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M Chartrain

Publications and source records attributed to M Chartrain.

At least 19 recordsLinked to original sources
PubMed2003

Measurement of strain-dependent toxicity in the indene bioconversion using multiparameter flow cytometry.

The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains. This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection. The toxic concentrations of the substrate, product, and by-products for each strain have been determined. The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested. The Gram-negative P. putida (421-5 and GM 730) and E. coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms. P. putida GM 730 and the E. coli TDO 123 strains responded similarly to toxic effects, and the E. coli TDO 123 strain was more resistant than the P. putida 421-5 strain. In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches.

Bioreactors↗
PubMed2002

Application of multi-parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene bioconversion.

The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.

Adenosine Triphosphate↗
PubMed2001

Evaluation of an electrochemical bioreactor system in the biotransformation of 6-bromo-2-tetralone to 6-bromo-2-tetralol.

Biotransformation of 6-bromo-2-tetralone (Br-beta-tetralone) to 6-bromo-2-tetralol (Br-beta-tetralol) by yeast cells of Trichosporon capitatum (ATCC 74312) and its partially purified Br-beta-tetralone reductase was evaluated in an electrochemical bioreactor. The biotransformation rates and final product formation were significantly affected by substrate concentration, biomass and electric potential. At 2 g/l of substrate, the initial reaction rate and final product were increased by 35% and 15%, respectively, with -1.5 V of electric potential compared to without electric potential. Additional substrate (2 g/l) provided by pulse feeding to the reaction mixture at different intervals resulted in 2.1 g/l Br-beta-tetralol compared to a total of 1.2 g/l without feeding. However, the increased production was not proportionate to the amount of additionally fed substrate. Increased substrate availability by the addition of 5% (v/v) ethanol resulted in the highest reaction rate and product formation, but addition of ethanol at a concentration higher than 5% decreased the reaction rate. At low biomass, the initial reaction rates were enhanced significantly when electric potential was high, but a higher biomass was necessary to obtain a similar reaction rate when electric potential was reduced. The highest initial reaction rate (59.2 mg/l per min) was achieved with a two-fold biomass concentration of 15.6 g of dry cell weight/l, substrate at 4 g/l and electric potential at -6 V. The conversion of Br-beta-tetralone to Br-beta-tetralol with partially purified Br-beta-tetralone reductase was slow in the presence of electric potential.

Biomass↗
PubMed2000

Biocatalysis for pharmaceuticals--status and prospects for a key technology.

In reviewing how biocatalysis can be applied to improve chiral synthesis for pharmaceuticals it becomes clear that there will be many opportunities using a simple enzyme system but that many of the more useful applications will require the whole cell because of the requirement for cofactors. An assessment is made of the opportunities to apply metabolic engineering to construct de novo metabolic pathways for the biosynthesis of useful advanced intermediates and a conceptual example is provided for the biosynthesis of cis-aminoindanol. We predict that in the future novel pathways will be assembled for a one-step biosynthesis of many semisynthetic natural products.

Animals↗
PubMed2000

Metabolic engineering and directed evolution for the production of pharmaceuticals.

The tools of metabolic and enzyme engineering have been well developed in academic laboratories and are now being applied for the optimization of biocatalysts used in the production of a wide range of pharmaceutically important molecules. Engineered microorganisms with a diverse set of modified or non-native enzyme activities are being used both to generate novel products and to provide improved processes for the manufacture of established products, such as in the production of precursors, intermediates, and complete compounds of importance to the pharmaceutical industry, including polyketides, nonribosomal peptides, steroids, vitamins, and unnatural amino acids. The use of directed evolution has rapidly emerged to be the method of choice for the development and selection of mutated enzymes with improved properties. A variety of such methods have been used to alter the activity, stability and availability of an array of enzymes. The industrial practice of these technologies at large scale is, however, in its infancy and stands as an exciting challenge for process scientists today.

Amino Acids↗
PubMed2000

Implementation of a rapid microbial screening procedure for biotransformation activities.

