Aprotic polar solvents inducing chromosomal malsegregation in yeast interfere with the assembly of porcine brain tubulin in vitro

A number of aprotic solvents which had previously been found to induce mitotic aneuploidy in yeast were tested for their effects on re-assembly of twice recycled tubulin from pig brain. Some of the solvents which were strong aneuploidy-inducing mutagens in yeast slowed down tubulin assembly in vitro at concentrations lower than those required for aneuploidy induction. Ethyl acetate, methyl acetate, diethyl ketone and acetonitrile fell into this category. Other strong aneuploidy-inducing agents like acetone and 2-methoxyethyl acetate accelerated tubulin assembly. Non-genetically active methyl isopropyl ketone and isopropyl acetate both accelerated assembly, whereas methyl n-propyl ketone and n-propyl acetate were weak inducers of aneuploidy and slowed down the rate and extent of assembly. Those chemicals which slowed down the assembly rate also reduced the extent of assembly. Most chemicals which accelerated assembly also led to an increased extent of assembly, with the exception of isopropyl acetate. At the higher concentrations, however, a maximum assembly rate was reached which was followed by a slow decline. Although a perfect correlation between effects on the induction of chromosomal malsegregation and the interference with tubulin assembly in vitro was not seen, the experiments with tubulin were carried out using this class of chemicals because some of them strongly induced mitotic aneuploidy under conditions which suggested tubulin to be the prime target. The lack of a perfect coincidence might be due to species differences between the porcine brain and the yeast spindle tubulin, or the test for aneuploidy induction may have been negative because the concentrations required for an effect on yeast tubulin may be greater than the general lethal toxicity limit. Bearing this reservation in mind, the results suggest that the yeast aneuploidy test has a considerable predictive value for mammalian mutagenicity.     

Reaction of yeast carboxypeptidase C1 with group-specific reagents.

The reactions between yeast carboxypeptidase C and the group-specific reagents, phenylglyoxal and iodoacetamide, have been studied in detail and the reactions of residue at the active site with N-tosyl-L-phenylalanine chloromethyl ketone and diisopropyl phosphorofluoridate have been confirmed. Modification of the enzyme by either phenylglyoxal or iodoacetamide results in the loss of peptidase activity, while esterase activity remains unchanged. Inactivation by phenylglyoxal appears to be the result of the modification of a single arginine residue, whereas inhibition by iodoacetamide can be correlated with the modification of a single methionine residue. Inactivation of the enzyme by either N-tosyl-L-phenylalanine chloromethyl ketone or diisopropyl phosphorofluoridate is the result of the modification of a single histidine and a single serine residue, respectively. The pattern of inhibition indicates certain analogies in the mechanism of yeast carboxypeptidase C to pancreatic chymotrypsin, on the one hand, and to carboxypeptidase A, on the other.     

Microbial production of natural raspberry ketone

Raspberry ketone is an important compound for the flavour industry. It is frequently used in products such as soft drinks, sweets, puddings and ice creams. The compound can be produced by organic synthesis. Demand for "natural" raspberry ketone is growing considerably. However, this product is extremely expensive. Consequently, there is a remaining desire to better understand how raspberry ketone is synthesized in vivo, and which genes and enzymes are involved. With this information we will then be in a better position to design alternative production strategies such as microbial fermentation. This article focuses on the identification and application of genes potentially linked to raspberry ketone synthesis. We have isolated candidate genes from both raspberry and other plants, and these have been introduced into bacterial and yeast expression systems. Conditions have been determined that result in significant levels of raspberry ketone, up to 5 mg/L. These results therefore lay a strong foundation for a potentially renewable source of "natural" flavour compounds making use of plant genes.     

Genetic change may be caused by interference with protein-protein interactions

Several aprotic polar solvents were shown to induce mitotic aneuploidy in yeast: diethyl ketone, γ-valerolactone, pyridine, pivalinic acid nitrile, phenylacetonitrile and fumaric acid dinitrile. Only fumaric acid dinitrile also strongly induced other types of genetic effects including mitotic crossing-over, mitotic gene conversion and point mutation. The other substances only induced aneuploidy and this only over a very narrow dose range.

