Synthesis of saturated long chain fatty acids from sodium acetate-1-C14 by Mycoplasma

Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C(14). The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C(14) and methyl palmitate-1-C(14). This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate.     

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N′-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.     

Biosynthesis of the nargenicin A<Subscript>1</Subscript> pyrrole moiety from <Emphasis Type="Italic">Nocardia</Emphasis> sp. CS682

A number of structurally diverse natural products harboring pyrrole moieties possess a wide range of biological activities. Studies on biosynthesis of pyrrole ring have shown that pyrrole moieties are derived from l-proline. Nargenicin A₁, a saturated alicyclic polyketide from Nocardia sp. CS682, is a pyrrole-2-carboxylate ester of nodusmicin. We cloned and identified a set of four genes from Nocardia sp. CS682 that show sequence similarity to the respective genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens, clorobiocin in Streptomyces roseochromogenes subsp. Oscitans, coumermycin A₁ in Streptomyces rishiriensis, one of the pyrrole rings of undecylprodigiosin in Streptomyces coelicolor, and leupyrrins in Sorangium cellulosum. These genes were designated as ngnN4, ngnN5, ngnN3, and ngnN2. In this study, we presented the evidences that the pyrrole moiety of nargenicin A₁ was also derived from l-proline by the coordinated action of three proteins, NgnN4 (proline adenyltransferase), NgnN5 (proline carrier protein), and NgnN3 (flavine-dependent acyl-coenzyme A dehydrogenases). Biosynthesis of pyrrole moiety in nargenicin A₁ is initiated by NgnN4 that catalyzes ATP-dependent activation of l-proline into l-prolyl-AMP, and the latter is transferred to NgnN5 to create prolyl-S-peptidyl carrier protein (PCP). Later, NgnN3 catalyzes the two-step oxidation of prolyl-S-PCP into pyrrole-2-carboxylate. Thus, this study presents another example of a pyrrole moiety biosynthetic pathway that uses a set of three genes to convert l-proline into pyrrole-2-carboxylic acid moiety.     

Characterization of sulphate-reducing bacterial populations within marine and estuarine sediments with different rates of sulphate reduction

Viable counts of sulphate-reducing bacteria, able to use a range of different growth substrates were determined in sediments from two Sea Lochs (Etive and Eil) and an estuarine site (Tay), in Scotland. The composition of the sulphate-reducing bacterial population, in terms of substrate utilization, broadly corresponded to the in situ substrates for sulphate reduction and concentration of substrates at each site. Addition of acetate, lactate, propionate, butyrate, hydrogen and glutamate/serine (20 mM) to replicate slurries from each site resulted in stimulation of the corresponding population of sulphate-reducing bacteria and the in situ rates of sulphate reduction. The metabolism of the added substrates and changes in bacterial phospholipid fatty acids (PLFA) were quantified. With the exception of acetate and hydrogen, added substrates were incompletely oxidised, producing a mixture of further substrates, which predominantly were sequentially oxidised, and resulted in the stimulation of a mixed population of sulphate-reducing bacteria. There were significant changes in the PLFA of slurries with added substrate compared to controls. Acetate was completely removed at all sites and the small increase in even chain PLFA together with the absence of stimulation of any other biomarker, indicated that acetate was oxidised by sulphate-reducing bacteria distinctly different from those using other substrates. A biomarker for Desulfobacter, 10 Methyl 16:0, was not stimulated in any of the acetate slurries or in slurries where acetate was produced. Biomarkers for the propionate utilizing Desulfobulbus sp (17:1w6, 15:1w6) were always stimulated in propionate slurries and also in lactate slurries, where partial lactate fermentation produced propionate and acetate. In lactate and glutamate / serine slurries from the Tay estuary and lactate and hydrogen slurries from Loch Etive the biomarker for Desulfovibrio sp (i17:1w7) as well as those for Desulfobulbus were stimulated. This provides direct evidence for the significance of Desulfovibrio sp. within sediment slurries and demonstrates the competitive interaction between members of this genus and Desulfobulbus sp. for lactate, hydrogen and amino acid metabolism. At the estuarine site, sulphate reduction was limited at higher sulphate concentrations (about 3.5 mM) than the Sea Loch sites (<2 mM) and this had a significant effect on propionate and butyrate metabolism, as well as on methane production. These results demonstrate that although the sulphate-reducing bacterial population at each site could metabolise identical substrates, the types of sulphate-reducing bacteria involved and their sulphate thresholds were characteristically different.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Propionate and butyrate metabolism in rat or sheep hepatocytes

