Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.     

Production of 3-hydroxy-γ-decalactone, the precursor of two decenolides with flavouring properties, by the yeast Yarrowia lipolytica

3-Hydroxy-γ-decalactone is the precursor of dec-2 and dec-3-en-4-olides which are valuable aroma compounds not yet produced. To promote the accumulation of this lactone, the yeast Yarrowia lipolytica was placed in different environmental conditions aiming at altering β-oxidation fluxes. The concentration of substrate, pH, aeration and dissolved oxygen level were modified. We observed an important accumulation at low aeration (0.40 molar yields) and, to a lesser extent, at lower pH (0.15). As oxygen played a key-role, we evaluated its effect at fixed dissolved oxygen and at the pH which was the most favourable to the biotransformation (pH 4.5). At 5% and 30% dissolved oxygen, yields reached 0.50. β-Oxidation fluxes are very dependent on the presence of oxygen and conditions of accumulation of 3-hydroxy-γ-decalactone with very high yields were identified. These results are an important step in the production of the two decenolides. Moreover, they show the high dependence of β-oxidation fluxes on environmental conditions and relate these conditions to the accumulation of intermediates, results that are of interest to all the processes using yeast on lipids or alkanes.     

Sesquiterpene lactones from Algerian Artemisia herba-alba

The phytochemical investigation of the methylene chloride/methanol extract of the aerial parts of Artemisia herba-alba afforded two new natural sesquiterpene lactones 1β,9β-diacetoxyeudesm-3-en-5α,6β,11βH-12,6-olide (1) and 1β,9β-diacetoxyeudesm-4-en-6β,11βH-12,6-olide (2). The structures of the compounds were determined by comprehensive NMR studies, including DEPT, COSY, NOESY, HMQC, HMBC and HRMS.     

Microbial transformations of gelomulide G: a member of the rare class of diterpene lactones

Microbial transformation of gelomulide G (3beta,6beta-diacetoxy-8beta,14beta-epoxyabiet-13(15)-en-16,12-olide, 1) was carried out. Incubation of 1 with Aspergillus niger afforded two new metabolites, 3beta,6beta-diacetoxy-8beta,14beta-dihydroxyabiet-13(15)-en-16,12-olide (2) and 3beta,6beta-diacetoxy-14beta-hydroxyabieta-8(9),13(15)-dien-16,12-olide (3). While Cunninghamella elegans afforded the 14-epimer of 2, i.e., 3beta,6beta-diacetoxy-8beta,14alpha-dihydroxyabiet-13(15)-en-16,12-olide (4), along with 3beta-acetoxy-6beta-hydroxy-8beta,14beta-epoxyabiet-13(15)-en-16,12-olide (5). The structures of the transformed products 2-5 were deduced to be new on the basis of MS and NMR data.     

Optimization of culture conditions for production of antimalarial menisporopsin A by the seed fungus Menisporopsis theobromae BCC 4162

AIMS: The aim of this work was to optimize the production of a novel antimaralial menisporopsin A by the seed fungus Menisporopsis theobromae BCC 4162. METHODS AND RESULTS: Fungal cultures were grown in shake flasks at 25 degrees C in the basal medium with varying carbon and nitrogen sources, aeration rates and initial pH levels. The optimal carbon and nitrogen sources that improved the production of menisporopsin A were 1% fructose and 2.5% meat extract respectively. The production was further enhanced when the culture incubated on a shaker at 200 rev min(-1) with an initial pH of 8. The yield of menisporopsin A cultured under the optimized conditions was increased from 348.30 (obtained from basal medium) to 889.02 mg l(-1), and the cultivation time was reduced from 28 to only 4 days. As a result, the productivity of menisporopsin A was greatly enhanced to 222.26 mg l(-1) day(-1) which is 18-fold higher than that of basal conditions. Larger scale production in a fermenter was also achieved, yielding menisporopsin A at a maximal level of 594.32 mg l(-1) in 4 days. CONCLUSIONS: The optimized culture conditions for menisporopsin A production by M. theobromae BCC 4162 was the cultivation under shaking or agitation at 25 degrees C in fructose-meat extract medium with an initial pH of 8. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of menisporopsin A in a fermenter with a relatively short incubation period could be valuable for further utilization for chemical structure modification and derivatization.     

