Enoate reductases from non conventional yeasts: Bioconversion, cloning, and functional expression in Saccharomyces cerevisiae

Old yellow enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the stereoselective trans-hydrogenation of the double bond, representing a promising alternative to metal-based catalysis. Bioconversion of ketoisophorone (KIP) by 28 non-conventional yeasts belonging to 16 different species was investigated. Growing cells of most of the strains reduced KIP via OYE and showed high stereoselectivity, producing R-levodione as major product. Competition by carbonyl reductase (CR) activity was observed in several strains. The best performing yeasts belong to Candida castellii, Kazachstania spencerorum and Kluyveromyces marxianus exhibited yields of levodione ≥77% up to 95% e.e., and. Candida freyschussii, the sole strain lacking the OYE gene, reduced KIP only to unsaturated alcohols via CR. Nine unedited OYE genes were cloned, sequenced, and heterologously expressed in Saccharomyces cerevisiae BY4741ΔOye2, a mutant that showed negligible OYE and CR activities. Compared with the corresponding wild-type yeasts, growing cells of the recombinant strains bioconverted KIP with improved yields of OYE products, minor competition by CR activity, and lower enantioselectivity. In particular, resting cells of recombinant S. cerevisae presented the best performance in KIP bioconversion. Based on the results herein reported, selected strains of non-conventional yeasts and novel OYE genes can be profitably used as innovative biocatalysts in asymmetric reductions.     

Cloning and overexpression of the old yellow enzyme gene of Candida macedoniensis, and its application to the production of a chiral compound

The gene encoding old yellow enzyme (OYE), which catalyzes the conversion of ketoisophorone (KIP; 2,6,6-trimethyl-2-cyclohexen-1,4-dione) to (6R)-levodione (2,2,6-trimethylcyclohexane-1,4-dione), of Candida macedoniensis was cloned and sequenced. A 1212bp nucleotide fragment (oye) was confirmed to be the gene encoding OYE based on the agreement of internal amino acid sequences. Oye encodes a total 403 amino acid residues, and the deduced amino acid sequence shows a high degree of similarity to those of other microbial OYE family proteins. An expression vector, pETOYE, that contains the full length of oye was constructed. Escherichia coli harboring pETOYE exhibited an about six-fold increase in specific KIP-reducing activity under the control of the T7 promoter as compared with that of C. macedoniensis. (6R)-Levodione formed with washed cells of the transformant and a cofactor regeneration system amounted to 638 mM (98.2 mg ml(-1)), the a molar yield being 96.9%. The asymmetric reduction of KIP to (6R)-levodione with E. coli cells, which co-expressed both oye and the glucose dehydrogenase gene (gdh), as a catalyst was investigated. The (6R)-levodione formed amounted to 627 mM (96.6 mg ml(-1)), the a molar yield being 95.4%. Since the use of E. coli BL21 (DE3) cells co-expressing oye and gdh as a catalyst is simple and does not require the addition of glucose dehydrogenase, it is highly advantageous for the practical synthesis of (6R)-levodione.     

非传统酵母代谢工程研究进展

近30年来解脂耶氏酵母、克鲁维酵母、毕赤酵母、假丝酵母、汉逊酵母等非传统酵母因其具有天然的生理代谢优势,如快速生长、多底物利用、胁迫耐受性等,在代谢工程领域得到了广泛关注,多种基因工程改造工具正逐渐被开发用于非传统酵母的特性拓展,使其成为合成重组蛋白、生物可再生化学物质的高效细胞工厂.文中总结了非传统酵母中基因编辑工具...     

Old yellow enzymes, highly homologous FMN oxidoreductases with modulating roles in oxidative stress and programmed cell death in yeast

