A Keto Reductase Involved in Steroid Degradation in Mycolicibacterium neoaurum

Phytosterols can be used by microorganisms as carbon and energy sources and completely degraded into CO₂ and H₂O. The catabolic pathway of phytosterols was well characterized in many microorganisms. Blocking the steroid core ring degradation by deletions of fadE30 and fadD3 genes, two important steroid intermediates, 3aα-H-4α-(3’-Propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone (sitolactone, or HIL) and 3aα-H-4α-(3’-propionic acid)-7aβ-methylhexahydro-1,5-indanedione (HIP) can be accumulated. They are currently used to synthesize nor-steroid drugs with an α-methyl group or without the methyl group at the C₁₀-position, such as estrone and norethindrone. In this study, a key gene involved in the bioconversion of HIP to HIL was identified in Mycolicibacterium neoaurum. Through heterologous expression, gene hipR was found to be involved in the reduction of the C₅ keto group of HIP to a hydroxy group, leading to spontaneously lactonization into HIL in vitro. Through gene complementation and knockout, HipR functions were verified and two HIP degradation pathways in vivo were elucidated. The finding of this research facilitated the understanding of the metabolic pathway of sterols, and was directly applied to engineering robust production strains by overexpression or knockout of related genes.     

Identification of Flavone Compounds in Eighteen Gramineae Species

Conditions for tryptophan synthesis from pyruvic acid, indole and NH₄Cl by Enterobacter aerogenes AHU 1540 having a high tryptophanase activity, were investigated using a reaction mixture containing 1.7% of pyruvic acid. Under optimum conditions, 16.4g/liter of tryptophan was accumulated after 24 hr of incubation.

Agaricus campestris AHU 9382 produced pyruvic acid in amounts of 22 ~ 26.5 g/liter from 5% of glucose after 3-days shaking culture. When E. aerogenes was added to this fermentation broth together with indole and NH₄Cl, pyruvic acid produced was rapidly converted to tryptophan and yields of tryptophan as high as 15 g/liter were obtained after 12 hr of incubation. Furthermore, pyruvic acid fermentation by Saccharomyces exiguus AHU 3110 or Corynebacterium sp. 37-3A could also be used as a pyruvic acid source for subsequent tryptophan production.     

NUTRITIONAL REQUIREMENTS OF FRAXINUS CALLUS CULTURES

A satisfactory synthetic medium has been developed for continuous growth of Fraxinus pennsylvanica Marsh. callus cultures. The medium contains modified Reinert and White (1956) inorganic nutrient solution with 5 mg/liter Fe as NaFe-EDTA and supplemented with myoinositol 10 mg/liter, pyridoxine HCl 0.1 mg/liter, 2,4-dichlorophenoxyacetic acid 0.04 mg/liter, kinetin 1 mg/liter, sucrose 20 g/liter, and agar 10 g/liter. myo-Inositol, pyridoxine and an auxin are essential. α-Naphthaleneacetic acid is an effective alternate auxin. Kinetin and to some extent gibberellic acid improve the yields. Thiamine has no effect.     

Sterols of Testudodinium testudo (formerly Amphidinium testudo): Production of the Δ8(14) sterol gymnodinosterol and chemotaxonomic relationship to the Kareniaceae

Testudodinium testudo is a peridinin-containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ⁸⁽¹⁴⁾ sterols, such as 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ⁸⁽¹⁴⁾ sterol, (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α-methyl-5α-ergosta-8,14,22-trien-3β-ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.     

Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.     

THE LIPID COMPOSITION OF THORACOSPHAERA HEIMII: EVIDENCE FOR INCLUSION IN THE DINOPHYCEAE1

The fatty acid, sterol and chlorophyll composition of the calcified, unicellular alga Thoracosphaera heimii (Lohmann) Kamptner are reported. The presence of 4,23,24-termethyl-5α-cholest-22E-en-3β-ol (dinosterol), 4,23,24-trimethyl-5α-cholest-22E-en-3-one (dinosterone) and the predominance of C₁₈, C₂₀ and C₂₂ unsaturated fatty acids, including the acid 18:5ω3, indicates that T. heimii is a dinoflagellate. The fatty acid: sterol ratio (1.3), is typical of dinoflagellates. The geochemical significance of dinosterone, the high relative concentration of 4-desmethyl-5α-stanols and the role of 23-methyl-5α-cholest-22E-en-3β-ol in the biosynthesis of dinosterol in T. heimii are also discussed.     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

