Engineering the robustness of Clostridium acetobutylicum by introducing glutathione biosynthetic capability

To improve the aero- and solvent tolerance of the solvent-producing Clostridium acetobutylicum, glutathione biosynthetic capability was introduced into C. acetobutylicum DSM1731 by cloning and over-expressing the gshAB genes from Escherichia coli. Strain DSM1731(pITAB) produces glutathione, and shows a significantly improved survival upon aeration and butanol challenge, as compared with the control. In addition, strain DSM1731(pITAB) exhibited an improved butanol tolerance and an increased butanol production capability, as compared with the recombinant strains with only gshA or gshB gene. These results illustrated that introducing glutathione biosynthetic pathway, which is redundant for the metabolism of C. acetobutylicum, can increase the robustness of the host to achieve a better solvent production.     

Crystal structure of the bifunctional chorismate synthase from Saccharomyces cerevisiae

Chorismate synthase (EC 4.2.3.5), the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi, and plants. The chorismate synthase reaction involves a 1,4-trans-elimination of phosphoric acid from EPSP and has an absolute requirement for reduced FMN as a cofactor. We have determined the three-dimensional x-ray structure of the yeast chorismate synthase from selenomethionine-labeled crystals at 2.2-A resolution. The structure shows a novel betaalphabetaalpha fold consisting of an alternate tight packing of two alpha-helical and two beta-sheet layers, showing no resemblance to any documented protein structure. The molecule is arranged as a tight tetramer with D2 symmetry, in accordance with its quaternary structure in solution. Electron density is missing for 23% of the amino acids, spread over sequence regions that in the three-dimensional structure converge on the surface of the protein. Many totally conserved residues are contained within these regions, and they probably form a structured but mobile domain that closes over a cleft upon substrate binding and catalysis. This hypothesis is supported by previously published spectroscopic measurements implying that the enzyme undergoes considerable structural changes upon binding of both FMN and EPSP.     

Expression of the farnesyldiphosphate synthase gene of Saccharomyces cerevisiae in tobacco

The coding region of the farnesyldiphosphate synthase (FDP synthase) gene from Saccharomyces cerevisiae has been inserted into a pBin19 vector, downstream of the cauliflower mosaic virus (CaMV) 35S promoter, in order to allow its expression in the genome of a higher plant, Nicotiana tabacum. We have produced transgenic tobacco in which the expression of the foreign gene leads to functional FDP synthase activity. In these transgenic plants, total FDP synthase-specific activity is increased 12-fold compared to controls. This increase of FDP synthase activity has been correlated to a clear increase of both sterol and carotenoid biosynthesis. This heterologous expression is also related to an increased resistance of transformed plants to R172117, a specific inhibitor of FDP synthase, and to sterol biosynthesis inhibitors such as flusilazol and fenpropimorph.Abbreviations: AP, Annick Petit; BAP, benzylaminopurine; CaMV, cauliflower mosaic virus; CTAB, cetyltrimethylammonium bromide; DMAEDP, dimethylamino ethyl diphosphate; DMADP, dimethylallyl diphosphate; DTT, dithiothreitol; ERG12, mevalonate kinase yeast gene; ERG20, FDP synthase yeast gene; FDP, farnesyl diphosphate; GGDP, geranylgeranyl diphosphate; GDP, geranyl diphosphate; IDP, isopentenyl diphosphate; LB, Luria Bertani; MS, Murashige and Skoog; NAA, Naphtaleneacetic acid; PVP, polyvinyl pyrrolidone; SBI, sterol biosynthesis inhibitor.     

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.     

Medium optimization for glutathione production by Saccharomyces cerevisiae

Glucose, peptone and magnesium sulphate were found to be suitable components for the cell growth and glutathione (GSH) production in the yeast strain. Saccharomyces cerevisiae CCRC 21727. The Box–Behnken design and response surface methodology were employed to derive a statistical model to investigate the effects of glucose, peptone and magnesium sulphate concentrations on GSH production. Neural networks were compared with a second-order-polynomial model in predicting the effects of component concentrations on the production of GSH and dry cell weight (DCW). Neural network models can predict cell growth and GSH production more precisely than second-order-response-surface models.     