A rapid and efficient microbial screening procedure was developed utilizing a 24-well plate format in conjunction with an automated liquid handling system and an HPLC. For the evaluation of this miniaturized and automated screening system, we selected the bioreduction of 6-bromo-beta-tetralone to 6-bromo-beta-tetralol. This procedure employed both yeast and rhodococci libraries, representing a culture collection comprised of several hundred strains, from which to screen for desirable bioconversion activity. Most of these strains had demonstrated bioreducing activity during previous screens to insure a "hit rate" as high as possible. The cultivation of microbes in the plate format was facile, time saving, and efficient compared to the standard method of screening utilizing larger volumes, such as test tubes or shake flasks. This improved method of screening for bioconversion activity, employing pre-selected microbial libraries based on microtiter plates and a fully roboticized analytical system, proved to rapidly yield valuable leads which compared advantageously with a more classical approach. A total of 192 yeast strains and 48 rhodococci strains were screened using this procedure. Analytical data revealed that 78% of the strains tested bioconverted the tetralone to the desired alcohol.

Journal Article↗
PubMed2000

Production of cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene (toluene cis glycol) by Rhodococcus sp. MA 7249.

An attractive method for producing cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene (toluene cis glycol) was developed employing a cis dihydrodiol dehydrogenase "deficient" strain of Rhodococcus (MA 7249). The toluene cis glycol produced was found to have optical rotations of [alpha]D25 = +25.8 (c 0.45, CH3OH) and +72.8 (c 0.42, CHCl3) which indicated an absolute configuration of (1S,2R) when compared with previously published values. When cultivated in laboratory fermentor in the presence of toluene vapors, MA 7249 reached a toluene cis glycol concentration up to 18 g/l in 110 h. Culture MA 7249 also accumulated cis (1S,2R) dihydrodiols from dihydronaphthalene, biphenyl, chlorobenzene, and styrene.

Journal Article↗
PubMed1999

Asymmetric direduction of 1,2-indanedione to cis (1S,2R) indanediol by Trichosporon cutaneum MY 1506.

Cis (1S,2R) indanediol is a potential precursor to (-)-cis (1S,2R)-1-aminoindan-2-ol, a key chiral synthon for a leading HIV protease inhibitor, Crixivan (Indinavir). A potential route to the biosynthesis of this important precursor, the microbial asymmetric direduction of 1,2-indanedione to its corresponding diol, cis (1S,2R) indanediol, was investigated. The screening of 32 yeast strains yielded Trichosporon cutaneum MY 1506 as a suitable biocatalyst. At the 2-l shake-flask scale, 1,2-indanedione (charged at 1.0 g/l) was bioconverted to cis (1S,2R) indanediol at a final bioconversion yield of 99.1% and an enantiomeric excess of >99%. When scaled up in a 23-l bioreactor, T. cutaneum produced 8.4 g of pure cis (1S,2R) indanediol, and the isolated yield of cis (1S,2R) indanediol was 52%. Purification of the scale-up also yielded 0.9 g of the more polar trans (1S,2R) indanediol diastereomer, a minor bioreduction product. Supercritical fluid chromatography analyses of the purified cis (1S,2R) and trans (1S,2S) indanediol demonstrated that the enantiomeric excesses during this bioconversion scale-up were 99% and 26%, respectively.

Journal Article↗
PubMed1999

Porcine liver esterase-catalyzed enantioselective hydrolysis of a prochiral diester into its optically pure (S)-ester acid, a precursor to a growth hormone secretagogue.

A limited screen of several commercially-available and internally-produced lipases and esterases identified porcine liver esterase as a suitable biocatalyst for the enantioselective hydrolysis of a diester into its (S)-ester acid with high optical purity (99%). This (S)-ester acid is a precursor to an experimental growth hormone secretagogue. After identifying xanthan gum as the best emulsifier and optimizing the reaction conditions, hydrolysis rates of 1 g/l.h and final (S)-ester acid (ee > 99%) titers of about 8.5 g/l were routinely achieved. This process supported the production of preparative amounts of optically pure (S)-ester (ee > 99%) with a high reaction yield of 82%. Upon purification, the (S)-ester was successfully used in the subsequent synthetic steps to yield the growth hormone secretagogue.

Journal Article↗
PubMed1996

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus.

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-1 bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-1 fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.

Cholinesterase Inhibitors↗
PubMed1996

Asymmetric bioreduction of a ketosulfone to the corresponding trans-hydroxysulfone by the yeast Rhodotorula rubra MY 2169.