Sterols of Amphidinium species in the Operculatum Clade: Predominance of cholesterol instead of Δ8(14) sterols previously considered Amphidinium-specific biomarkers

The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ⁸⁽¹⁴⁾-nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3β-ol (C_28:1) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol; C_29:2) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3β-ol; C_27:1), which is found in many other dinoflagellate genera, rather than Δ⁸⁽¹⁴⁾ sterols. While the Δ⁸⁽¹⁴⁾ sterols 4α-methyl-5α-cholest-8(14)-en-3β-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3β-ol (C_30:2) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol; C_30:1), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ⁸⁽¹⁴⁾ sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ⁸⁽¹⁴⁾ sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.     

Utilization of Hydrocarbons by Microorganisms

As already reported, Corynebacterium hydrocarboclastus S10B1 was able to accumulate a good deal of l-glutamate in a thiamine-deficient medium at the sole expense of n-alkanes, but unable to form l-glutamate in a thiamine-sufficient medium though an abundant cell growth was observed.

α-Ketoglutaric acid and dl-alanine were found to be produced in the same thiamine-deficient medium in which l-glutamate was accumulated. Both products formed from n-tetradecane by this organism were isolated from culture broth, purified and identified. The optimum concentration of thiamine in the culture medium was 3 to 5 µg per liter for their production. The maximum yields of α-ketoglutaric acid and dl-alanine reached 16 g and 1.5 g per liter in the calcium carbonate-added medium, respectively. However, the addition of more than 30 μg per liter of thiamine extremely repressed their accumulation.     

Sterol chemical configuration influences the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayers containing 5α-cholestan-3β- and 3α-ol

It is commonly believed that all membrane sterols are rigid all-trans ring systems with a fully extended alkyl side-chain and that they similarly influence phospholipid bilayer physical properties. Here, we report the sterol concentration-dependent, thermotropic phase behaviour of binary dipalmitoylphosphatidylcholine (DPPC)/sterol mixtures containing two similar 5α-H sterols with different functional group orientations (3α-OH or 3β-OH), which adopt an ideal all-trans planar ring conformation but lack the deformed ring B conformation of cholesterol (Chol) and epicholesterol (Echol), using differential scanning calorimetry (DSC). Our deconvolution of the DSC main phase transition endotherms show differences in the proportions of sterol-poor (sharp) and sterol-rich (broad) domains in the DPPC bilayer with increasing sterol concentration, which delineate gel/liquid-crystalline (P_β′/L_α) and disordered gel (L_β)/liquid-ordered (l_o) phase regions. There are similarities in the DPPC main phase transition temperature, cooperativity and enthalpy for each 3β-ol and 3α-ol pair with increasing sterol concentration and differences in the parameters obtained for both the sterol-poor and sterol-rich regions. The sterol-poor domain persists over a greater concentration range in both 3α-ol/DPPC mixtures, suggesting that either those domains are more stable in the 3α-ols or that those sterols are less miscible in the sterol-rich domain. Corresponding parameters for the sterol-rich domain show that at sterol concentrations up to 20 mol%, the 5α-H,3β-ol is more effective at reducing the phase transition enthalpy of the broad component () than Chol, but is less effective at higher concentrations. Although mixtures containing Echol and 5α-cholestan-3α-ol have similar positive slopes below 7 mol% sterol, suggesting that they abolish the L_β/l_o phase transition equally effectively at low concentrations, Echol is more effective than the saturated 3α-ol at higher sterol concentrations. A comparison of obtained for the saturated and unsaturated pairs suggests that the latter sterols stabilize the l_o phase and broaden and abolish the DPPC main phase transition more effectively than the saturated sterols at physiologically relevant concentrations, supporting the idea that the double bond of Chol and Echol promotes greater sterol miscibility and the formation of l_o phase lipid bilayers relative to corresponding saturated sterols in biological membranes.     

