甾醇侧链切除的微生物转化技术

甾体化合物是医药工业中具有巨大市场的产品之一,甾醇侧链切除的微生物转化是解决甾体医药原料的基础,像甾醇这一类疏水性底物的微生物转化技术是产业化的关键。本文综述了甾醇侧链切除微生物转化技术的新进展,详细介绍了我们最近开发的浊点系统两相分配生物反应器新技术。     

n-Chloroaniline transformation by Escherichia coli under anaerobic conditions

In a medium without oxygen in the presence of nitrates, E. coli transforms p-chloranilin (p-CA) to yield a more hydrophilic compound which cannot be extracted with an organic solvent from water. The conditions for consecutive transformation of p-nitro-chlorobenzene (p-NCB) and p-CA have been determined: the reaction p-NCB leads to p-CA is inhibited by nitrates, p-CA transformation occurs in the presence of nitrates in the medium and depends on their concentration.     

Sterol formation by Canida boidinii

The content of sterols in the yeast Candida boidinii is low: 0.35--0.40% in a mineral medium with methanol as a sole carbon source; 0.55--0.60% in a medium with ethanol; 0.50--0.60% in a medium with glucose; 0.50--0.55% on wort--agar. Ergosterol is the main sterol in all of the cases. sterols are found mainly in the bound state as esters, and constitute about 90% of the total sterol content. Free sterols constitute only 10%. The level of sterols in this culture is rather constant and hardly changes within the first days of cultivation. The content of sterols increases only slightly (by 30--40%) when the culture ages.     

Microwave improved Escherichia coli transformation

Aims: The calcium chloride chemical transformation of Escherichia coli is still the most widley used cloning method in small laboratories. Therefore, any practicable improvement in its transformation efficiency seems to be of general interest. Methods and Results: We found that giving calcium chloride competent cells a 1 min microwave pulse at the lowest power setting (180 W), instead of the classic 1–2 min 42°C heat‐shock step, increases the transformation efficiency around threefold (3.3 ± 0.5). Moreover, when both treatments were given in a 2‐min 42°C ? 5 min on ice ? 1 min microwave pluse sequence, an additional improvement of 1.6 was obtained, resulting in an overall increase in efficiency of approximately 5.3‐fold compared to classical heat shock. Conclusions: This transformation method significantly improves the classical heat shock treatment. Significance and Impact of the Study: This method might be useful to those laboratories that cannot afford an electroporation apparatus.     

Sterol hydroperoxide metabolism by Salmonella typhimurium

In order to rationalize multiphasic dose-response data evincing mutagenicity towards Salmonella typhimurium TA1537 for sterol hydroperoxides 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide and 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide their metabolism by the bacterial test strain was investigated. The 5 alpha-hydroperoxide was isomerized to the 7 alpha-hydroperoxide and reduced to 5 alpha-cholest-6-ene-3 beta,5-diol; the 7 alpha-hydroperoxide was reduced to cholest-5-ene-3 beta,7 alpha-diol and transformed to 3 beta-hydroxycholest-5-en-7-one. The 3 beta,5 alpha-diol and 3 beta,7 alpha-diol were not interconverted nor was either transformed to the 7-ketone.     

Sterol absorption by the small intestine

PURPOSE OF REVIEW: Cholesterol absorption is a selective process in that plant sterols and other non-cholesterol sterols are absorbed poorly or not at all. Recent research on the sterol efflux pumps adenosine triphosphate-binding cassette transporter G5 and adenosine triphosphate-binding cassette transporter G8 has not only provided an explanation for this selectivity, but also, together with the discovery of a new class of cholesterol absorption inhibitor, has yielded new insights into the mechanisms that potentially regulate the flux of cholesterol across the enterocyte. This review discusses these recent developments and their importance to the regulation of whole body cholesterol homeostasis. RECENT FINDINGS: Adenosine triphosphate-binding cassette transporters G5/8 regulate plant sterol absorption and also the secretion into bile of cholesterol and non-cholesterol sterols. Loss of adenosine triphosphate-binding cassette transporter G5/8 function results in sitosterolemia. Ezetimibe, a novel, potent and selective inhibitor of cholesterol absorption which is effective in milligram doses, lowers plasma plant sterol concentrations in sitosterolemic subjects, thus suggesting that this drug might be inhibiting the activity of a putative sterol permease in the brush border membrane of the enterocyte that actively facilitates the uptake of cholesterol as well as other non-cholesterol sterols. SUMMARY: Intestinal cholesterol absorption represents a major route for the entry of cholesterol into the body's miscible pools and therefore can potentially impact the plasma LDL-cholesterol concentration. The combined use of agents that inhibit the absorption and synthesis of cholesterol provides a powerful new approach to the prevention and treatment of atherosclerosis.     

