Secondary metabolite scale-up to minimize homolog impurity levels

A mutant strain of Streptomyces hygroscopicus was found to produce up to 9.0 units/L of an immunoregulant precursor, immunomycin, with up to 3.5% of a lower homolog impurity under either dual fed-batch or batch conditions. Glycerol and valine were key nutrients influencing productivity and impurity levels. Soybean oil was successfully substituted for glycerol as a carbon source to minimize shot additions to batch culture. The remainder of the production medium was composed largely of defined components with the exception of yeast extract. Valine limitation increased lower homolog formation while decreasing higher homolog formation; excess valine decreased lower homolog formation below 2-3% while increasing higher homolog formation. Higher homolog formation in the presence of valine seemed to be slower than lower homolog formation in the absence of valine. Valine was believed to be the major butyrate precursor; consequently its availability influenced the impurity profile. A preliminary cost analysis suggests that elimination of added valine from the cultivation and replacement of glycerol with soybean oil can result in a 6.6-fold reduction in media costs relative to the original fed-batch process. Copyright 1998 John Wiley & Sons, Inc.

Pneumocandins from Zalerion arboricola. I. Discovery and isolation.

HPLC bioautography of the directed biosynthesis of Zalerion arboricola led to the discovery of pneumocandin B0 (L-688,786), a new antifungal and anti-Pneumocystis carinii lipopeptide. Isolation techniques were developed to separate this component from pneumocandin A0 (L-671,329) in fermentations of a mutant of Zalerion arboricola. A number of related compounds were also isolated, which differ from pneumocandins A0 and B0 in the hydroxylation patterns on the ornithine, homotyrosine, and proline.

Use of soybean oil and ammonium sulfate additions to optimize secondary metabolite production.

A valine-overproducing mutant (MA7040, Streptomyces hygroscopicus) was found to produce 1.5 to 2.0 g/L of the immunoregulant, L-683,590, at the 0.6 m3 fermentation scale in a simple batch process using soybean oil and ammonium sulfate-based GYG5 medium. Levels of both lower (L-683,795) and higher (HH1 and HH2) undesirable homolog levels were controlled adequately. This batch process was utilized to produce broth economically at the 19 m3 fermentation scale. Material of acceptable purity was obtained without the multiple pure crystallizations previously required for an earlier culture, MA6678, requiring valine supplementation for impurity control. Investigations at the 0.6 m3 fermentation scale were conducted, varying agitation, pH, initial soybean oil/ammonium sulfate charges, and initial aeration rate to further improve growth and productivity. Mid-cycle ammonia levels and lipase activity appeared to have an important role. Using mid-cycle soybean oil additions, a titer of 2.3 g/L of L-683,590 was obtained, while titers reached 2.7 g/L using mid-cycle soybean oil and ammonium sulfate additions. Both higher and lower homolog levels remained acceptable during this fed-batch process. Optimal timing of mid-cycle oil and ammonium sulfate additions was considered a critical factor to further titer improvements.

Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN.

Indene is oxidized to mixtures of cis- and trans-indandiols and related metabolites by Pseudomonas putida and Rhodococcus sp. isolates. Indene metabolism is consistent with monooxygenase and dioxygenase activity. P. putida resolves enantiomeric mixtures of cis-1,2-indandiol by further selective oxidation of the 1R, 2S-enantiomer yielding high enantiomeric purity of cis-(1S, 2R)-indandiol, a potential intermediate in the synthesis of indinavir sulfate (CRIXIVAN), a protease inhibitor used in the treatment of AIDS. Molecular cloning of P. putida toluene dioxygenase in Escherichia coli confirmed the requirement for the dihydrodiol dehydrogenase in resolving racemic mixtures of cis-indandiol. Rhodococcus sp. isolates convert indene to cis-(1S, 2R)-indandiol at high initial enantiomeric excess and one isolate also produces trans-(1R, 2R)-indandiol, suggesting the presence of monooxygenase activity. Scale up and optimization of the bioconversions to these key synthons for chiral synthesis of potential intermediates for commercial manufacture of indinavir sulfate are described.