Volatiles released by a Streptomyces species isolated from the North Sea

The North Sea Streptomyces strain GWS-BW-H5 was investigated by analyzing headspace extracts of agar-plate cultures (HE) or liquid cultures (LCE), obtained with a closed-loop stripping apparatus (CLSA), by GC/MS (Table 1). The volatile profile of the HE is dominated by the known volatiles (-)-geosmin (4) and 2-methyisoborneol (1). Small amounts of sesquiterpenes occur, which are present in a more-diverse structural variety and in higher quantities in the LCE. The different structures can be rationalized by few cationic intermediates along their biosynthetic pathway. The most-prominent difference between the two culture methods were the presence of the Me-branched gamma- and delta-lactones 31-38, not previously reported from nature, in the LCE. Major components were 10-methyldodecan-5-olide (34), 10-methyldodec-2-en-4-olide (36), and 10-methyldodec-3-en-4-olide (38). The structures of all new lactones were verified by synthesis. Furthermore, more volatiles in higher amounts were produced by the liquid culture than by to the agar plate culture. Since 36 showed inhibitory growth effects against strain GWS-BW-H5, growth inhibition against twelve other strains isolated from the same habitat was tested. Antagonistic activity against four of the strains was observed, with a slightly higher threshold level than found for penicillin G, which was used in control experiments (Table 2).     

Cytotoxic versus anti-inflammatory effects in HeLa,Jurkat T and human peripheral blood cells caused by guaianolide-type sesquiterpene lactones

Four guaianolide type sesquiterpene lactones (SL), namely the new 1,2-dihydro-3-oxo-costic acid guaianyl ester 3beta-O-(1,2-didehydro-3-oxo-costoyloxy)-4beta,10beta-dihydroxy-guaia-1(2)-en-6beta,12-olide (1) and 3beta-O-(1,2-didehydro-3-oxo-costoyloxy)-4beta,10beta-dihydroxy-guaia-1(2)-en-6alpha,12-olide (2), as well as the known moroccolide A [5alphaH-2beta,4-epoxy-3beta-hydroxy-guaia-1(10),11(13)-dien-6beta,12-olide, 3] and 3beta-O-(2-methylbutyryl)-moroccolide A [5alphaH-2beta,4-epoxy-3beta-(2-methylbutyryloxy)-guaia-1(10),11(13)-dien-6beta,12-olide, 4] were examined for their cytotoxic and anti-inflammatory effects in HeLa, Jurkat T and human peripheral blood mononuclear cells. Compounds 1, 2 and 4 were found to exert a strong cytotoxicity similar in potency in all investigated cell types, whereas 3 was significantly less active. Along with the cytotoxic effect compounds 1 and 4 showed a potent and comparable down-regulation of the mRNAs of the house-keeping genes beta-actin and GAP-DH in PBMCs after 20 h. In contrast, the down-regulation of the PMA-induced mRNA levels of the NF-kappaB-driven pro-inflammatory genes IL-2, IL-6, GM-CSF, TNF-alpha, and IL-1beta in PBMCs is significantly stronger with compound 4. Compound 3 did not significantly modulate cytokine mRNAs levels at biochemically relevant concentrations. The electromobility shift assay (EMSA), revealed a stronger inhibition of NF-kappaB for 1 (IC(50) 2.5 microM) than for 4 (IC(50) 5 microM). Both compounds were also subjected to an IL-6 luciferase reporter gene assay and showed IC(50) values of 1.0 (1) and 1.2 microM (4). Thus, the NF-kappaB inhibition measured by EMSA, as well as the IL-6 luciferase assay did not reflect the differential modulation of pro-inflammatory genes measured with RT-rt-PCR.     

Decalactone Production by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> under increased O<Subscript>2</Subscript> Transfer Rates

Yarrowia lipolytica converts methyl ricinoleate to γ-decalactone, a high-value fruity aroma compound. The highest amount of 3-hydroxy-γ-decalactone produced by the yeast (263 mg l^-1) occurred by increasing the k_La up to 120 h⁻¹ at atmospheric pressure; above it, its concentration decreased, suggesting a predominance of the activity of 3-hydroxyacyl-CoA dehydrogenase. Cultures were grown under high-pressure, i.e., under increased O₂ solubility, but, although growth was accelerated, γ-decalactone production decreased. However, by applying 0.5 MPa during growth and biotransformation gave increased concentrations of dec−2-en-4-olide and dec-3-en-4-olide (70 mg l⁻¹).     