The capacities of isolated hepatocytes to metabolize volatile fatty acids have been compared in rat and sheep hepatocytes. In both species, acetate utilization in vitro was quite limited. Significant species differences for propionate and butyrate consumption were found: propionate utilization by rat hepatocytes was relatively limited and plateaued at about 0.8-1.0 mM, whereas butyrate utilization was approx. 2-times higher. In contrast, ruminant hepatocytes exhibited a lower rate of butyrate utilization, but propionate metabolism was much more active than in rat liver cells. With relatively low concentrations of substrates (max. 2 mM), only propionate, compared to lactate or alanine, had a significant glucogenicity with hepatocytes from fed sheep. In both species, butyrate inhibited propionate consumption, although to a larger extent in sheep. The conversion of [2-14C]propionate to glucose by sheep hepatocytes was inhibited by 2 mM butyrate (60%) or ammonia (30%); 1 mM oleate or 10 mM glucose were ineffective. The basal rate of ammonia utilization by sheep hepatocytes was much lower than in rat and was unaffected upon addition of ornithine. Ammonia metabolism was markedly enhanced by butyrate and, in contrast to rat liver cells, also by propionate.     

Metabolism of propionate to acetate in the cockroach Periplaneta americana

Carbon-13 NMR and radiotracer studies were used to determine the precursor to methylmalonate and to study the metabolism of propionate in the cockroach Periplaneta americana. [3,4,5-13C3]Valine labeled carbons 3, 4, and 26 of 3-methylpentacosane, indicating that valine was metabolized via propionyl-CoA to methylmalonyl-CoA and served as the methyl branch unit precursor. Potassium [2-13C]propionate labeled the odd-numbered carbons of hydrocarbons and potassium [3-13C]propionate labeled the even-numbered carbons of hydrocarbons in this insect. This labeling pattern indicates that propionate is metabolized to acetate, with carbon-2 of propionate becoming the methyl carbon of acetate and carbon-3 of propionate becoming the carboxyl carbon of acetate. In vivo studies in which products were separated by HPLC showed that [2-14C]propionate was readily metabolized to acetate. The radioactivity from sodium [1-14C]propionate was not incorporated into succinate nor into any other tricarboxylic acid cycle intermediate, indicating that propionate was not metabolized via methylmalonate to succinate. Similarly, [1-14C]propionate did not label acetate. An experiment designed to determine the subcellular localization of the enzymes involved in converting propionate to acetate showed that they were located in the mitochondrial fraction. Data from both in vivo and in vitro studies as a function of time indicated that propionate was converted directly to acetate and did not first go through tricarboxylic acid cycle intermediates. These data demonstrate a novel pathway of propionate metabolism in insects.     

Biosynthesis of candicidin by phosphate-limited resting cells ofStreptomyces griseus

Summary A phosphate-limited resting cell system ofStreptomyces griseus in a synthetic medium has been developed in which biosynthesis of the polyene macrolide, candicidin, is linear for at least 36 h without cell growth. Glucose and to a lesser degree sucrose, but not lactose, support antibiotic synthesis. Glucose is utilized at a constant rate for antibiotic synthesis without affecting mycelial dry weight. Acetate and propionate, the building units of the macrolide aglycone, stimulate candicidin biosynthesis in cultures supplemented with glucose but do not support its synthesis in the absence of glucose. Maximal stimulation of candicidin biosynthesis was produced by 40 mM propionate or 250 mM acetate. The biosynthetic intermediate, methyl malonate, and the analog, 1-propanol, were more stimulatory than propionate at the same concentration.     

Concomitant changes in progesterone catabolic enzymes, cytochrome P450 2C and 3A, with plasma insulin concentrations in ewes supplemented with sodium acetate or sodium propionate