The treatment protocol used suggested that these chemicals acted via interference with tubulin assembly and disassembly causing a malfunctioning of spindle fiber microtubules. This hypothesis was tested using twice recycled porcine brain tubulin. Diethyl ketone, γ-valerolactone, pyridine and phenylacetonitrile inhibited GTP-promoted assembly of porcine brain tubulin in vitro in the concentration range needed for the induction of mitotic aneuploidy in yeast. Pivalinic acid nitrile accelerated tubulin aggregation whereas fumaric acid dinitrile had no effect even at concentrations 18 times higher than the lowest tested concentration effective in yeast.

The in vitro experiments with porcine brain tubulin further suggest that genetic change can result from interference with specific protein-protein interactions. Fumaric acid dinitrile was the only exception since it did induce aneuploidy but had no effects on the assembly of porcine brain tubulin. This could be caused either by interference with protein-protein interactions other than between molecules during assembly and disassembly of microtubules or species-specific differences in susceptibility between yeast spindle and porcine brain tubulin.     

Hydrogen transfer pathways of the asymmetric reduction of alpha,beta-unsaturated ketone mediated by baker's yeast

The hydrogen transfer mechanism of cofactor reduction and recycling processes in the yeast reduction of alpha,beta-unsaturated ketone was studied by using quantitative isotope tracing close to natural abundance measured by (2)H NMR. In the reaction, the active cofactor is NADPH. The cofactor-transferred hydride attacks the beta sp(2) carbon of the enone carbonyl while water hydrogen is transferred to the alpha position. The reductant involved in the reaction depends on the quantity of yeast. When the amount of yeast is very large, the enzymes use preferentially certain unidentified substance stored in the yeast cells rather than the added glucose as electron donor. In this case, the hydrogen transferred by the cofactor is mainly of water origin. When the yeast amount is low, the added glucose is more efficiently used by the enzymes as electron donor and its hydrogen atoms bound to C-1 and C-3 are delivered to the substrate.     

Functional studies of aldo-keto reductases in Saccharomyces cerevisiae

We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast.     

Aneuploidy-inducing chemicals in yeast evaluated by the micronucleus test

Chinese hamsters were exposed to acetone, methyl ethyl ketone, ethyl acetate and 2-methoxy ethyl acetate, known to be strong inducers of aneuploidy in the yeast Saccharomyces cerevisiae. All solvents yielded negative results in the micronucleus test, whereas the vinca alkaloid vindesine--used as a positive control substance--proved to act as a spindle poison in mammals in vivo.     

Next issue (October 2007): Multi-enzyme Systems for the Synthesis of Complex Natural Compounds

Edited by Dr. Stefan Martens, Marburg, Germany Highlight articles: – Flavonoids and other phenylpropanoid-derived natural products – Glucosylation of phenolic compounds using cultured plant cells – Ketocarotenoid biosynthesis in tobacco – Microbial production of natural raspberry ketone – A factorial approach to lactone production in yeast – Fermentative production of methylated stilbens – Biosynthesis of deoxyflavanones ... and much more: Read the next issue of BTJ!     

Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevisiae

A diploid yeast strain D61.M was used to study induction of mitotic chromosomal malsegregation, mitotic recombination and point mutation. Several ketones (including acetone and methyl ethyl ketone) and some organic acid esters (including the methyl, ethyl and 2-methoxyethyl esters of acetic acid) and acetonitrile strongly induced aneuploidy but not recombination or point mutation. Only diethyl ketone induced low levels of recombination and point mutation in addition to aneuploidy. Related compounds were weak inducers of aneuploidy: methyl n-propyl ketone, the methyl esters of propionic and butyric acid, acetic acid esters of n- and iso-propanol and ethyl propionate. No mutagenicity was found with n-butyl and isoamyl acetate, ethyl formate, acetyl acetone (2,5-dipentanone) and dioxane. Methyl isopropyl ketone induced only some recombination and point mutation but no aneuploidy. Efficient induction was only observed with a treatment protocol in which growing cells were exposed to the chemicals during a growth period of 4 h at 28 degrees C followed by incubation in ice for more than 90 min, usually overnight for 16-17 h. Aneuploid cells could be detected in such cultures during a subsequent incubation at growth temperature if the chemical was still present. Detailed analysis showed that there was a high incidence of multiple events of chromosomal malsegregation. It is proposed that the mutagenic agents act directly on tubulin during growth so that labile microtubules are formed which dissociate in the cold. When cells are brought back to temperatures above the level critical for reassembly of tubulin and allowed to grow, faulty microtubules are formed.     

