Neal Connors

Isolation and Structure Elucidation of Thiazomycin —A Potent Thiazolyl Peptide Antibiotic from Amycolatopsis fastidiosa

Thiazolyl peptides are a class of rigid macrocyclic compounds richly populated with thiazole rings. They are highly potent antibiotics but none have been advanced to clinic due to poor aqueous solubility. Recent progress in this field prompted a reinvestigation leading to the isolation of a new thiazolyl peptide, thiazomycin, a congener of nocathiacins. Thiazomycin possesses an oxazolidine ring as part of the amino-sugar moiety in contrast to the dimethyl amino group present in nocathiacin I. The presence of the oxazolidine ring provides additional opportunities for chemical modifications that are not possible with other nocathiacins. Thiazomycin is extremely potent against Gram-positive bacteria both in vitro and in vivo. The titer of thiazomycin in the fermentation broth was very low compared to the nocathiacins I and III. The lower titer together with its sandwiched order of elution presented significant challenges in large scale purification of thiazomycin. This problem was resolved by the development of an innovative preferential protonation based one- and/or two-step chromatographic method, which was used for pilot plant scale purifications of thiazomycin. The isolation and structure elucidation of thiazomycin is herein described.

Effects of amino acid and trace element supplementation on pneumocandin production by Glarea lozoyensis: impact on titer, analogue levels, and the identification of new analogues of pneumocandin B0

Addition of the amino acids threonine, serine, proline, and arginine to fermentations of the fungus Glarea lozoyensis influenced both the pneumocandin titer and the spectrum of analogues produced. Addition of threonine or serine altered the levels of the “serine analogues” of pneumocandins B0 and B5 and allowed for their isolation and identification. Proline supplementation resulted in a dose-dependent increase in the levels of pneumocandins B0 and E0, whereas pneumocandins C0 and D0 decreased as a function of proline level. Moreover, proline supplementation resulted in an overall increase in the synthesis of both trans-3- and trans-4-hydroxyproline while maintaining a low trans-4-hydroxyproline to trans-3-hydroxyproline ratio compared to the unsupplemented culture. Pneumocandin production and the synthesis of hydroxyprolines was also affected by addition of the proline-related amino acid arginine but not by the addition of glutamine or ornithine. Zinc, cobalt, copper, and nickel, trace elements that are known to inhibit α-ketoglutarate-dependent dioxygenases, affected the pneumocandin B0 titer and altered the levels of pneumocandins B1, B2, B5, B6, and E0, analogues that possess altered proline, ornithine, and tyrosine hydroxylation patterns. Journal of Industrial Microbiology & Biotechnology (2001) 26, 216–221.

Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1

Two mutation and selection methods were used to isolate mutants of Pseudomonas putida F1 which convert indene to cis-(1S),(2R)-indandiol in a toluene-independent fashion. Using soybean or silicone oil as a second phase to deliver indene to the culture, cis-(1S),(2R)-indandiol, cis-(1R),(2S)-indandiol, 1,2-indenediol (or the keto-hydroxy indan tautomer), and the monooxygenation products 1-indenol and 1-indanone were produced from indene as a function of time. Similarly the enantiomeric excess of the cis-(1S),(2R)-indandiol produced also increased with increasing time. In addition, mutants were isolated which produced cis-(1S),(2R)-indandiol of lower optical purity which corresponded to reduced levels of 1,2-indenediol. These data suggest this toluene dioxygenase produces cis-(1S),(2R)-indandiol of low optical purity and that cis-glycol dehydrogenase plays a role in resolving the two cis-1,2-indandiol enantiomers.

Novel proline hydroxylase activities in the pneumocandin-producing fungus Glarea lozoyensis responsible for the formation of trans 3- and trans 4-hydroxyproline

Novel proline 3-hydroxylase (P3H) and proline 4-hydroxylase (P4H) activities that convert free l-proline to both trans 3- and trans 4-hydroxy-l-proline were detected in protein extracts of the anamorphic fungus Glarea lozoyensis. The enzymatic conversion of l-proline to trans 3- and trans 4-hydroxy-l-proline was strictly dependent on α-ketoglutarate, ascorbate, and dithiothreitol. Ferrous iron was required for optimal P3H and P4H activity. These substrate and co-factor requirements indicate these enzyme activities belong to the class of 2-oxoglutarate-dependent dioxygenases. Both P3H and P4H were inhibited by zinc and other trace metals. The addition of proline to the fermentation medium resulted in an increase in the specific activity of P4H and a decrease in the synthesis of pneumocandin C0. Additionally, the synthesis of trans 3- and trans 4-hydroxy-l-proline in vivo was affected differently by the proline concentration in the medium. This result suggested that two enzymes may be responsible for the regio- and stereospecific hydroxylation of l-proline.