β-Methylmuconolactone, a key intermediate in the dissimilation of methylaromatic compounds by a modified 3-oxoadipate pathway evolved in nocardioform actinomycetes

Abstract p -Toluate-grown cells of Rhodococcus ruber N75, R. corallinus N657, R. rhodochrous N5 and Rhodococcus strains BCN1, BCN2 and 4PH1 metabolized 4-methylcatechol by a modified 3-oxoadipate pathway. Steps in the conversion of this compound to 4-methyl-3-oxoadipic acid were investigated. The conversion of 4-carboxymethyl-3-methylbut-2-en-1, 4-olide to 4-carboxymethyl-3-methylbut-2-en-1, 4-olide by a new enzyme is described.     

Mechanisms underlying the toxicity of lactone aroma compounds towards the producing yeast cells

AIMS: To study the fundamental mechanisms of toxicity of the fruity aroma compound gamma-decalactone, that lead to alterations in cell viability during its biotechnological production by yeast cells; Yarrowia lipolytica that is able to produce high amounts of this metabolite was used here as a model. METHODS AND RESULTS: Lactone concentrations above 150 mg l-1 inhibited cell growth, depolarized the living cells and increased membrane fluidity. Infrared spectroscopic measurements revealed that the introduction of the lactone into model phospholipid bilayers, decreased the phase transition temperature. Moreover, the H+-ATPase activity in membrane preparations was strongly affected by the presence of the lactone. On the other hand, only a slight decrease in the intracellular pH occurred. CONCLUSIONS: We propose that the toxic effects of gamma-decalactone on yeast may be initially linked to a strong interaction of the compound with cell membrane lipids and components. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings may enable the elaboration of strategies to improve yeast cell viability during the process of lactones bioproduction.     

离舌橐吾中艾里莫酚烷型倍半萜类化学成分的研究

为分析菊科橐吾属植物离舌橐吾Ligularia veitchiana(Hemsl.)Greenm中艾里莫酚烷型倍半萜类的化学成分,并对其进行抗肿瘤活性研究,实验综合运用硅胶柱色谱、反相硅胶柱色谱以及制备型高效液相等色谱方法,从其根茎的95%乙醇提取物的乙酸乙酯部位中分离得到了13个艾里莫酚烷型倍半萜类化合物,根据化合物的理化性质及其波谱学数据鉴定为:eremophilenolide(1),eremophila-7(11),9-dien-8-one(2),eremophil-6-en-11-ol(3),8-oxo-eremophil-6-en-11-one(4),(6α,8α)-6-hydroxyeremophil-7(11)-en-12,8-olide(5),8β-hydroxyeremophil-7(11)-en-12,8α-olide(6),6β-hydroxy-8α-methoxyeremophil-7(11)-12,8β-olide(7),2α-hydroxyeremophil-11-en-9-one(8),6β-methoxy-8β-hydroxyeremophil-7(11)-en-12,8α-olide(9),6β-hydroxyeremophil-7(11)-en-12,8β-olide(10),6β-hydroxy-8β-methoxyeremophil-7(11)-12,8α-olide(11), 6β,8β-dihydroxyeremophil-7(11)-en-12,8α-olide(12)和6β,8α-dihydroxyeremophil-7(11)-en-12,8β-olide(13)。其中,化合物5和10、7和11～13为三对非对映异构体。除化合物3和5外,所有化合物均为首次从该植物中分离得到。运用MTT法对所有化合物进行体外抗肿瘤细胞活性的筛选,结果表明其对胃癌细胞HGC-27和宫颈癌细胞Caski均未显示细胞毒作用。     

Short-cut nitrification of domestic wastewater in a pilot-scale A/O nitrogen removal plant