In a genetic screen to identify modifiers of Bax-dependent lethality in yeast, the C terminus of OYE2 was isolated based on its capacity to restore sensitivity to a Bax-resistant yeast mutant strain. Overexpression of full-length OYE2 suppresses Bax lethality in yeast, lowers endogenous reactive oxygen species (ROS), increases resistance to H(2)O(2)-induced programmed cell death (PCD), and significantly lowers ROS levels generated by organic prooxidants. Reciprocally, Delta oye2 yeast strains are sensitive to prooxidant-induced PCD. Overexpression and knock-out analysis indicate these OYE2 antioxidant activities are opposed by OYE3, a highly homologous heterodimerizing protein, which functions as a prooxidant promoting H(2)O(2)-induced PCD in wild type yeast. To exert its effect OYE3 requires the presence of OYE2. Deletion of the 12 C-terminal amino acids and catalytic inactivation of OYE2 by a Y197F mutation enhance significantly survival upon H(2)O(2)-induced PCD in wild type cells, but accelerate PCD in Delta oye3 cells, implicating the oye2p-oye3p heterodimer for promoting cell death upon oxidative stress. Unexpectedly, a strain with a double knock-out of these genes (Delta oye2 oye3) is highly resistant to H(2)O(2)-induced PCD, exhibits increased respiratory capacity, and undergoes less cell death during the adaptive response in chronological aging. Simultaneous deletion of OYE2 and other antioxidant genes hyperinduces endogenous levels of ROS, promoting H(2)O(2)-induced cell death: in Delta oye2 glr1 yeast high levels of oxidized glutathione elicited gross morphological aberrations involving the actin cytoskeleton and defects in organelle partitioning. Altering the ratio of reduced to oxidized glutathione by exogenous addition of GSH fully reversed these alterations. Based on this work, OYE proteins are firmly placed in the signaling network connecting ROS generation, PCD modulation, and cytoskeletal dynamics in yeast.     

Candida potacharoeniae sp. nov. and Candida spenceri sp. nov., two novel galactose-containing ascomycetous anamorphic yeast species isolated in Thailand

Fifteen strains of anamorphic yeasts isolated from various natural substrates collected in various places in Thailand were found to represent two novel species of anamorphic yeast genus Candida based on the sequence analysis of the D1/D2 domain of the large subunit rRNA genes, chemotaxonomic and conventional properties used for the classification of yeasts. These strains are located in the clade including Candida etchellsii and Candida magnoliae. Fourteen strains represented by ST-490(T) (BCC 15176(T)=NBRC 106439(T)= CBS 11674(T)) are closely related to Candida sorbosivorans in the D1/D2 sequences but 11 nucleotides (2.4%) were substituted. The remaining strain, ST-594(T) (=BCC 15278(T)=NBRC 106446(T)=CBS 11673(T)) showed a close relationship to Candida geochares but 21 nucleotides (4.7%) were substituted. Apparently, these strains represent two novel Candida species of the Starmerella clade. The two species are described as Candida potacharoeniae sp. nov. and Candida spenceri sp. nov. in the present paper. Like the most species of this clade, the two species contain galactose in the cells in addition to glucose and mannose and have high mol% G + C of 54.4-55.9 and 54.9, respectively.     

A comparison of phospholipase activity, cellular adherence and pathogenicity of yeasts

Phospholipase A and lysophospholipase activities were measured in the culture fluid and in the blastospores of Candida albicans. When phospholipase activity was measured in six yeasts (four strains of C. albicans and a single strain each of Candida parapsilosis and Saccharomyces cerevisiae) a correlation was found between this activity and two potential parameters of pathogenicity. The C. albicans isolates which adhered most strongly to buccal epithelial cells and were most pathogenic in mice had the highest phospholipase activities. Non-pathogenic yeasts, including C. albicans isolates which did not adhere and did not kill mice, had lower phospholipase activities.     

In vitro comparison of antifungal activity of oxiconazole and econazole against yeast.

Oxiconazole was compared in vitro with econazole in tests with 400 yeasts strains. Tests were performed by measurement of growth inhibition by both microdilution in Sabouraud broth and a Shadomy' agar diffusion systems. The sensitivity/resistance percentages were similar in Candida albicans strains. Oxiconazole showed a higher activity than that of econazole in Candida spp. and yeasts other than Candida.     

Distribution of L-azetidine-2-carboxylate N-acetyltransferase in yeast

The budding yeast Saccharomyces cerevisiae Sigma1278b contains the MPR1 gene encoding N-acetyltransferase, which detoxifies the L-proline analog L-azetidine-2-carboxylate (AZC). Of 131 yeasts tested, AZC acetyltransferase activity was detected in 17 strains of 41 strains that showed AZC resistance. Degenerate-PCR analysis revealed that two strains, i.e., Candida saitoana AKU4533 and Wickerhamia fluorescens AKU4722, contained a DNA fragment highly homologous to MPR1. This indicates that AZC acetyltransferases are widely distributed in yeasts.     