Polyhydroxybutyrate-enhanced transformation of log-phase Escherichia coli

Transformation of Escherichia coli plays an important role in recombinant DNA technology. Most current transformation protocols require that the cells be treated to attain a particular physiological state known as "competence," and this makes transformation procedures lengthy and arduous. Here we describe a protocol for transforming log-phase E. coli using dimethyl sulfoxide (DMSO) solutions of poly-(R)-3-hydroxybutyrate (PHB) to facilitate the transfer of plasmid DNA into cells, and certain reagents and temperature shocks to promote DNA uptake. The protocol was optimized using factorial design techniques across variables that included PHB molecular weight and concentration, DMSO concentration, monovalent and divalent salts, glucose, cold and heat shocks, cell density, and pH. Using 10 ng DNA, the optimized protocol produces approximately 1000 colony-forming units (CFUs) from 100 microL early log-phase cell culture or approximately 300 CFU from a 21-24 h single colony, sufficient for many applications. The total volume of the transformation reaction mixture is only 150 microL suggesting that the procedure may be adapted for use in microplates or automated transformation technologies.     

Recombination following transformation of Escherichia coli by heteroduplex plasmid DNA molecules

Circular heteroduplex DNA molecules introduced into Escherichia coli-competent cells are converted to new recombinant plasmids as a result of enzymatic actions in vivo. A pair of plasmids with partial sequence homology were each linearized at a different position with restriction enzymes, and the termini were made flush with the single-strand-specific S1 nuclease. Duplex molecules were then formed by melting and annealing these plasmid DNAs together. In contrast to linear homoduplex molecules, heteroduplexes circularize and therefore transform E. coli efficiently. Unique DNA sequences on each of the parental strands in the transforming heteroduplexes can be selectively incorporated or deleted as a result of in vivo enzymatic activities in transformed cells. This method permits the generation of new recombinant sequences in vivo without relying solely on the presence of convenient restriction sites for manipulation of DNA fragments in vitro.     

Sterol affinity of Candida albicans cells resistant to polyene antibiotics]

The affinity levels of sterols in the sensitive and resistant cultures of C. albicans for polyenic antibiotics were studied comparatively. The affinity level was determined by liberation of potassium under the effect of the antibiotic participating in interaction with the sterol. The protective effect of the sterol suspended in solution and included into the composition of the liposomes from egg lecithin was studied. It was found that the sterols of the resistant cultures of C. albicans had the same (or even somewhat higher) affinity to amphotericin B as those from the sensitive cultures. The data indicate that resistance of the strains studied is not based on the loss of the sterol capacity for binding polyenic antibiotics.     

Cholesterol synthesis by several strains of Escherichia]

The results of the study of lipids in the media used for growing different Escherichia strains are presented. Donor plasma with carbon-labeled sodium acetate was used as culture medium. Those strains which induced an increase in cholesterol content in the medium after 48-hour incubation were considered cholesterin-synthetizing. During the growth of these strains the radioactive marker became incorporated into the lipids accumulated in the medium: phospholipids, cholesterol, fatty acids. The degree of this incorporation depended on the dose of labeled sodium acetate and the amount of the inoculated culture. Cholesterol-synthetizing activity of Escherichia is characteristic of only freshly isolated strains.     

Transfer of transformation and conjugation plasmids from Escherichia coli to Bacillus subtilis and Azospirillum brasilense]

The conjugative transfer of RP4 plasmid from Escherichia coli to Azospirillum brasilense was detected after introduction and subsequent incubation of these microorganisms in soil. The plasmid transfer via transformation from Escherichia coli to Bacillus subtilis was observed in case both bacteria were growing together in sand containing sucrose solution. The possible reason for low frequency interspecies plasmid transformation under conditions close to natural habitats is poor survival of "domesticated" rather than wild type Bacillus subtilis strains and lack of competence state in this case.     

Liposome-mediated transformation of tobacco mesophyll protoplasts by an Escherichia coli plasmid.

