Cloning, characterization and heterologous expression of epoxide hydrolase-encoding cDNA sequences from yeasts belonging to the genera Rhodotorula and Rhodosporidium

Epoxide hydrolase-encoding cDNA sequences were isolated from the basidiomycetous yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031 in order to evaluate the molecular data and potential application of this type of enzymes. The deduced amino acid sequences were similar to those of the known epoxide hydrolases from Rhodotorula glutinis CBS 8761, Xanthophyllomyces dendrorhous CBS 6938 and Aspergillus niger LCP 521, which all correspond to the group of the microsomal epoxide hydrolases. The epoxide hydrolase encoding cDNAs of the Rhodosporidium and Rhodotorula species were expressed in Escherichia coli. The recombinant strains were able to hydrolyze trans-1-phenyl-1,2-epoxypropane with high enantioselectivity.     

Leucine biosynthesis and its regulation in the basidiomycete Rhodosporidium toruloides

Complementation tests and enzyme analyses separated 29 leucine auxotrophs of the Basidiomycete Rhodosporidium toruloides into three groups, each deficient in one of the leucine biosynthetic enzymes. The following differences are suggested between the organization of the leucine pathway in R. toruloides and the Ascomycetes Saccharomyces cerevisiae and Neurospora crassa: (1) isopropylmalate, the product of the first enzymic reaction appears not to be an internal inducer of the later enzymes of the pathway; this is consistent with the apparent lack of mutants homologous to the leu3 class in N. crassa and S. cerevisiae; (2) as in S. cerevisiae, but unlike N. crassa, isopropylmalate synthase is under the control of a general cross pathway control system; (3) unlike S. cerevisiae, but like N. crassa, R. toruloides appears to possess only one gene encoding isopropylmalate synthase.Abbreviations IPM Isopropylmalate - EMS methanesulphonic acid, ethyl ester     

Expression of a cephalosporin C esterase gene in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> for the direct production of deacetylcephalosporin C

A recombinant fungal microorganism capable of producing deacetylcephalosporin C was constructed by transforming a cephalosporin C esterase gene from Rhodosporidium toruloides into Acremonium chrysogenum. The cephalosporin C esterase gene can be expressed from its endogenous R. toruloides promoter or from the Aspergillus nidulans trpC promoter under standard Acremonium chrysogenum fermentation conditions. The expression of an active cephalosporin C esterase enzyme in A. chrysogenum results in the conversion of cephalosporin C to deacetylcephalosporin C in vivo, a novel fermentation process for the production of deacetylcephalosporin C. The stability of deacetylcephalosporin C in the fermentation broth results in a 40% increase in the cephalosporin nucleus.     

The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose

A Pseudomonas putida S12 strain was constructed that efficiently produced the fine chemical cinnamic acid from glucose or glycerol via the central metabolite phenylalanine. The gene encoding phenylalanine ammonia lyase from the yeast Rhodosporidium toruloides was introduced. Phenylalanine availability was the main bottleneck in cinnamic acid production, which could not be overcome by the overexpressing enzymes of the phenylalanine biosynthesis pathway. A successful approach in abolishing this limitation was the generation of a bank of random mutants and selection on the toxic phenylalanine anti-metabolite m-fluoro-phenylalanine. Following high-throughput screening, a mutant strain was obtained that, under optimised culture conditions, accumulated over 5 mM of cinnamic acid with a yield (Cmol%) of 6.7%.     

Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane

Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane. The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells. The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity. An increase in both the alginate and CaCl₂ concentrations used for bead preparation led to a decrease in enzyme stability. An increase in the alginate concentration led to an increase in bead diameter. The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl₂ concentrations higher than 0.4 M were utilised for bead preparation.     