A microbial screen identified the yeast Rhodotorula rubra MY 2169 as a suitable biocatalyst for the asymmetric bioreduction of a ketosulfone (5,6 dihydro-6(s)-propyl-4H-thieno[2,3b] thiopyran-4-one-7,7-dioxide) to the corresponding trans-hydroxysulfone. This synthesizer is a precursor to the carbonic anhydrase inhibitor L-685,393, a new drug candidate targeted for the treatment of ocular glaucoma. Process development studies revealed that the rate of bioreduction was sensitive to temperature, pH, solvent concentration and the physiological state of the yeast cells. The maximum specific bioreduction rate was achieved by employing cells harvested in the stationary phase of growth. The diastereomeric excess of the trans-hydroxysulfone produced was found to be only affected by the residual amount of ketosulfone present in the bioconversion medium; therefore, when close monitoring of the residual ketosulfone was implemented, the desired enantiomeric excess was achieved at harvest. When scaled up, this bioreduction process supported the production of gram quantities of highly optically pure trans-hydroxysulfone (diastereomeric excess > 96%).

Alcohols↗
PubMed1991

Bioconversion of the sodium salt of simvastatin (MK-733) to 6-desmethyl-6-alpha-hydroxymethyl simvastatin.

An actinomycete (MA 6474, ATCC 53828) isolated from a soil sample (Mutare, Zimbabwe) was found to biotransform the sodium salt of Simvastatin (MK-733) to 6-alpha-hydroxymethyl MK-733, 6-beta-hydroxymethyl MK-733, and 6-ring-hydroxy MK-733. The bioconversion efficiency to the desired compound, 6-alpha-hydroxymethyl MK-733, was enhanced by optimizing the physico-chemical parameters of the process. In shake flask cultures, addition of magnesium (0.125 mg/l Mg SO4.7H2O) to the medium resulted in a five-fold increase in the rate of bioconversion to the alpha diastereomer. The ratio of bioconversion products (6-alpha-hydroxymethyl,6-beta-hydroxymethyl, and 6-ring-hydroxy MK-733) was regulated by pH. Process improvements and scale up in 23-1 fermentors, which consisted of a controlled addition of substrate (MK-733), resulted in a 2-fold increase in alpha diastereomer production (42 vs. 79 U/ml) and a 23-fold rate increase in the formation of alpha-diastereomer. A high diastereomeric ratio (alpha: beta = 9:1) facilitated downstream processing.

Actinomycetales↗
PubMed1991

Biochemical and physiological characterization of the efrotomycin fermentation.

An efrotomycin fermentation was characterized through physical, chemical and biochemical studies. Growth of the actinomycete, Nocardia lactamdurans occurred during the first 50 h of the fermentation cycle at the expense of glucose, protein, and triglycerides. The initiation of efrotomycin biosynthesis was observed when glucose dropped to a low concentration. Upon glucose depletion, cell growth ceased and a switch in the respiratory quotient occurred. Efrotomycin biosynthesis was supported by the utilization of soybean oil and starch. Analysis of triglyceride metabolism showed that no diglycerides or monoglycerides accumulated during the fermentation. The activity of extracellular enzymes (lipase, protease, and amylase) increased during the cell growth phase and decreased significantly after 150 h. The concentrations of DNA, tetrahydro-vitamin K2 (a membrane component), and free amino acids in the supernatant increased dramatically late in the fermentation cycle (225 h), indicating massive cell lysis. During this same time period, a reduction in cellular respiratory activity and efrotomycin biosynthesis were observed.

Amino Acids↗
PubMed1990

Bioconversion of avermectin into 27-OH avermectin.

The bioconversion of avermectin to its 27-hydroxy derivative is achieved with Nocardia autotrophica subsp. canberrica. The approach of increasing bioconversion productivity rather than efficiency was adopted in these studies. Process improvement studies focused on the physico-chemical conditions of the fermentation, examined initially at the shake-flask scale. Bioconversion yields were affected by pH, substrate concentration, time of substrate addition, substrate solubilization, carbon to nitrogen ratio, and medium strength. Optimization of these parameters resulted in a 8-fold process improvement. During pre scale-up studies, the sensitivity of this bioconversion to the antifoam employed was demonstrated and lard oil was selected as giving the best results. Additional process changes were required during scale-up efforts in larger vessels, including replacement of the original substrate solvent with dimethylsulfoxide.

Anthelmintics↗
PubMed1988

Control of Interspecies Electron Flow during Anaerobic Digestion: Role of Floc Formation in Syntrophic Methanogenesis.