Some Physicochemical Properties of Invertase of Candida utilis

This study examined the microbial transformation of carbazole (CZ) by an isolated bacterium that can use CZ as a sole carbon and nitrogen source. The strain identified as Pseudomonas stutzeri produced a large amount of anthranilic acid (AA) from CZ in the medium containing a nonionic surfactant. In dialysis culture using ion-exchange resin, 7.9 g/liter (58mm) of AA was accumulated from 15g/liter (90mm) of CZ and the molar yield of AA reached about 64%.     

Enhanced oxygen transfer rates in fermentation using soybean oil-in-water dispersions

Summary The effect of soybean oil on the volumetric oxygen transfer coefficient during the cultivation ofAerobacter aerogenes cells is presented. For our aeration-agitation conditions (0.278 vvm and 500 rpm), it has been demonstrated that the use 19% (v/v) of soybean oil enabled a 1.85-fold increase of thek _l a coefficient (calculated on a per liter aqueous phase basis). For smaller volumetric oil fractions,k _L a increased linearly with the oil loading. Because of the oxygen-vector properties of soybean oil, this oil is able to significantly increase thek _L a of a bioreactor.Nomenclature C^*, C saturation and actual dissolved oxygen concentrations respectively (g/m³) - K_La volumetric oxygen transfer coefficient (h^–1) - K_La_initial k _La measured before the oil addition (h^–1) - M_O2 molar mass of oxygen (dalton) - N oxygen transfer rate (g/m³. h) - P_O2. P_N2 partial pressures ofO ₂ andN ₂ in the gas (atm) - P_H2OT partial pressure of water in air at the temperatureT (atm) - P_T total pressure (atm) - Q₀ volumetric flow rate of outlet air before seeding (m³/h) - Sp spreading coefficient (dynes/cm) - T absolute temperature of outlet gas (K) - V_i volume of the liquidi in the fermentor (m³) - V_M molar volume at 273 K and 1 atm (m³/mole) - _ij interfacial tension betweeni andj componants (dynes/cm) - _v volumetric fraction of the oil (v/v) - G gas - O oil - W water - i inlet - o outlet     

Pathway engineering of Escherichia coli for α-ketoglutaric acid production

α-Ketoglutaric acid (α-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for α-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for α-KG production in Escherichia coli. First, the TCA cycle was rewired for α-KG production starting from pyruvate, and the engineered strain E. coli W3110Δ4-PCAI produced 15.66 g/L α-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in α-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to α-KG production up to 28.54 g/L. Finally, the engineered strain E. coli W3110Δ4-P_(H)CAI_(H)A was able to produce 32.20 g/L α-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of α-KG.     

Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

STEROL DISTRIBUTION IN THE GENUS HETEROCAPSA (PYRRHOPHYTA)1

The sterol composition of seven strains of marine peridinioid dinoflagellates comprising the four known species of Heterocapsa Stein was examined by gas chromatography-mass spectrometry to determine the utility of these compounds in systematics. Cholest-5-en-3β-ol (cholesterol), 24-methyl-cholest-5-en-3β-ol (24-methylcholesterol), 4α,24(S)-dimethyl-5α-cholestan-3β-ol (4,24-dimethylcholestanol), 4α,23,24(R)-trimethyl-5α-cholest-22-en-3β-ol (dinosterol), 4α,23ξ,24ξ-trimethyl-5α-cholestan-3β-ol (dihydrodinosterol), and an unknown sterol were detected. Sterol composition does not vary significantly from species to species within the genus Heterocapsa and thus cannot be used for species differentiation. Sterols may, however, have value in defining the properties of dinoflagellate taxa above the family level. Over the course of the growth curve for Heterocapsa niei (Loeblich) Morrill & Loeblich 4,24-dimethylcholestanol and dinosterol covaried, suggesting that 4,24-dimethylcholestanol is converted into dinosterol by a previously proposed bioalkylation scheme.     