Assimilation of benzoate byRhodotorula rubra,Rhodotorula glutinis andRhodosporidium toruloides,as affected by glucose or xylose

Xylose or glucose (5 g/l) was utilized simultaneously with benzoate (5 g/l) byRhodosporidium toruloides andRhodotorula rubra in batch culture. At a higher glucose concentration, benzoate was utilized only after glucose was depleted from the media. Both yeasts preferentially utilized benzoate before xylose even if there were more than 5 g xylose/l.Rhodotorula glutinis preferentially utilized glucose (10 g/l) before benzoate but utilized xylose and benzoate simultaneously.The authors are with the Department of Biochemical Technology, Faculty of Chemistry, Slovak Technical University, Radlinského 9, 812 37 Bratislava, Slovak Republic     

Stabilization of d-amino acid oxidase from Rhodosporidium toruloides by immobilization onto magnetic nanoparticles

d-Amino acid oxidase from Rhodosporidium toruloides was immobilized onto glutaraldehyde-activated magnetic nanoparticles. Approximately four enzyme molecules were attached to one magnetic nanoparticle when the weight ratio of the enzyme to the support was 0.12. After immobilization, the T _m was increased from 45°C of the free form to 55°C. In the presence of 20 mM H₂O₂, the immobilized form retained 93% of its activity after 5 h while the free form was completely inactivated after 3.5 h.     

Some physiological observations on the uptake of d-glucose and 2-deoxy-d-glucose by starving and exponentially-growing yeasts

Some methods for measuring the uptake of sugars by yeasts were investigated critically. A study was made of the effects of starvation of Pichia pinus, Candida utilis, Saccharomyces cerevisiae and Rhodosporidium toruloides on their uptake of d-glucose and 2-deoxy-d-glucose. Marked changes in the rates of uptake of these sugars occurred during 10 h of starvation, including (a) an immediate increase of up to 75% above that for growing cells and (b) a continuous decline to as little as 4%. Each yeast behaved differently. The rates did not remain constant during the periods of starvation often used for studies on the transport of sugars into yeasts. For Pichia pinus, there were striking differences, associated with starvation, between the transport of 2-deoxy-d-glucose and d-glucose, despite evidence that the two sugars enter this yeast by means of the same carrier. Some physiological explanations for these findings are discussed.     

Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides

Summary The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity. Furthermore, the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme. Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported. We have inserted the entire uninterupted pal gene from R. toruloides into the Escherichia coli expression vector pKK 223-3. E. coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity. The potential implications of this finding are discussed.Offprint requests to: H. Ørum     

Phylogenetic analysis of basidiomycetous yeasts by means of 18S ribosomal RNA sequences: relationship ofErythrobasidium hasegawianum and other basidiomycetous yeast taxa

The basidiomycetous yeast genusErythrobasidium Hamamoto, Sugiyama & Komagata, based on the type speciesE. hasegawianum Hamamoto et al., is characterized by filobasidiaceous basidia and the Q-10 (H₂) system as its major ubiquinone. It is tentatively placed in the Filobasidiaceae. The molecular characterization is based on 18S ribosomal RNA sequence comparisons among the basidiomycetous yeasts, and the ultrastructural characterization on the cell wall and hyphal septal pores inE. hasegawianum clearly indicate a close relationship with the teliospore-forming yeastsRhodosporidium toruloides andLeucosporidium scottii. Our molecular phylogeny with statistical analysis suggests that the existing taxonomic system of basidiomycetous yeasts, based primarily on the morphology of basidia including the teliospores (probasidia), should be revised.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

圆红冬孢酵母基因编辑及天然产物合成的研究进展

圆红冬孢酵母(Rhodotorula toruloides)是一种能够天然合成多种类胡萝卜素和油脂的非模式酵母。该菌能够利用各种廉价原料,耐受甚至同化利用多种有毒木质纤维素水解副产物。目前,该酵母被广泛用于微生物油脂、萜烯类化合物、各种高价值酶、糖醇和聚酮化合物的生产研究。鉴于其广阔的工业应用前景,研究人员对其开展了多维度的理论和技术的探索,包括基因组、转录组、蛋白组、遗传操作平台等。本文着重阐述近年来圆红冬孢酵母的代谢工程和天然产物合成的研究进展,并展望其细胞工厂构建中面临的挑战和可能的应对决策。     

Genomic organization of multiple genes coding for rhodotorucine A,a lipopeptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides

Rhodotorucine A, a lipopeptide mating pheromone, is secreted from mating type A cells of Rhodosporidium toruloides and induces sexual differentiation of the opposite mating type a cells. Genome of A-type cells contains three homologous genes (RHA1, RHA2, and RHA3) encoding rhodotorucine A. Genomic Southern blot analysis using RHA1 DNA as a probe showed that RHA1 strongly hybridize with A-type genomic DNA but weakly with a-type, suggesting that the sequences of RHA genes were dissimilar in the opposite a-type genome. The range of dissimilar regions in a-type genome was searched using RHA-flanking DNA segments as probes. The result suggests that a-type genome lacks sequences coding for rhodotorucine A and its 5 upstream but contains its 3 non-coding sequences. The absence of mating pheromone genes in the opposite mating type genome suggests that the expression of mating-type-specific genes in R. toruloides is not controlled trans-criptionally, as shown in the yeast Saccharomyces cerevisiae.     

一株降解N-酰基高丝氨酸内酯酵母菌菌株的分离鉴定及其降解特性

利用N酰基高丝氨酸内酯(N-acyl-homoserine lactone,简称AHL)为唯一碳源和能源,筛选得到一株能够降解AHL的菌株R1。常规鉴定和18S rDNA序列分析表明,菌株R1属于红冬孢酵母菌(Rhodosporidium toruloides),定名为R.toruloidesR1。结果显示R.toruloidesR1能利用所测试的3种AHL作为唯一碳源和能源生长,具有降解AHL的能力,其对AHL依赖型胡萝卜欧文氏软腐病菌(Erwinia carotovora subsp.carotovora)的致病有一定的抑制作用。     

Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia lyase

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.     

Silencing the genes for dopa decarboxylase or dopachrome conversion enzyme reduces melanization of foreign targets in Anopheles gambiae

The production of melanin is a complex biochemical process in which several enzymes may play a role. Although phenoloxidase and serine proteases are clearly key components, the activity of other enzymes, including dopa decarboxylase and dopachrome conversion enzyme may also be required. We tested the effect of knockdown of gene expression for these two enzymes on melanization of abiotic targets in the mosquito, Anopheles gambiae. Knockdown of dopa decarboxylase and dopachrome conversion enzyme resulted in a significant reduction of melanization of Sephadex beads at 24 h after injection. Knockdown of a third enzyme, phenylalanine hydroxylase, which is involved in endogenous production of tyrosine, had no effect on bead melanization. Quantitative analysis of gene expression demonstrated significant upregulation of phenylalanine hydroxylase, but not the other two genes, following injection.     

The clustering on thePseudomonas putida chromosome of genes specifying dissimilatory functions

Summary Two previously demonstrated linkage groups containing genes with catabolic function inPseudomonas putida have been shown to cotransduce with a third cluster of catabolic genes, namely those specifying enzymes of nicotinic acid dissimilation. Thus enzymes of the following dissimilatory pathways are coded by genes clustered in one small (10-15% of the chromosome) genetic region:p-hydroxy-benzoic, quinic, shikimic, benzoic, mandelic, phenylacetic, and nicotinic acids; and histidine, tyrosine and phenylalanine. We propose that this clustering is a consequence of the selection, in natural populations of bacteria, of gene arrangements which permit simultaneous transfer of the genetic determinants of a variety of dissimilatory pathways.     

A pAO1-encoded molybdopterin cofactor gene (moaA) ofArthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein

A gene homologous tomoaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor ofEscherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 ofArthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encodedmoaA gene fromA. nicotinovorans was expressed inE. coli as an active protein that functionally complementedmoaA mutants. Its reduced amino acid sequence shows 43% identity to theE. coli MoaA, 44% to the NarAB gene product fromBacillus subtilis, and 42% to the gene product of two contiguous ORFs fromMethanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in thefixZ gene product fromRhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement themoaA mutation inE. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy ofmoaA may not be unique in theA. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.     

Stabilization of native and double <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidases from <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> and <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis> by immobilization on streptavidin-coated magnetic beads

Double d-amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k_cat/K_M) of immobilized DAOs toward d-alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward d-alanine was decreased by 56%. After immobilization, the T_m value for RtDAO was shifted 15°C higher to 60°C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5–8°C to 56, 60 and 60°C, respectively. In the presence of 10 mM H₂O₂, immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin–streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.