Primary structure and disruption of the phosphatidylinositol synthase gene of Saccharomyces cerevisiae

The wild-type yeast nuclear gene, PIS, encodes phosphatidylinositol synthase (CDPdiacylglycerol-inositol 3-phosphatidyltransferase, EC 2.7.8.11) (Nikawa, J., and Yamashita, S. (1984) Eur. J. Biochem. 143, 251-256). We now report the sequence of the cloned 2, 129-base pair DNA and the location of the PIS coding region within the sequence. The PIS coding frame is capable of encoding 220 amino acid residues with a calculated molecular weight of 24,823. On Northern blot analysis, an RNA species that hybridized with the coding region was detected in the total poly(A)+ RNA of the wild-type yeast. The primary translation product contains a region showing local sequence homology with yeast phosphatidylserine synthase (EC 2.7.8.8) and Escherichia coli 3-phosphatidyl-1'-glycerol-3'-phosphate synthase (EC 2.7.8.5), suggesting that these three enzymes are evolutionarily related. The PIS gene was disrupted in vitro through insertion of the yeast HIS3 gene into the coding region. A heterozygous diploid, PIS/pis::HIS3, constructed from a PIS/PIS his3/his3 diploid by replacing one of the wild-type PIS genes with the disrupted PIS gene, showed no segregation of viable His+ spores on tetrad analysis, indicating that disruption of the PIS gene is lethal. The nonviable spores were in an arrested state with a characteristic terminal phenotype, suggesting that the function of the PIS gene is essential for progression of the yeast cell cycle.     

Continuous production of glutathione by immobilized Saccharomyces cerevisiae cells

Summary Glutathione was continuously produced by an immobilized Saccharomyces cerevisiae IFO 2044 cell column. The production of glutathione was strongly influenced by the level of activity of the glycolytic pathway. This activity was maintained constant by the addition of NAD.Abbreviations ADP adenosine-5-diphosphate - ATP adenosine-5-triphosphate - NAD nicothinamide adenine dinucleotide     

Production of glutathione in extracellular form by Saccharomyces cerevisiae

The present research was aimed at inducing, in a post fermentative procedure (biotransformation) and by modifying cell permeability, glutathione (GSH) accumulation and subsequent release from cells of Saccharomyces cerevisiae. With the aim of limiting process costs, research considered the possibility of employing baker's yeasts (S. cerevisiae), inexpensive cells source available on the market, in comparison with a collection strain. The tested yeasts showed different sensitivity to the chemical/physical treatments performed to alter cell permeability. Modest effects were evidenced with Triton, active only on Zeus yeast samples (1.7 g GSH/l, near 60% of which in extracellular form). Lauroyl sarcosine showed an interesting action on GB Italy sample (2.8 g GSH/l, near 80% extracellular). Lyophilization evidenced good performance with Lievitalia yeast strain (2.9 g GSH/l, 90% extracellular). The possibility of obtaining GSH directly in extracellular form represents an interesting opportunity of reducing GSH production cost and furthering the range of application of this molecule.     

Disruption of the CHO1 gene encoding phosphatidylserine synthase in Saccharomyces cerevisiae

A Saccharomyces cerevisiae mutant that lacked phosphatidylserine synthase [EC 2.7.8.8] (CDP-1,2-diacyl-sn-glycerol: L-serine O-phosphatidyltransferase) completely was constructed by disrupting its structural gene, CHO1. Over two-thirds of its coding region, from the starting to the 200th codon, was replaced with a LEU2 DNA fragment. This new cho1 mutant showed no detectable synthesis of phosphatidylserine but grew slowly in a medium that contained either ethanolamine or choline. These results indicate that phosphatidylserine synthase and most probably phosphatidylserine are dispensable in S. cerevisiae but necessary for its optimal growth. Additional supplementation with myo-inositol raised the cellular content of phosphatidylinositol and improved the growth of the mutant, suggesting the importance of the negative charges of the membrane surface. The CHO1-disrupted mutant, when grown on choline, accumulated phosphatidylethanolamine to a significant level even after extensive dilution of the initial culture. It segregated prototrophic revertants that could synthesize phosphatidylethanolamine without recovery of phosphatidylserine synthesis. These results imply the presence of a route(s) for the formation of ethanolamine or its phosphorylated derivative in S. cerevisiae.     

