Microbial fermentation-derived inhibitors of efflux-pump-mediated drug resistance.

A library of 85000 microbial fermentation extracts was screened for inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa and Candida albicans. New compounds EA-371alpha and EA-371delta were isolated and demonstrated to be potent and specific inhibitors of the MexAB-OprM pump in P. aeruginosa. Two series of fungal metabolites, enniatins and beauvericins, were found to be ubiquitous and potent inhibitors of ABC transporters. Milbemycins were rediscovered as potent inhibitors of the CDRI pump in C. albicans, and demonstrated to potentiate effectively the antifungal activity of fluconazole and SCH-56592 against a wide variety of Candida clinical isolates.

The production of cephalosporin C by Acremonium chrysogenum is improved by the intracellular expression of a bacterial hemoglobin.

A DNA vector for expressing an oxygen-binding heme protein (Vitreoscilla hemoglobin, or VHb) in filamentous fungi was constructed and introduced into a cephalosporin C-producing strain of Acremonium chrysogenum. Expression of VHb in transformants was demonstrated by Western immunoblot analysis and by increased carbon monoxide binding activity of cell extracts. Several VHb-expressing transformants produced significantly higher yields of cephalosporin C than control strains in batch culture experiments. Using the same vector system, VHb was also expressed in the related fungus Penicillium chrysogenum.

Actinorhodin production by Streptomyces coelicolor and growth of Streptomyces lividans are improved by the expression of a bacterial hemoglobin.

Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which can be limiting in large-scale fermentations. In an attempt to improve oxygen utilization by the cells, we expressed a heterologous bacterial hemoglobin gene in Streptomyces coelicolor and Streptomyces lividans. Hemoglobin expression was demonstrated by immunoblot analysis and carbon monoxide binding activity. In batch fermentations run under reduced aeration, the expression of hemoglobin in S. coelicolor resulted in a ten-fold increase in specific yields of the aromatic polyketide, actinorhodin. Actinorhodin yields were also much less sensitive to aeration conditions in the hemoglobin-expressing strain. In addition, hemoglobin-expressing S. lividans cells grown under reduced aeration had higher final cell densities and exhibited greater oxygen consumption rates than non-expressing cells.

Growing Saccharomyces cerevisiae in calcium-alginate beads induces cell alterations which accelerate glucose conversion to ethanol.

Nongrowing Saccharomyces cerevisiae cells previously grown in alginate exhibit ethanol production rates 1.5 times greater than cells previously grown in suspension. Analysis of glucose, ethanol, and glycerol formation data using quasi-steady-state pathway stoichiometry shows that alginate-grown cells possess phosphofructokinase (PFK), ATPase, and polysaccharide synthesis maximum activities which are approximately two-, two-, and ninefold larger, respectively, than in suspension-grown cells. The estimated change in PFK maximum velocity is consistent with in vitro assays of PFK activity in extracts of suspension- and alginate-grown yeast. Estimation of ethanol production flux control coefficients using in vivo nuclear magnetic resonance (NMR) spectroscopy measurements of intracellular metabolite concentrations and a previously proposed detailed kinetic model of ethanol fermentation in yeast shows that glucose uptake dominates flux control in alginate-grown cells in suspension while earlier research revealed that PFK and ATPase exert significant flux control in suspension-grown cells. When placed in a calcium alginate matrix, alginate-grown cells produced ethanol 1.8 times more rapidly and accumulated substantially more polyphosphate than suspension-grown cells placed in alginate. Cells growing in alginate elicit responses at the genetic level which substantially alter pathway rates and flux control when these cells are used as either a suspended or an immobilized biocatalyst. These responses in gene expression to growth in alginate serve to reconfigure flux controls in alginate to a pattern which is similar to that obtained for suspended-grown cells in suspension.

In vivo nuclear magnetic resonance analysis of immobilization effects on glucose metabolism of yeast Saccharomyces cerevisiae.

Fermentation rates and intracellular compositions have been determined for alginate-entrapped Saccharomyces cerevisiae and for identical cells in suspension. Glucose uptake and ethanol and glycerol production are approximately two times faster in immobilized cells than in suspended cells. Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy of fermenting immobilized and suspended cells shows differences in intermediate metabolite levels such as fructose-1,6 diphosphate, glucose-6-phosphate, and 3-phosphoglycerate and in internal pH. Carbon-13 NMR shows an increase in polysaccharide production. These data suggest that immobilization has accelerated the rate of glucose transport or of glucose phosphorylation. These effects of immobilization upon cell metabolism are observed in a very short period of time under conditions in which negligible DNA, RNA, or protein synthesis takes place.