Carbohydrate and energy-yielding metabolism in non-conventional yeasts

Sugars are excellent carbon sources for all yeasts. Since a vast amount of information is available on the components of the pathways of sugar utilization in Saccharomyces cerevisiae it has been tacitly assumed that other yeasts use sugars in the same way. However, although the pathways of sugar utilization follow the same theme in all yeasts, important biochemical and genetic variations on it exist. Basically, in most non-conventional yeasts, in contrast to S. cerevisiae, respiration in the presence of oxygen is prominent for the use of sugars. This review provides comparative information on the different steps of the fundamental pathways of sugar utilization in non-conventional yeasts: glycolysis, fermentation, tricarboxylic acid cycle, pentose phosphate pathway and respiration. We consider also gluconeogenesis and, briefly, catabolite repression. We have centered our attention in the genera Kluyveromyces, Candida, Pichia, Yarrowia and Schizosaccharomyces, although occasional reference to other genera is made. The review shows that basic knowledge is missing on many components of these pathways and also that studies on regulation of critical steps are scarce. Information on these points would be important to generate genetically engineered yeast strains for certain industrial uses.     

Asymmetric alkene reduction by yeast old yellow enzymes and by a novel Zymomonas mobilis reductase

The genes encoding yeast old yellow enzymes (OYE 1, 2, and 3) and NAD(P)H-dependent 2-cyclohexen-1-one reductase from Zymomonas mobilis (NCR) were expressed separately in Escherichia coli. All four recombinant strains reduced the carbon double bond in alpha,beta-unsaturated alkenals and alkenones, however rates and enantio-specificities differed. Which of the two possible enantiomers was predominantly formed, was not only dependent on the choice of enzyme but also on the substrate: In addition to a dependency on methylation in alpha- or beta-position, the data of this study illustrate that firstly the E- or Z-configuration (cis- or trans-) of the carbon double-bond and secondly the remainder of the substrate molecule play roles in determining enantio-specificity. Based on the currently accepted mechanism of flavin mediated anti-hydrogenation of the carbon double bond, the data in this study may be explained by a flipped orientation of some of the substrates in the active center of OYE.     

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.     

Ultrastructure of methanol-utilizing yeast cells: appearance of microbodies in relation to high catalase activity.

Nine strains of methanol-utilizing yeasts belonging to the genera Candida, Hansenula, Kloeckera, Pichia, and Torulopsis were examined with respect to the interrelationship between their catalase content and ultrastructure. Methanol-grown cells of all the yeasts tested showed higher catalase activities than the respective ethanol- and glucose-grown cells. In connection with this, occurrence of a specific organelle surrounded by a single-unit membrane ("microbodies") was observed only in the methanol-grown cells. Several morphological differences were observed between the microbodies of methanol-utilizing yeasts and those of hydrocarbon-utilizing yeasts such as Candida tropicalis. That is, microbodies of methanol utilizers were large in size, existed in closely associated forms, and had crystalloid structures. Localization of catalase activity in these microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidene. Especially, 3,3'-diaminobenzidine reaction product accumulated heavily in crystalloids of yeast microbodies.     

从海蜇中筛选抗真菌活性的海洋细菌菌株

从中国北部湾海域采集的海蜇样品中分离到577株海洋细菌,以条件致病真菌白色念珠菌(Candida albicans)作为敏感测试菌株,采用琼脂块法测定了577株海洋细菌的抗真菌活性,结果表明有119株具有抗白色念珠菌活性,占总数的20.6%。对其中的40株高活性菌株进行抗真菌和抗细菌活性研究,结果表明,有21株海洋细菌对3种或3种以上的真菌有抑制活性,23株对细菌有不同程度的抑制作用,其中1株海洋细菌I0s4-18具有广谱的抗菌作用,对革兰氏阴性细菌、革兰氏阳性细菌、酵母菌及丝状真菌均具有抑制作用。海蜇共附生的微生物中能产生抗真菌活性的微生物的比例是很高的,I0s4-18菌株具有良好的开发前景。     

Enhancement of the Gene Targeting Efficiency of Non-Conventional Yeasts by Increasing Genetic Redundancy

In contrast to model yeasts, gene targeting efficiencies of non-conventional yeasts are usually low, which greatly limits the research and applications of these organisms. In this study, we aimed to enhance the gene targeting efficiency of non-conventional yeasts by improving the fitness of mutant strains, particularly by increasing the genetic redundancy of host cells. To demonstrate this process, OCH1 gene deletion in Pichia pastoris was performed. Extra copies of the OCH1 gene on a helper plasmid were provided for the P. pastoris GS115 strain before the native OCH1 gene in the genomic DNA was knocked out. The redundancy in OCH1 gene significantly eliminated the growth defects of the och1 mutant and increased the deletion efficiency of the OCH1 gene by two orders of magnitude with the same length of homologous flanks. The same strategy was used to delete the KU70 and SGS1 genes. The targeting efficiencies of KU70 and SGS1 were increased by 1- and 23-fold, respectively. Therefore, this study provided an efficient strategy for the deletion of “stubborn” genes in non-conventional yeasts. This study further showed that cellular fitness is potentially an important factor that can limit the efficiency of gene targeting.     