An Escherichia coli plasmid, pLGV23neo, carrying a kanamycin resistance gene expressed in plant cells, was encapsulated into negatively charged liposomes prepared by the reverse phase evaporation technique. These liposomes were induced to fuse with tobacco mesophyll protoplasts by polyethyleneglycol treatment. Kanamycin-resistant clones were reproducibly isolated from transfected cultures at an average frequency of 4 X 10(-5). Plants regenerated from these resistant colonies were confirmed to be transformed according to three criteria. Protoplasts isolated from their leaves were resistant to 100 micrograms/ml kanamycin. The enzyme aminoglycoside 3'-phosphotransferase II encoded by the plasmid pLGV23neo was detected in leaf extracts. Approximately 3-5 copies of the gene encoding for kanamycin resistance were inserted in the genome of at least one of the studied transformants. The restriction pattern of inserted DNA was best explained by assuming a tandem integration of the pPLGV23neo sequences, implying an homologous recombination event between these sequences during transformation. Kanamycin resistance was transmitted as a single dominant nuclear marker to the progeny of resistant plants after selfing or cross-pollination with the wild-type.     

Reductive transformation of TNT by Escherichia coli resting cells: kinetic analysis

Microbial 2,4,6-trinitrotoluene (TNT) biotransformation via sequential nitro-reduction appears a ubiquitous process, but the kinetics of these transformations have been poorly understood or described. TNT transformation by Escherichia coli was monitored and a kinetic model for reductive TNT depletion was developed and experimentally calibrated in this report. Using resting cells of aerobically pregrown E. coli, TNT was quickly reduced to hydroxylaminodinitrotoluenes. The standard Michaelis–Menten model was modified to include three additional parameters: product toxicity (T _c), substrate inhibition (K _i), and intracellular reducing power (RH) limitation. Experimentally measured product toxicity (5.2 μmol TNT/mg cellular protein) closely matched the best-fit model value (2.84 μmol TNT/mg cellular protein). Parameter identifiability and reliability (k _m, K _s, T _c, and K _i) was evaluated and confirmed through sensitivity analyses and via Monte Carlo simulations. The resulting kinetic model adequately described TNT reduction kinetics by E. coli resting cells in the absence or presence of reducing power limitation.     

Chrysotile asbestos fibers mediate transformation of Escherichia coli by exogenous plasmid DNA

The ability of chrysotile asbestos fibers to introduce the exogenous plasmid pUC18 into Escherichia coli JM109 cells was tested. Cells were transformed with pUC18 DNA although the frequency of transformation was quite low: 759+/-301 transformants were obtained per microgram of pUC18. Plasmids were purified from E. coli which had been transformed by mediation with chrysotile asbestos. Following this, the plasmids were confirmed to be pUC18 by Southern hybridization. This asbestos-mediated transformation was optimal within 5 min when 10 mg ml(-1) of asbestos was used. Plasmids up to 7.69 kb were introduced by this method.     

Escherichia coli cell competence induced by calcium cations]

Escherichia coli K-12 cells grown to early and late exponential, and early and late stationary phases were treated with CA2+ and analysed for the ability of exogenous 14C-DNA uptake and genetic transformation. DNA-membrane complexes were detected detected by isopicnic centrifugation of cell lysates in sucrose density gradient. It is found that 1) during all the tested phases of the growth cycle, E. coli cells attain the ability of enhanced DNA uptake after Ca2+ treatment; 2) exogenous and host DNAs are associated with the cell membrane during all tested growth phases; 3) nevertheless, the ability to form transformants is strongly time-dependent: the cells can be transformed during logarithmic phase only; 4) Ca2+ protects exogenous DNA against its degradation by bovine pancreatic DNAase. The peculiarities of Ca2+-induced competence, actual and possible interference of Ca2+ at different transformation steps are briefly discussed.     

Sterol biosynthesis by the sea urchin Echinus esculentus

1. The 4-demethyl sterols of Echinus esculentus consisted of cholesterol as the major component, with lower concentrations of nine other C(26), C(27), C(28) and C(29) Delta(5) sterols. 2. [2-(14)C]Mevalonic acid was readily incorporated by the urchin into squalene, lanosterol and desmosterol but only to a small extent into cholesterol. 3. [26-(14)C]Desmosterol did not appear to be reduced to give cholesterol, but conversion of 5alpha-[2-(3)H(2)]lanost-8-en-3beta-ol into cholesterol was observed. 4. No C-24 dealkylation of [4-(14)C]sitosterol or metabolism of [4-(14)C]cholesterol could be detected.     

Sterol Uptake in Saccharomyces cerevisiae Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.