酚酮类对树干毕赤酵母乙醇发酵及脂肪酸组成的影响

木质素降解产物对微生物产生的抑制作用,是燃料乙醇生物炼制的主要瓶颈之一。本文以树干毕赤酵母为发酵菌株,研究木质素降解产物中3种酚酮类(4-羟基苯乙酮、4-羟基-3-甲氧基苯乙酮、4-羟基-3,5-二甲氧基苯乙酮)对其木糖乙醇发酵及酵母细胞脂肪酸组成的影响。采用高效液相色谱(HPLC)和气相色谱-质谱联用(GC/MS)技术对乙醇发酵性能和酵母细胞脂肪酸组成进行分析。研究结果表明,酚酮类物质对乙醇发酵呈现抑制作用且其分子量越低抑制作用越明显,当4-羟基苯乙酮浓度为1.50 g/L时,发酵24 h的木糖利用率、乙醇得率和乙醇浓度分别下降了42.47%、5.30%和9.76 g/L;培养基中存在酚酮类物质时,酵母细胞中的不饱和脂肪酸的比例上升,添加1.50 g/L的3种酚酮类物质后,树干毕赤酵母细胞不饱和脂肪酸和饱和脂肪酸的比例从原来的2.58分别上升到3.03、3.06和3.61,酵母细胞膜的流动性随之上升,不稳定性提高。因此,酚酮类物质能够降低酵母生长、提高不饱和脂肪酸的比例以及降低乙醇发酵能力,有效降低或去除木质素降解产物含量是提高木质纤维原料生物炼制的关键。     

The baker's yeast-mediated reduction of conjugated methylene groups in an organic solvent

The baker's yeast-mediated reduction of a series of conjugated methylene compounds was conducted in a petroleum ether reaction system. The methylene ketone, 3-phenyl-3-buten-2-one (1), was stereoselectively reduced to (R)-3-phenyl-2-butanone; no reduction of the ketone carbonyl was observed. Reduction of 2-phenyl-2-propenenitrile (7) also occurred stereoselectively to give (R)-2-phenylpropanenitrile (8) in good yield. The yeast mediated reduction of the methylene aldehyde, 2-phenyl-2-propenal (3), gave a mixture of products arising from reduction of both the methylene and carbonyl groups; stereoselective reduction of the methylene group gives (R)-2-phenylpropanal (6) which is rapidly reduced to (R)-2-phenyl-1-propanol (5), whereas reduction of the carbonyl gives 2-phenyl-2-propen-1-ol (4) which is slowly reduced to racemic (5).     

Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis)

(+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144 ± 10 μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases.     

Effects of chemical combinations on the induction of aneuploidy in Saccharomyces cerevisiae

Nocodazole, ethyl acetate, acetone and methyl ethyl ketone all are known to induce aneuploidy. Treatment of yeast strain D61.M with mixtures containing ineffective low levels of nocodazole and ineffective low levels of these solvents was highly effective in inducing aneuploidy. Ineffective low levels of nocodazole mixed with ineffective low levels of methyl 2-benzimidazolecarbamate also gave elevated frequencies of aneuploidy. Dimethyl formamide, a solvent that does not induce aneuploidy, mixed with low levels of nocodazole gave no increase in aneuploidy frequency above those levels seen in controls.     

优良菌种发酵葡萄酒优化条件的探讨

以诱变耐低温果酒酵母菌种YU2.28和产香酵母S15.3为发酵菌株,进行了葡萄酒发酵条件优化的试验研究.探讨了菌种生长温度、通氧量等因素,通过对菌种的生长情况和发酵醪液中总酯含量的变化分析,确定了自选酵母酿制葡萄酒的最佳技术参数,并对优化条件下发酵得到的葡萄酒进行GC/MS分析.结果显示：YU2.28和S15.3以1：3比例的混合发酵,接种量3%,调节醪液pH值为4.0,SO2添加量40 mg/L,发酵温度20℃,主发酵6 d内控制以230r/min的摇床转速进行摇瓶发酵,并进行9 h（每天1.5 h）供氧处理,后发酵30 d,酿造出的葡萄酒品质较佳,具有酒体丰盈,酒液澄清透亮,香气醇和的特征.成品酒香气成分共检测出醇类9种,酯类8种,酸类6种和少量的醛类、酮类等成分.     

Crystallographic binding studies with triosephosphate isomerases: conformational changes induced by substrate and substrate-analogues.

