalyte line of insect control products, are produced by fermentation of the actinomycete Saccharopolyspora spinosa. Spinosyns are macrolides ( Fig. 1) consisting of a 21-carbon tetracyclic lactone to which are attached two deoxysugars: tri-O-methylated rhamnose and forosamine (6). The most active components of the spinosyn family of compounds are spinosyns A and D, which differ from each other by a single methyl substituent at position 6 of the polyketide. Other factors in this family have different levels of methylation and are significantly less active. Both the rhamnose and forosamine moieties are essential for the insecticidal activity of spinosyns (2). Spinosad is highly effective against target insects and has an excellent environmental and mammalian toxicological profile (2,13,14).Spinosyn biosynthesis occurs via the nonglycosylated intermediate, the aglycone (AGL). Rhamnose is the first sugar attached and is tri-O-methylated to yield the intermediate pseudoaglycone.Only after the rhamnose is attached can the forosamine sugar be incorporated (M. C. Broughton, M. L. B. Huber, L. C. Creemer, H. A. Kirst, and J. R. Turner, Abstr. 91st Annu. Meet. Am. Soc. Microbiol. 1991, abstr. K-58, p. 224, 1991. Both trimethyl rhamnose and forosamine are believed to be synthesized from glucose-1-phosphate via the common intermediate 4-keto-6-deoxy-D-glucose (Fig. 2). The biosynthetic pathway for rhamnose (Fig. 2) has been elucidated in enteric bacteria, where the deoxysugar is an element of surface antigens (8, 18). The first step, activation of glucose by addition of a nucleotidyl diphosphate (NDP), is catalyzed by an NDP-glucose synthase (the gtt gene product). The second step, dehydration to NDP-4-keto-6-deoxyglucose, is catalyzed by glucose dehydratase (the gdh gene product). 4-Keto-6-deoxy-D-glucose is the common intermediate to many deoxysugar biosynthetic pathways, and the enzymes encoded by the gtt and gdh genes may supply the precursors for all of them. Rhamnose synthesis requires two additional enzymes, a 3Ј5Ј epimerase (encoded by epi) and a 4Ј ketoreductase (encoded by kre), that are unique to the pathway. They convert the NDP-4-keto-6-deoxyglucose to NDP-L-rhamnose, the activated sugar that is the substrate of th...

Modification of glycopeptidolipids by an O-methyltransferase of Mycobacterium smegmatis a aThe GenBank accession number for the sequence determined in this work is AY138899.

“…Rhamnose biosynthesis has been delineated in M. tuberculosis and M. smegmatis and genes encoding the key enzymes have been characterized in M. tuberculosis (Cole et al, 1998 ;Ma et al, 1997). Unlike the situation in enteric bacteria the mycobacterial genes for rhamnose biosynthesis are dispersed around the chromosome in a similar fashion to that described in Saccharopolyspora spinosa (Madduri et al, 2001). Rhamnose is also required for glycosylation of GPLs in M. smegmatis, and genes for glucose-1-phosphate thymidylyltransferase (rmlA) and dTDP glucose 4,6-dehydrogenase (rmlB) occur in the GPL biosynthetic locus.…”

Chalcomycin Biosynthesis Gene Cluster from Streptomyces bikiniensis : Novel Features of an Unusual Ketolide Produced through Expression of the chm Polyketide Synthase in Streptomyces fradiae

“…The next step after deoxygenation is reduction of the 3-keto group of the pathway intermediate. SpnQ is believed to serve this role in the biosynthesis of the forosamine component of spinosad (23). A homolog of SpnQ was not found within the Ͼ80-kb sequenced segment from the chalcomycin producer.…”

Functional Characterization and Substrate Specificity of Spinosyn Rhamnosyltransferase by in Vitro Reconstitution of Spinosyn Biosynthetic Enzymes

“…The tetracyclic aglycone scaffold is delicately constructed first by type I polyketide (PK-I) 2 synthases, followed by proposed postmodification events, including FAD-oxidation at C-15, Michael addition between C-3 and C-14, ␥-dehydration at C-11, and Diels-Alder cyclization (4,5). The spinosyn biosynthesis genes recently cloned and sequenced are located in an ϳ80-kb cluster of the S. spinosa genome, except that the four genes involved in L-rhamnose (L-Rha) biosynthesis, gtt (NDP-glucose synthase), gdh (NDP-glucose 4,6-dehydratase), epi (NDP-4-keto-6-deoxyglucose epimerase), and kre (NDP-4-ketorhamnose reductase), are located in three different regions of the genome (6). Previous genetic experiments have suggested that the polyketide aglycone is biosynthesized prior to immediate attachment of L-Rha by the rhamnosyltransferase SpnG, followed by consecutive tri-O-methylation of the L-Rha on resulting spinosyn rhamnosyl pseudoaglycone (SRPG) and then by the addition of D-forosamine to give spinosyns (Scheme 1) (4, 7).…”