Progesterone is essential for maintaining pregnancy, and several authors have suggested that low peripheral concentrations of progesterone may be responsible for high rates of embryonic loss. The primary organ involved in the catabolism of progesterone is the liver, and cytochrome P450 2C and 3A sub-families account for a large proportion of this catabolism. Elucidating a mechanism to decrease progesterone catabolism, thereby increasing embryonic and uterine exposure to progesterone, seems a logical approach to ameliorate high rates of embryonic loss. The objectives of the current experiment were to determine the pattern of insulin secretion after supplementing feed with either sodium acetate or sodium propionate and to determine any association between the differential patterns of insulin secretion with the hepatic activity of cytochrome P450 2C and 3A and progesterone clearance. Sixteen ovariectomized ewes were fed 3 kg/day for 10 days of a diet consisting of 50% corn silage, 38% triticale haylage, 12% soybean meal and 600 ml of 3.5 M sodium acetate (energy control; n = 8) or 2.0 M sodium propionate (gluconeogenic substrate; n = 8). Equal portions of the ration (1 kg as-fed basis along with 200 ml of 3.5 M sodium acetate or 2.0 M sodium propionate) were offered three times daily at 0600, 1400 and 2200 h. Concentrations of insulin in plasma were determined immediately before feeding and at 15, 30, 60, 90, 120, 180, 240 and 300 min after feeding. Progesterone clearance from peripheral circulation (ng/ml per min) was measured by giving a 5 mg injection of progesterone into the left jugular vein and collecting blood via the right jugular vein at 0, 2, 4, 6, 8, 10, 15, 20 and 30 min afterwards. Liver biopsies were taken 1 h after feeding to determine cytochrome P450 2C and 3A activities. Insulin concentrations in ewes supplemented with sodium propionate were elevated at 15, 30 and 60 min after feeding compared to the sodium acetate group. Cytochrome P450 2C and 3A activities were decreased 1 h after feeding in the sodium propionate-treated ewes relative to sodium acetate. Insulin appears to down-regulate cytochrome P450 activity, which could be used to decrease the catabolism of progesterone during early gestation, thereby increasing peripheral concentrations of progesterone and, consequently, embryonic exposure to progesterone.     

Mechanisms of growth inhibition by propionate and restoration of the growth by sodium bicarbonate or acetate in Rhodopseudomonas sphaeroides S

Mechanisms of growth inhibition by propionate on the growth of Rhodopseudomonas sphaeroides were studied. Partially purified pyruvate dehydrogenase complex (PDC) from R. sphaeroides was inhibited by propionyl-CoA, one of the metabolic intermediates of propionate, while propionate itself did not inhibit the enzyme. This suggests that the inhibitor of the growth in vivo is not propionate but propionyl-CoA. The inhibition by propionyl-CoA was competitive with respect to coenzyme A concentration. The K1 value for propionyl-CoA was 0.84 mM. Addition of NaHCO3, which restored the growth of this bacterium in the presence of propionate, increased the rate of propionate incorporation by 1.7-fold and decreased the intracellular level of propionyl-CoA by half. These findings suggest that HCO3-ion lowers the level of propionyl-CoA by accelerating its carboxylation reaction, which is catalyzed by propionyl-CoA carboxylase. Effects of NaHCO3 and acetate on the growth restoration were also studied by the use of propionyl-CoA carboxylase-deficient mutants. NaHCO3 did not restore the growth of the mutants, indicating an essential role of propionyl-CoA carboxylase on the restoration of growth by NaHCO3 as suggested above. Addition of acetate restores the growth of the mutants in the presence of propionate. Acetate probably restores the growth by supplying acetyl-CoA.     

Changes of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Activity in Stressed Pinus Sylvestris Needles

Stimulation of ethylene biosynthesis in pine needles by hydrogen peroxide and sodium bisulfite coincided with the activation of ACC oxidase at the level of protein synthesis. Decrease in ethylene production at high concentrations of sodium bisulfite (above 7 mM) was apparently due to inhibition of ACC oxidase activity. Treatment of pine needles with aminotriazole caused an inhibition of both ethylene production and ACC oxidase activity. Both methylviologen and methyl jasmonate stimulated ACC oxidase activity in a concentration-dependent manner with no parallel changes in ethylene production. The presented results suggest that ACC oxidase plays an important role in regulation of ethylene formation in pine needles in response to different stimuli.     

Microbial conversion of distillery waste to bioenergy: Effects of media enrichment with low chain fatty acids and Candida sp.

During microbial methanogensis of diluted distillery waste or spent wash (initial COD 25,000 mg/l) in the presence of sodium acetate, sodium propionate, or sodium butyrate at the concentration of 2000 mg/l, biogas containing 22.0% to 39.4% methane was produced in 20 days in a semicontinuous fermentation system. The analysis of the volatile fatty acid spectrum of the effluent showed accumulation of 46.3% branched chain fatty acids. When the fermentation medium was supplemented with modified Smith and Mah (SM) medium containing electrolytes and 1% sodium acetate, production of methane went up to 59.0% in 18 days. With the addition of a strain of Candida sp. the coculture produced about the same volume of methane (61%) but the time required was reduced (14 days). COD (16600.8 mg/l) was reduced and the effluent contained only 3% branched chain fatty acids adn 96% straight chain fatty acids. Fortification with the sodium salts of three branched-chain fatty acids as sources of carbon in SM medium (alone) reduced methane production. Only 0.0% to 14.2% methane was recorded. By combining these acids with straight chain fatty acids in SM medium, methane production increased significantly (about 4-fold) in proportion to the concentration of straight chain fatty acids added.     

Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis

Acetate kinase was purified 102-fold to a specific activity of 656 mumol of ADP formed/min/mg of protein from acetate-grown Methanosarcina thermophila. The enzyme was not intrinsically membrane bound. The native enzyme (Mr 94,000) was an alpha 2 homodimer with a subunit Mr of 53,000. The activity was optimum between pH 7.0 and 7.4. A pI of 4.7 was determined. The enzyme was stable to O2 and stable to heating at 70 degrees C for 15 min but was rapidly inactivated at higher temperatures. The apparent Km for acetate was 22 mM and for ATP was 2.8 mM. The enzyme phosphorylated propionate at 60% of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. The enzyme required one of several divalent cations for activity; the maximum rate was obtained with Mn2+. Western blots of cell extract proteins showed that acetate grown cells synthesized higher quantities of the acetate kinase than did methanol grown cells.     

Synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli

Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc.     

Seed Dormancy in Red Rice (Oryza sativa) (IX. Embryo Fructose-2,6-Bisphosphate during Dormancy Breaking and Subsequent Germination)

Fructose-2,6-bisphosphate (Fru-2,6-bisP) was evaluated as a potential marker for the dormancy-breaking phase or the germination phase before pericarp splitting in red rice (Oryza sativa). During 4 h of imbibition at 30[deg]C, Fru-2,6-bisP of dehulled dormant and nondormant seeds increased to 0.26 and 0.38 pmol embryo-1, respectively. In nondormant seeds, embryo Fru-2,6-bisP content remained stable until the onset of pericarp splitting (12 h) and increased rapidly thereafter. In dormant seeds, Fru-2,6-bisP declined to 0.09 pmol embryo-1 at 24 h. Embryo Fru-2,6-bisP was correlated with O2 uptake of dormant and nondormant seeds. A 24-h exposure of dehulled, water-imbibed, dormant seeds to treatments yielding >90% germination (sodium nitrite [4 mM], propionic acid [22 mM], methyl propionate [32 mM], propanol [75 mM], and propionaldehyde [40 mM]) led to changes in embryo Fru-2,6-bisP that were unrelated to the final germination percentages. Furthermore, a 2-h pulse of propionaldehyde increased Fru-2,6-bisP 4-fold but did not break dormancy. Whereas nitrite and propionaldehyde increased Fru-2,6-bisP to 0.33 pmol embryo-1 after 2 h of contact, propionic acid and methyl propionate did not increase Fru-2,6-bisP above the untreated control. In all cases, further increases in Fru-2,6-bisP occurred after pericarp splitting. However, the plateau Fru-2,6-bisP attained during chemical contact was inversely correlated with elapsed time to 30% germination (r = -0.978). Therefore, although Fru-2,6-bisP is not a universal marker for dormancy release, its rapid increase during nitrite and propionaldehyde treatments suggests that events associated with dormancy breaking can occur within 2 h of chemical treatment.     

Effect of Environmental Parameters on Bacterial Degradation of Bunker C Oil, Crude Oils, and Hydrocarbons

Mixed microbial cultures, previously enriched on Bunker C fuel oil, grew on and degraded Bunker C fuel oil at temperatures ranging from 5 to 28 C. At 15 C, 41 to 85% of the benzene-soluble components of Bunker C disappeared after incubation for 7 days; at 5 C the values ranged from 21 to 52% after 14 days of incubation. A Nocardia sp. isolated from a culture enriched on Bunker C oil grew on Venezuelan crude oil, Bunker C, hexadecane, and a hydrocarbon mixture at temperatures of 5 and 15 C. The 10-C decrease in temperature resulted in an average 2.2-fold decrease in generation time of the bacteria. Gas-liquid chromatographic measurements of Venezuelan and Arabian crude oils which had been incubated with the Nocardia sp. showed significant degradation of the n-alkane portion and the chromatographically unresolved components of the oils. The concentration of elemental nitrogen required to bring about the disappearance of 1 mg of hexadecane by the Nocardia sp. was 0.5 mg. The results confirm suggestions that the rate of natural biodegradation of oil in marine temperate-to-polar zones is probably limited by low temperatures and phosphorus concentrations, but suggest that the concentrations of nitrogen occurring naturally are probably not rate-limiting factors.