Synthesis of 3beta, 7alpha, 11alpha-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives and their cytotoxic activities.

A series of novel 3beta, 7alpha, 11alpha-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives were synthesized and characterized by NMR, HRMS. The pregnenolone (1) was first biotransformed by Mucor circinelloides var lusitanicus to 3beta, 7alpha, 11alpha-trihydroxy-pregn-5-en-20-one (3), then 3 was treated with various benzaldehydes to produce 3beta, 7alpha, 11alpha-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives. These derivatives showed remarkable activity against EC109 cells. The absolute configuration of 3 was also confirmed by signal-crystal X-ray analysis.

Recent advances in the biochemistry of spinosyns.

Spinosyn and its analogs, produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. They are macrolides with a 21-carbon, 12-membered tetracyclic lactones that are attached to two deoxysugars, tri-O-methylrhamnose and forosamine. Labeling studies, analysis of the biosynthetically blocked mutants, and the genetic identification of the spinosyn gene cluster have provided detailed information concerning the mechanism of spinosyn biosynthesis and have enabled combinatorial biosynthesis of a large group of new spinosyns. The following developments have recently impacted the field of spinosyn biology: (1) A second-generation spinosyn called spinetoram (XDE-175) was launched in late 2007; it is a semisynthesized spinosyn derivative produced through the modification of 3'-O-methyl group of rhamnose and the double bond between C5 and C6 of spinosyn J and L. This molecule was shown to have improved insecticidal activity, enhanced duration of control, and an expanded pest spectrum. (2) A new class of spinosyns, the butenyl-spinosyns, was discovered from Saccharopolyspora pogona. The butenyl-spinosyns are similar to spinosyns, but differ in the length of the side chain at C-21. In addition to structural similarities with the spinosyns, the butenyl-spinosyns exhibit a high level of similarity in insecticidal activity to spinetoram. (3) Spinosyn analogs, 21-cyclobutyl-spinosyn A and 21-cyclobutyl-spinosyn D were generated by metabolic engineering of the spinosyn biosynthetic gene cluster. They showed better insecticidal activities against cotton aphid and tobacco budworm than that of spinosyn A and D. Future progress toward the development of more potent spinosad analogs, as well as enhancements in production yields will likely result from these recent advances in the genetics and biochemistry of spinosyns.

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

Deoxysugar, 2', 3', 4'-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4'-O-methylation of 2', 3'-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4'-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4'-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4'-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.

Large scale production of Bacillus thuringiensis PS149B1 insecticidal proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens.

The 14kDa (Cry34Ab1) and 44kDa (Cry35Ab1) binary insecticidal proteins are produced naturally by Bacillus thuringiensis PS149B1 as parasporal inclusion bodies. Here, we show production of these two insecticidal proteins in recombinant Pseudomonas fluorescens and their subsequent purification to near homogeneity to provide large quantities of protein for safety-assessment studies associated with the registration of transgenic corn plants. The gene sequence specific for each protein was expressed in P. fluorescens and fermented at the 75-L scale. For Cry34Ab1, the protein accumulated as insoluble inclusion bodies, and was purified by extraction directly from the cell pastes at pH 3.4 with a sodium acetate buffer, selective precipitation at pH 7.0, and differential centrifugation. For Cry35Ab1, the protein was extracted from the purified inclusion bodies with sodium acetate buffer (pH 3.5) containing 0.5M urea, followed by diafiltration. No chromatography steps were required to produce over 30g of lyophilized protein powder with purity greater than 98%, while retaining full insecticidal activity against Western corn rootworm larvae. The proteins were further characterized to assure identity and suitability for use in safety-assessment studies.

Heterologous expression of the Saccharomyces cerevisiae alcohol acetyltransferase genes in Clostridium acetobutylicum and Escherichia coli for the production of isoamyl acetate.

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1 and ATF2 in bacteria for ester production.

Sequences affecting the regulation of solvent production in Clostridium acetobutylicum.

The high solvent phenotype of Clostridium acetobutylicum mutants B and H was complemented by the introduction of a plasmid that contains either an intact or partially-deleted copy of solR, restoring acetone and butanol production to wild-type levels. This demonstrates that the solR open reading frame on pSOLThi is not required to restore solvent levels. The promoter region upstream of alcohol dehydrogense E (adhE) was examined in efforts to identify sites that play major roles in the control of expression. A series of adhE promoter fragments was constructed and the expression of each in acid- and solvent-phases of growth was analyzed using a chloramphenicol acetyl-transferase reporter system. Our results show that a region beyond the 0A box is needed for full induction of the promoter. Additionally, we show that the presence of sequences around a possible processing site designated S2 may have a negative role in the regulation of adhE expression.

Isolation and characterization of 14 additional genes specifying the anaerobic biosynthesis of cobalamin (vitamin B12) in Propionibacterium freudenreichii (P. shermanii).

A search for genes encoding enzymes involved in cobalamin (vitamin B12) production in the commercially important organism Propionibacterium freudenreichii (P. shermanii) has resulted in the isolation of an additional 14 genes encoding enzymes responsible for 17 steps of the anaerobic B12 pathway in this organism. All of the genes believed to be necessary for the biosynthesis of adenosylcobinamide from uroporphyrinogen III have now been isolated except two (cbiA and an as yet unidentified gene encoding cobalt reductase). Most of the genes are contained in two divergent operons, one of which, in turn, is closely linked to the operon encoding the B12-dependent enzyme methylmalonyl-CoA mutase. The close linkage of the three genes encoding the subunits of transcarboxylase to the hemYHBXRL gene cluster is reported. The functions of the P. freudenreichii B12 pathway genes are discussed, and a mechanism for the regulation of cobalamin and propionic acid production by oxygen in this organism is proposed.