Three novel species of the anamorphic yeast genus Candida in the Candida intermedia clade found in Japan, Thailand and Taiwan

Four strains of yeasts isolated in Japan, Thailand and Taiwan were found to represent three novel species of the genus Candida. The three species are located in a clade including Candida tsuchiyae, Candida thailandica and Candida akabanensis in a tree based on the D1/D2 domain sequences of the large subunit rRNA genes but clearly differentiated from these relative species. Three novel species are proposed for these strains, i. e., Candida berkhoutiae sp. nov., for strains ST-49(T) (=BCC 7749(T)=NBRC 106733(T)=CBS 11722(T)) isolated from insect frass in Thailand and SA13S01 (=NBRC 106053) isolated from soil in Taiwan, Candida ezoensis sp. nov., for strain Y07-1601-2(T) (=NBRC 105019(T)=CBS 11753(T)) isolated from forest soil in Japan, and Candida inulinophila sp. nov., for ST-369(T) (=BCC 15081(T)=NBRC 106735(T)=CBS 11725(T)) isolated from an unidentified wild mushroom from Thailand.     

Molecular cloning and characterization of NAD(+)-dependent xylitol dehydrogenase from Candida tropicalis ATCC 20913

Induction of xylitol dehydrogenase of Candida tropicalis ATCC 20913 by various carbon sources was investigated. The enzyme activity was induced when the yeast was grown on l-arabinose and d-xylose. A novel gene encoding the enzyme was cloned and characterized. The 1,095-bp coding sequence of the gene encodes a polypeptide of 364 amino acids, with a molecular mass of 39.4 kDa. Sequence analysis of the putative protein showed it to be a member of the zinc-containing alcohol dehydrogenase family and to have homology to xylitol dehydrogenase genes from other yeasts and fungi. The recombinant xylitol dehydrogenase expressed in Escherichia coli oxidized polyols such as xylitol and d-sorbitol and reduced ketoses such as d-xylulose and d-fructose. It required exclusively NAD or NADH as a cofactor.     

两株希木龙假丝酵母14α-去甲基化酶基因(ERG11)的克隆及其功能初步验证

为探讨希木龙假丝酵母(假丝酵母又称念珠菌)的耐药机制,首先克隆出两株希木龙念珠菌ERG11基因,初步验证其功能,从而为后续研究奠定基础。从美国国家生物技术信息中心(National Center of Biotechnology Information,NCBI)基因数据库中获取白念珠菌、热带念珠菌、近平滑念珠菌和光滑念珠菌Erg11蛋白的保守序列,设计简并引物,聚合酶链反应(polymerase chain reaction,PCR)扩增获得希木龙念珠菌ERG11cDNA部分片段;用快速cDNA末端扩增法(rapid amplification of cDNA ends,RACE)分别扩增其5′和3′端,获得完整的ERG11编码序列(coding sequence,CDS);将CDS克隆到pYES2表达载体中,在尿嘧啶营养缺陷型酿酒酵母中过表达ERG11;用微量液基稀释法检测转化后的酿酒酵母对氟康唑的敏感性,初步验证其功能。结果显示,简并PCR扩增获得预期708bp片段,5′RACE和3′RACE分别获得385bp和1 336bp片段,经纯化、克隆、测序、比对分析,获得两株菌的ERG11CDS;比对其编码的蛋白,与其他念珠菌的Erg11蛋白高度同源;分别检测克隆了这两株希木龙念珠菌ERG11CDS表达载体的酿酒酵母对氟康唑的敏感性,发现过表达ERG11明显降低其对氟康唑的敏感性。结果提示,简并PCR联合RACE能准确有效地克隆出希木龙念珠菌ERG11基因,用pYES2酿酒酵母表达系统能初步验证其功能。     

Old Yellow Enzyme from Candida macedoniensis catalyzes the stereospecific reduction of the C=C bond of ketoisophorone

Microorganisms were screened for ones that reduced 3,5,5-trimethyl-2-cyclohexene-1,4-dione (ketoisophorone; KIP), and several strains were found to produce (6R)-2,2,6-trimethylcyclohexane-1,4-dione (levodione). The enzyme catalyzing the reduction of the C=C bond of KIP to yield (6R)-levodione was isolated from Candida macedoniensis AKU4588. The results of primary structural analysis and its enzymatic properties suggested that the enzyme might be an Old Yellow Enzyme family protein.