The feasibility of nitrite accumulation in a pilot-scale A/O (anoxic/oxic) nitrogen removal plant treating domestic wastewater was investigated at various dissolved oxygen (DO) concentrations and pH levels. The results showed that the pH was not a useful operational parameter to realize nitrite accumulation. Significant nitrite accumulation was observed at the low DO concentration range of 0.3–0.8 mg/l and the maximum nitrite accumulation ratio of about 90% occurred at a DO concentration of 0.6 mg/l. This suggests a reduction of 22% in the oxygen consumption, and therefore a considerable saving in aeration. However, the nitrite accumulation was destroyed at the high DO concentration and the resumption was very slow. In addition, the average ammonia removal efficiency reached as high as 93% at the low DO level. Moreover, experimental results indicated that nitrogen could be removed by simultaneous nitrification and denitrification (SND) via nitrite in the aerobic zones at the low DO concentration, with the efficiency of 6–12%.     

Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.Abbreviations 3CB 3-chlorobenzoate - 4CB 4-chlorobenzoate - 3,5DCB 3,5-dichlorobenzoate - 2,4D 2,4-dichlorophenoxyacetate - NOE Nuclear-Overhauser-Effect     

Empirical model for the autotrophic biodegradation of thiocyanate in an activated sludge reactor

AIMS: The aim of this investigation was to develop an empirical model for the autotrophic biodegradation of thiocyanate using an activated sludge reactor. METHODS AND RESULTS: The methods used for this purpose included the use of a laboratory scale activated sludge reactor unit using thiocyante feed concentrations from 200 to 550 mg x l(-1). Reactor effluent concentrations of <1 mg x l(-1) thiocyanate were consistently achieved for the entire duration of the investigation at a hydraulic retention time of 8 h, solids (biomass) retention of 18 h and biomass (dry weight) concentrations ranging from 2 to 4 g x l(-1). A biomass specific degradation rate factor was used to relate thiocyanate degradation in the reactor to the prevailing biomass and thiocyanate feed concentrations. A maximum biomass specific degradation rate of 16 mg(-1) x g(-1) x h(-1) (mg thiocyanate consumed per gram biomass per hour) was achieved at a thiocyanate feed concentration of 550 mg x l(-1). The overall yield coefficient was found to be 0.086 (biomass dry weight produced per mass of thiocyanate consumed). CONCLUSION: Using the results generated by this investigation, an empirical model was developed, based on thiocyanate feed concentration and reactor biomass concentration, to calculate the required absolute hydraulic retention time at which a single-stage continuously stirred tank activated sludge reactor could be operated in order to achieve an effluent concentration of <1 mg x l(-1). The use of an empirical model rather than a mechanistic-based kinetic model was proposed due to the low prevailing thiocyanate concentrations in the reactor. SIGNIFICANCE AND IMPACT OF THE STUDY: These results represent the first empirical model, based on a comprehensive data set, that could be used for the design of thiocyanate-degrading activated sludge systems.     

Modeling and optimization of poly(3hydroxybutyrate-co-3hydroxyvalerate) production from cane molasses by Azohydromonas lata MTCC 2311 in a stirred-tank reactor: effect of agitation and aeration regimes

The effects of agitation and aeration rates on copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production by Azohydromonas lata MTCC 2311 using cane molasses supplemented with propionic acid in a bioreactor were investigated. The experiments were conducted in a three-level factorial design by varying the impeller (150-500 rev min(-1)) and aeration (0.5-1.5 vvm) rates. Further, the data were fitted to mathematical models [quadratic polynomial equation and artificial neural network (ANN)] and process variables were optimized by genetic algorithm-coupled models. ANN and hybrid ANN-GA were found superior for modeling and optimization of process variables, respectively. The maximum copolymer concentration of 7.45 g l(-1) with 21.50 mol% of 3HV was predicted at process variables: agitation speed, 287 rev min(-1); and aeration rate, 0.85 vvm, which upon validation gave 7.20 g l(-1) of P(3HB-co-3HV) with 21 mol% of 3HV with the prediction error (%) of 3.38 and 2.32, respectively. Agitation speed established a relative high importance of 72.19% than of aeration rate (27.80%) for copolymer accumulation. The volumetric gas-liquid mass transfer coefficient (k (L) a) was strongly affected by agitation and aeration rates. The highest P(3HB-co-3HV) productivity of 0.163 g l(-1) h(-1) was achieved at 0.17 s(-1) of k (L) a value. During the early phase of copolymer production process, 3HB monomers were accumulated, which were shifted to 3HV units (9-21%) during the cultivation period of 24-42 h. The enhancement of 7.5 and 34% were reported for P(3HB-co-3HV) production and 3HV content, respectively, by hybrid ANN-GA paradigm, which revealed the significant utilization of cane molasses for improved copolymer production.     

Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures

AIMS: To study the effect of sugars and sugar mixtures on the growth kinetics of Oenococcus oeni NCIMB 11648 in batch culture with the aim of producing a high cell productivity system for starter cultures. METHODS AND RESULTS: The growth of O. oeni was investigated on single sugars (glucose, fructose or sucrose) and their mixtures (glucose-fructose, glucose-sucrose or fructose-sucrose). Better growth was obtained on sugar mixtures compared with growth on a single sugar. The production system of O. oeni biomass was investigated in batch culture with or without pH control with respect to kinetics, specific growth rate and biomass yield. The effect of pH and substrate concentration on fermentation balances and ATP yield were determined. The optimal growth of O. oeni was achieved on the glucose-fructose mixture (9 g l(-1), 1 : 1) at pH 4.5 and 25 degrees C with pH control, with highest cell volumetric productivity (7.9 mg cell l(-1) h(-1)), biomass yield (0.041 g cell g(-1) sugar) and specific growth rate (0.066 h(-1)). CONCLUSIONS: The limitations to the growth of O. oeni were pH and inhibition by end product resulting in poor utilization of the medium with low cell yields. The cell productivity of the system can be improved by the appropriate use of mixed sugar growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uniquely showed that appropriate sugar mixtures with the correct environmental conditions can significantly improve the productivity of O. oeni cultures.     

Humirianthenolides,new degraded diterpenoids from Humirianthera rupestris

The tuber of Humirianthera rupestris (Icacinaceae) contains the degraded diterpenoids 3β,20-epoxy-30α- hydroxy- 14-oxo-9β-podocarpan-19,6β-olide (humirianthenolide A), 3β,20-epoxy-3α,14α-dihydroxy-9β-podocarpan-19,6β- olide (humirianthenolide B), 3β,20; 16,14-diepoxy-3α-hydroxy-17-nor-15-oxo-9β-abiet-13-en-19,6β-olide (humirianthenolide C), 3β,20-epoxy-3α,14-dihydroxy-13-oxo-9β-podocarp-8(14)-en-19,6β-olide (humirianthenolide D), 3β,20-epoxy-3α-hidroxy-14-oxo-8α,9β-podocarpan-19,6β-olide (humirianthenolide E) and 3β,20-epoxy-3α,14β- dihydroxy-8α,9β-podocarpan-19,6β-olide (humirianthenolide F). ¹H NMR and ¹³C NMR spectroscopy were efrective for the determination of the humirianthenolide structures.     

Purification and characterization of 4-methylmuconolactone methyl-isomerase, a novel enzyme of the modified 3-oxoadipate pathway in nocardioform actinomycetes.

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.     

Modified ortho-cleavage pathway in Alcaligenes eutrophus JMP134 for the degradation of 4-methylcatechol

Abstract 2,4-Dichlorophenoxyacetate-grown cells of Alcaligenes eutrophus JMP134 [1] metabolized 4-methylphenoxyacetate via a modified ortho -cleavage pathway. 4-Carboxymethyl-4-methylbut-2-en-1,4-olide (4-methyl-2-enelactone), 4-carboxymethyl-3-methylbut-2-en-1,4-olide (3-methyl-2-enelactone) and 4-methyl-3-oxoadipate, were identified as intermediates.     

Two sesquiterpene lactones from Trichogonia gardneri

The structures and stereochemistries of two sesquiterpene lactones from Trichogonia gardneri were established as (6R,7S,8S,9S,IOR)-4E-9,10-dihydroxy-8-tigloxygermacr-4-en-6,12-olide) and (5R*,6R*,7S*,8S*,9R*)-14-acetoxy-3-chloro-9-hydroxy-2-oxo-8-tigloxyguia-1(10),3-dien-6,12-of olide by a combination of NMR spectrometry and X-ray diffraction. The results show that the structures of several sesquiterpene lactones which were isolated previously from related species require revision.