Isolation and Identification of α-Spinasterol and α-Spinasteryl D-Glucoside from Sugar Beet Pulp

A sterol and a steryl glucoside were isolated from dried beet pulp. The sterol was identified with α-spinasterol, the glucoside possessed chemical and physical properties such as follows: The molecular formula C₃₅H₅₈O₆, m.p. 292°, [ α]¹⁹_D-34.1°, acetate; m.p. 168°, benzoate; m.p. 175-177°, and positive for Molish and Lieber-mann-Burchard reactions. When it was hydrolyzed with 1% sulfuric acid, the crystal of α-spinasterol and D-glucose detectable by paper chromatography were obtained. These results gave evidence that the glucoside was in question to be α-spinasteryl D-glucoside.     

Studies on the Utilization of Petrochemicals by Microorganisms

During the investigation of petrochemical-utilizing microorganisms, 60 strains of 1,2- propanediol-utilizing microorganisms were isolated from soil. Among these microorganisms, the strain PG-21-1 was found to produce lactic acid from 1, 2-propanediol and β-hydroxybutyric acid from 1, 3-butanediol. From the results of taxonomical studies, the strain PG-21-1 was identified to Arthrobacter oxydans.

The yield of lactic acid was increased up from 0,18 g/liter to 9.02 g/liter by improving the cultural conditions. The residual 1, 2-propanediol recovered from culture broth was found to be optically active.     

IDENTIFICATION AND PHYLOGENETIC IMPLICATIONS OF FATTY ACIDS AND STEROLS IN THREE GENERA OF AQUATIC PHYCOMYCETES

Allomyces macrogynus, A. arbuscula, A. javanicus, Allomyces male and female hybrid strains, Blastocladia ramosa and Monoblepharella sp. were examined for their fatty acid and sterol compositions by GLC and combined GLC/MS. All the organisms produce a range of fatty acids 12 to 20 carbon atoms in length. Palmitic, stearic, and arachidic acid represent the highest concentrations of saturated fatty acids; oleic, linoleic, and arachidonic acid the highest unsaturated fatty acids. B. ramosa synthesizes only two polyunsaturates, linoleic and linolenic, but Allomyces and Monoblepharella are capable of desaturation as far as arachidonic acid. Cholesterol is produced by all the isolates and is the dominant sterol in Allomyces. 24-Methyl and 24-ethyl derivatives of cholesterol are the dominant sterols of Monoblepharella. B. ramosa contains a more complex sterol mixture representing changes which occur in the formation of cholesterol from lanosterol: 24-dihydrolanosterol, 14α-methyl Δ⁸-cholestenol, Δ⁸⁽⁹⁾-cholestenol, 14α-methyl Δ⁷-cholestenol, Δ⁷-cholestenol and cholesterol. Δ⁷-cholestenol, 24-dihydrolanosterol, and 14α-methyl Δ⁸-choIestenol appear to be the major components. This is the first time that 14α-methyl Δ⁸ and 14α-methyl Δ⁷-cholestenol have been reported as naturally occurring sterols.     

Chemical synthesis of the (25R)- and (25S)-epimers of 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding glycine and taurine conjugates

The (25R)- and (25S)-epimers of C₂₇ 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding N-acylamidate conjugates with glycine or taurine were prepared starting from cholic acid in 14 steps. The principal reactions involved were (1) reduction of a key intermediary C₂₄allo-cholic acid performate with NaBH₄/triethylamine/ethyl chloroformate, (2) iodination of the resulting 3,7,12-triformyloxy-5α-cholan-24-ol with I₂/triphenylphosphine; (3) nucleophilic substitution of the iodo derivative with diethylmethyl malonate/NaH; and (4) hydrolysis of the resulting 3,7,12-triformyloxy-25-methyl-26,27-diethyl ester with KOH, followed by decarboxylation of the geminal dicarboxylic acid with LiCl. N-Acylamidation of the resulting (25R)/(25S)-3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid mixture with glycine or taurine afforded the corresponding epimeric mixtures of the glycine and taurine conjugates. The (25R)- and (25S)-epimers of the three variants of unconjugated and conjugated 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid were efficiently separated by HPLC on a reversed-phase C₁₈ column and their structural characteristics, particularly the chiral center at C-25, delineated using ¹H and ¹³C NMR. These synthetic compounds should be useful as authentic reference standards for establishing their presence in bile as well as being useful in studies on the biosynthesis of allo-bile acids from cholesterol.