TIM catalyses the interconversion of a triosephosphate aldehyde into a triosephosphate ketone. This is a simple chemical reaction in which only protons are transferred. The crystallographic studies of TIM from chicken, yeast and trypanosome complexed with substrate and substrate analogues are discussed. The substrate binds in a deep pocket. On substrate binding, large conformational changes are induced in three loops. As a result of these conformational changes in the liganded structure, the active site pocket is sealed off from bulk solvent and the sidechain of the catalytic glutamate becomes optimally positioned for catalysis.     

Stereoselective biocatalytic hydride transfer to substituted acetophenones by the yeast Metschnikowia koreensis

Freely suspended and variously entrapped viable cells of the yeast Metschnikowia koreensis were examined for the asymmetric reduction of prochiral acetophenone. A ketone substrate at 25 mM can be converted (92%) to the corresponding alcohol within 3 h using freely suspended cells [46 mg/mL dry cell weight (DCW)] at pH 9 (Tris buffer, 50 mM), 25 °C, in an agitated reactor (200 rpm). The reaction displayed an excellent stereoselectivity of >99%. Supplementation of the reaction mixture with glucose (20 g/L) greatly enhanced the rate of the bioreduction reaction likely because of improved cofactor recycling in the cells. The cells could successfully reduce various acetophenones substituted with electron withdrawing groups on the phenyl ring, particularly at the para-position compared to ortho- or meta-substituted acetophenones. The ketone reductase of M. koreensis showed Prelog-selectivity as the reaction exclusively yielded (S)-alcohols. The thermostability and the substrate tolerance of the yeast were improved by immobilization in calcium alginate beads. Immobilization reduced the effectiveness factor only slightly.     

Chitin synthase in Candida albicans: comparison of digitonin-permeabilized cells and spheroplast membranes.

The treatment of Candida albicans (yeast form) with digitonin or dimethyl sulfoxide permeabilized cells and caused the activation of chitin synthase in situ. Endogenous activation was completely prevented by the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid); partially prevented by the protease inhibitors antipain, leupeptin, and N alpha-tosyl-L-lysyl chloromethyl ketone; and also partially prevented by EDTA. Thus, a clostripain-like protease may be involved in the endogenous activation phenomenon. The pH activity profile, cofactor requirements, and kinetic parameters of the endogenously activated chitin synthase were identical to those of the trypsin-activated enzyme in protoplast membranes.     

Purification and characterization of an NADH-dependent alcohol dehydrogenase from Candida maris for the synthesis of optically active 1-(pyridyl)ethanol derivatives

A novel (R)-specific alcohol dehydrogenase (AFPDH) produced by Candida maris IFO10003 was purified to homogeneity by ammonium sulfate fractionation, DEAE-Toyopearl, and Phenyl-Toyopearl, and characterized. The relative molecular mass of the native enzyme was found to be 59,900 by gel filtration, and that of the subunit was estimated to be 28,900 on SDS-polyacrylamide gel electrophoresis. These results suggest that the enzyme is a homodimer. It required NADH as a cofactor and reduced various kinds of carbonyl compounds, including ketones and aldehydes. AFPDH reduced acetylpyridine derivatives, β-keto esters, and some ketone compounds with high enantioselectivity. This is the first report of an NADH-dependent, highly enantioselective (R)-specific alcohol dehydrogenase isolated from a yeast. AFPDH is a very useful enzyme for the preparation of various kinds of chiral alcohols.     

Asymmetric bioreduction of a bisaryl ketone to its corresponding (S)-bisaryl alcohol, by the yeast Rhodotorula pilimanae ATCC 32762

The screening of 310 microbial strains yielded eight as suitable biocatalysts for the asymmetric bioreduction of a highly hindered bisaryl ketone to its corresponding alcohols. The production of both enantiomers with elevated optical purity (ee>96%) was achieved by different microorganisms. When scaling up the asymmetric bioreduction process in laboratory bioreactors (23 l scale), the production of preparative amounts (1.5 g) of the (S) enantiomer with elevated optically purity (ee>96%) was achieved when employing the yeast Rhodotorula pilimanae ATCC 32762. Achieving this asymmetric bioreduction with enantiocomplementarity in employing such a hindered substrate is remarkable and highlights the potential of such biological approach.     

Endogenous Siderophore 2,5-Dihydroxybenzoic Acid Deficiency Promotes Anemia and Splenic Iron Overload in Mice

Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.