Bioconversion of indene to trans-2S,1S-bromoindanol and 1S,2R-indene oxide by a bromoperoxidase/dehydrogenase preparation from Curvularia protuberata MF5400

Abstract 1S,2R-Indene oxide is the precursor of cis -1S,2R-aminoindanol, a key intermediate for the Merck HIV–1 protease inhibitor, Crixivan®. As an alternative to the challenging chemical synthesis of this chiral epoxide from indene, the biotransformation route using an enzyme catalyst was examined. Approximately 3% of the 400 fungal cultures isolated from high salt environments were found to possess neutral haloperoxidase activities. Subsequent studies revealed that indene conversion by these positive cultures could only be obtained when both hydrogen peroxide and bromide ions were present. The products were generally racemic trans -bromoindanols which upon basification yielded racemic epoxides. Finally, it was found that a crude enzyme preparation from the fungal culture Curvularia protuberata MF5400 converted indene to the chiral 2S,1S-bromoindanol which can be chemically converted to the desired 1S,2R-epoxide through basification or used directly in the asymmetric synthesis of cis -1S,2R-aminoindanol. The bioconversion rate and the enantiomeric excess (ee) achieved with this cell-free system were heavily pH dependent. An initial 1.5-h reaction at pH 7.0 gave ∼10% yield of the chiral bromoindanol or epoxide from indene, and the yield was rapidly improved to >30% for trans -2S,1S-bromoindanol with an ee of 80%. Reaction mechanistic studies revealed that the stereoselectivity observed was apparently due to a specific dehydrogenase activity present in MF5400 which was also found to resolve chemically synthesized racemic trans -2-bromoindanols.

Actinomycetes scale-up for the production of the antibacterial, nocathiacin.

An Amycolatopsis fastidiosa culture, which produces the nocathiacin class of antibacterial compounds, was scaled up to the 15,000 L working volume. Lower volume pilot fermentations (600, 900, and 1,500 L scale) were conducted to determine process feasibility at the 15,000 L scale. The effects of inoculum volume, impeller tip speed, volumetric gas flow rate, superficial gas velocity, backpressure, and sterilization heat stress were examined to determine optimal scale‐up operating conditions. Inoculum volume (6 vs. 2 vol %) and medium sterilization (Ro of 68 vs. 92 min−1) had no effect on productivity or titer, and higher impeller tip speeds (2.1 vs. 2.9 m/s) had a slight effect (20% decrease). In contrast, higher backpressure, incorporating increased head pressure at the 15,000 L scale (1.2 vs. 0.7 kg/cm2) and low gas flow rates (0.25 vs. 0.8 vvm), appeared to be problematic (40–50% decrease). High off‐gas CO2 levels were likely reasons for observed lower productivity. Consequently, air flow rate for this 25‐fold scale‐up (600–15,000 L) was controlled to match off‐gas CO2 profiles of acceptable smaller scale batches to maintain levels below 0.5%. The 15,000 L‐scale fermentation achieved an expected nocathiacin I titer of 310 mg/L after 7 days. Other on‐line data (i.e., pH, oxygen uptake rate, and CO2 evolution rate) and off‐line data (i.e., analog production, glucose utilization, ammonium production, and dry cell weight) at the 15,000 L scale also tracked similarly to the smaller scale, demonstrating successful fermentation scale‐up.

Effects of medium sterilization on the production of zaragozic acids by the fungusLeptodontidium elatius

The production of zaragozic acids by fermentation of the fungusLeptodontidium elatius was examined at the 800-L fermentor scale under two different production medium batch sterilization conditions. Low production-medium heat input (R0=33.4 min) resulted in a 4′-desacetoxy zaragozic acid C:4′-O-desacetyl zaragozic acid C:zaragozic acid C ratio of 0.53:0.60:1.0. At a higher heat input (R0=50.5 min), the ratio shifted to 1.0:0.66:1.0 with a corresponding 26% increase in total zaragozic acid production. This higher total zaragozic acid titer resulted from an increase in the amount of 4′-desacetoxy zaragozic acid C produced while the levels of the other two analogues remained unchanged. Batch sterilization conditions also resulted in differences in growth, carbon substrate consumption, and oxygen uptake rates. The structures of the zaragozic acids produced suggest a precursor/end product relationship. A biosynthetic model describing the synthesis of the three zaragozic acids listed above is postulated and used to explain the effects of production-medium heat input during sterilization.

Crotonic acid-directed biosynthesis of the immunosuppressants produced by Streptomyces hygroscopicus var. ascomyceticus

Abstract The addition of 5.6 mM crotonic acid to Streptomyces hygroscopicus var. ascomyceticus fermentations producing immunomycin reduced the level (as a percentage of immunomycin) of the major analog impurity L-683,795 from 6.7% for the control to 2.5%. Crotonic acid supplementation did not increase the titer of immunomycin and appeared to suppress production at higher concentrations. The addition of butyrate or valine, which can be catabolized to butyrate, at the concentrations used for crotonic acid were not as effective in reducing the percentage of L-683,795. Importantly, 5.6 mM crotonic acid supplementation reduced the L-683,795 level from 7.0% to 4.5% at the 23-l fermentor scale indicating that the effect of crotonic acid supplementation is scaleable to at least the laboratory bioreactor scale. Moreover, crotonyl-coenzyme A reductase (crotonyl-CoA reductase) activity could be detected in cell extracts. This work suggests that crotonic acid can be converted to butyrate through the activity of crotonyl-CoA reductase and can serve as a source of exogenously added C-4 precursor for macrolide biosynthesis.