Butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes

Abstract

Spinosyns, a novel class of insect active macrolides produced by Saccharopolyspora spinosa, are used for insect control in a number of commercial crops. Recently, a new class of spinosyns was discovered from S. pogona NRRL 30141. The butenyl-spinosyns, also called pogonins, are very similar to spinosyns, differing in the length of the side chain at C-21 and in the variety of novel minor factors. The butenyl-spinosyn biosynthetic genes (bus) were cloned on four cosmids covering a contiguous 110-kb region of the NRRL 30141 chromosome. Their function in butenyl-spinosyn biosynthesis was confirmed by a loss-of-function deletion, and subsequent complementation by cloned genes. The coding sequences of the butenyl-spinosyn biosynthetic genes and the spinosyn biosynthetic genes from S. spinosa were highly conserved. In particular, the PKS-coding genes from S. spinosa and S. pogona have 91-94% nucleic acid identity, with one notable exception. The butenyl-spinosyn gene sequence codes for one additional PKS module, which is responsible for the additional two carbons in the C-21 tail. The DNA sequence of spinosyn genes in this region suggested that the S. spinosa spnA gene could have been the result of an in-frame deletion of the S. pogona busA gene. Therefore, the butenyl-spinosyn genes represent the putative parental gene structure that was naturally engineered by deletion to create the spinosyn genes.

The spinosyn family of insecticides: realizing the potential of natural products research

“…18,[36][37][38] More recently, another species of Saccharopolyspora, named S. pogona, was discovered that produces an even larger series of more than 30 structurally related compounds. 39,40 One distinctive feature of many compounds in this newer series is a C-21-(2-butenyl) substituent in place of the previous 21-ethyl group, leading to the name of butenyl-spinosyns (or pogonins). 2-Butenyl analogs of spinosyns A (3) and D (4) are depicted in Figure 3.…”

“…Some of the other components of this new series differ from the original spinosyn series with changes such as an 8b-hydroxyl group, a 17-O-sugar different from forosamine, a C-21 unsaturated substituent different from 2-butenyl or an expanded macrocyclic ring. 39,40 The large number and wide diversity of known biosynthesized structures suggest that an even broader range of biosynthetic variations might be possible and that additional structural variations of the spinosyns may still yet be found. In addition, very little effort on bioconversions of the spinosyns has yet been reported.…”

“…39 The C-21 substituent has also been altered by genetic engineering of the PKS starter module to accept a variety of non-natural substrates and then incorporate them into new unnatural C-21-modified spinosyn analogs. 50 A different genetically engineered mutant strain produced a new 17-a-O-L-olivosyl analog instead of the natural b-O-D-forosaminyl parent.…”

Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration?

“…Erythromycin, a historically important antibiotic which is also the starting material for the semi-synthetic antibiotics azithromycin, clarithromycin, dirithromycin, roxithromycin, and telithromycin, is produced by Saccharopolyspora erythraea, but not by Streptomyces species. Similarly, spinosyns, important insecticidal macrolides used in agriculture and animal health [2], are produced by Saccharopolyspora spinosa [27] and Saccharopolyspora pogona [11], but are not known to be produced by Streptomyces species or other actinomycete genera. An antibiotic that selects against Streptomyces species and for Saccharopolyspora species might lead to the discovery of novel Saccharopolyspora strains that produce additional important compounds not produced by the abundant streptomycetes.…”

SpnH from Saccharopolyspora spinosa encodes a rhamnosyl 4′-O-methyltransferase for biosynthesis of the insecticidal macrolide, spinosyn A

“…Permethylated Lrhamnose is unusual in its highly unreactive chemical nature, and its infrequent occurrence in nature. This completely O-methylated sugar has been reported only two other times in nature: (a) in the antitumor polyketide, elloramycin, which is produced by S. olivacus [7] and (b) in the structurally similar macrolide, butenyl-spinosyn, which is produced by Saccharopolyspora pogona [25]. The initial functional assignment of spnI, spnK and spnH to the Omethyltransferase family came from sequence homology comparisons to other established methyltransferases from antibiotic-producing microorganisms [5,15].…”