The flora of an anaerobic whey-processing chemostat was separated by anaerobic sedimentation techniques into a free-living bacterial fraction and a bacterial floc fraction. The floc fraction constituted a major part (i.e., 57% total protein) of the total microbial population in the digestor, and it accounted for 87% of the total CO(2)-dependent methanogenic activity and 76% of the total ethanol-consuming acetogenic activity. Lactose was degraded by both cellular fractions, but in the free flora fraction it was associated with higher intermediary levels of H(2), ethanol, butyrate, and propionate production. Electron microscopic analysis of flocs showed bacterial diversity and juxtapositioning of tentative Desulfovibrio and Methanobacterium species without significant microcolony formation. Ethanol, an intermediary product of lactose-hydrolyzing bacteria, was converted to acetate and methane within the flocs by interspecies electron transfer. Ethanol-dependent methane formation was compartmentalized and closely coupled kinetically within the flocs but without significant formation of H(2) gas. Physical disruption of flocs into fragments of 10- to 20-mum diameter initially increased the H(2) partial pressure but did not change the carbon transformation kinetic patterns of ethanol metabolism or demonstrate a significant role for H(2) in CO(2) reduction to methane. The data demonstrate that floc formation in a whey-processing anaerobic digestor functions in juxtapositioning cells for interspecies electron transfer during syntrophic ethanol conversion into acetate and methane but by a mechanism which was independent of the available dissolved H(2) gas pool in the ecosystem.

Journal Article↗
PubMed1987

Microbial ecophysiology of whey biomethanation: comparison of carbon transformation parameters, species composition, and starter culture performance in continuous culture.

Changes in lactose concentration and feed rate altered bacterial growth and population levels in a whey-processing chemostat. The bacterial population and methane production levels increased in relation to increased lactose concentrations comparable to those in raw whey (6%) and converted over 96% of the substrate to methane, carbon dioxide, and cells. Sequential increases in the chemostat dilution rate demonstrated excellent biomethanation performance at retention times as low as 25 h. Retention times shorter than 25 h caused prevalent bacterial populations and methane production to decrease, and intermediary carbon metabolites accumulated in the following order: acetate, butyrate, propionate, lactate, ethanol, and lactose. Bacterial species dominated in the chemostat as a function of their enhanced substrate uptake and growth kinetic properties. The substrate uptake kinetic properties displayed by the mixed chemostat population were equivalent to those of individual species measured in pure culture, whereas the growth kinetic properties of species in mixed culture were better than those measured in pure culture. A designed starter culture consisting of Leuconostoc mesenteroides, Desulfovibrio vulgaris, Methanosarcina barkeri, and Methanobacterium formicicum displayed biomethanation performance, which was similar to that of a diverse adapted mixed-culture inoculum, in a continuous contact digestor system to which 10 g of dry whey per liter was added. Preserved starter cultures were developed and used as inocula for the start-up of a continuous anaerobic digestion process that was effective for biomethanation of raw whey at a retention time of 100 h.

Journal Article↗
PubMed1986

Microbial ecophysiology of whey biomethanation: intermediary metabolism of lactose degradation in continuous culture.

The intermediary carbon and electron flow routes for lactose degradation during whey biomethanation were studied in continuous culture. The chemostat was operated under lactose-limited conditions with a 100-h retention time. The carbon balance observed for lactose degradation was 4.65 mmol of CH(4), 4.36 mmol of CO(2) and 1.15 mmol of cellular carbon per mmol of lactose consumed, with other intermediary metabolites (i.e., acetate, lactate, etc.) accounting for less than 2% of the lactose consumed. The carbon and electron recoveries for this biomethanation were 87 and 90%, respectively. C tracer studies demonstrated that lactose biomethanation occurred in three distinct but simultaneous phases. Lactose was metabolized primarily into lactate, ethanol, acetate, formate, and carbon dioxide. During this hydrolytic phase, 82% of the lactose was transformed into lactate. These metabolites were transformed into acetate and H(2)-CO(2) in a second, acetogenic, phase. Finally, the direct methane precursors were transformed during the methanogenic phase, with acetate accounting for 81% of the methane formed. A general scheme is proposed for the exact carbon and electron flow route during lactose biomethanation, which predicts the prevalent microbial populations in this ecosystem.

Journal Article↗