The three-dimensional structure of the bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase of Saccharomyces cerevisiae

In Saccharomyces cerevisiae and other fungi, the enzymes dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) are encoded by a polycistronic gene that is translated into a single polypeptide having all three functions. These enzymatic functions are essential to both prokaryotes and lower eukaryotes, and catalyse sequential reactions in folate biosynthesis. Deletion or disruption of either function leads to cell death. These enzymes are absent from mammals and thus make ideal antimicrobial targets. DHPS is currently the target of antifolate therapy for a number of infectious diseases, and its activity is inhibited by sulfonamides and sulfones. These drugs are typically used as part of a synergistic cocktail with the 2,4-diaminopyrimidines that inhibit dihydrofolate reductase. A gene encoding the S.cerevisiae HPPK and DHPS enzymes has been cloned and expressed in Escherichia coli. A complex of the purified bifunctional polypeptide with a pterin monophosphate substrate analogue has been crystallized, and its structure solved by molecular replacement and refined to 2.3A resolution. The polypeptide consists of two structural domains, each of which closely resembles its respective monofunctional bacterial HPPK and DHPS counterpart. The mode of ligand binding is similar to that observed in the bacterial enzymes. The association between the domains within the polypeptide as well as the quaternary association of the polypeptide via its constituent DHPS domains provide insight into the assembly of the trifunctional enzyme in S.cerevisiae and probably other fungal species.     

Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae

Aims: Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. Methods and Results: A dammarenediol‐producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT‐PCR. Fermentation was carried out in a 5‐L GRJY‐bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250 μg l^?1. However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Conclusions: Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. Significance and Impact of the Study: This study showed that the high‐value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene‐derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.     

Enhanced glutathione production by evolutionary engineering of Saccharomyces cerevisiae strains

Glutathione is an important natural tripeptide mainly used because of its antioxidative properties. Commercial glutathione is microbially synthesized by yeasts and the growing demand requires the development of new production strains. An adaptive laboratory evolution strategy using acrolein as a selection agent was employed to obtain strains with an enhanced glutathione accumulation phenotype accompanied by an acrolein resistance phenotype. Two particularly interesting isolates were obtained: one with a high volumetric productivity for glutathione reaching 8.3 mg_glutathione/L h, which is twice as high as the volumetric productivity of its parental strain. This strain reached an elevated intracellular glutathione content of 3.9%. A second isolate with an even higher acrolein tolerance exhibited a lower volumetric productivity of 5.8 mg_glutathione/L h due to a growth phenotype. However, this evolved strain accumulated glutathione in 3.3‐fold higher concentration compared to its parental strain and reached a particularly high glutathione content of almost 6%. The presented results demonstrate that acrolein is a powerful selection agent to obtain high glutathione accumulation strains in an adaptive laboratory evolution experiment.     

Effect of amino acids on glutathione production by Saccharomyces cerevisiae

Summary The constituent amino acids of the glutathione (GSH) tripeptide chain, glutamate, cysteine and glycine, were investigated for positive effects on GSH production in shake-flask cultures of Saccharomyces cerevisiae with glucose as the carbon source. Cysteine was confirmed as the key amino acid for increasing the specific GSH production rate, g, but showed some growth inhibition, especially in the second growth phase (ethanol-assimilation phase). An intracellular cysteine delivery agent, thiazolidine, showed a similar pattern of increased GSH production and growth inhibition, but to a slightly lesser degree, compared with free cysteine. The initial cysteine concentration affected both the specific growth rate, µ, and g, up to about 5 mm for µ and about 2–3 mm for g. Results of the [³⁵S]cysteine-labelling experiments suggest a complicated role of cysteine in increasing GSH production and further investigation may be necessary. Offprint requests to: S. Shioya     

Metabolic Engineering of Saccharomyces cerevisiae

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.     

乙酰辅酶A合成代谢对酿酒酵母生理功能的影响

【目的】研究酿酒酵母(Saccharomycesc erevisiae)中乙酰辅酶A合成酶基因ACS1和ACS2的生理作用。【方法】将来源于S.cerevisiae的ACS1和ACS2分别进行过量表达,研究过量表达ACS1和ACS2后S.cerevisiae胞内乙酰辅酶A含量、ATP水平、甲羟戊酸途径转录和乙醇耐受性等生理学特性变化。【结果】与出发菌株相比,过量表达ACS1和ACS2使得:(1)胞内乙酰辅酶A含量提高了2.19倍(ACS1)和5.02倍(ACS2);(2)胞内ATP含量提高了3.93倍(ACS1)和2.05倍(ACS2);(3)甲羟戊酸途径8个关键基因表达量显著上调;(4)S.cerevisiae对乙醇胁迫抵御能力显著增强。过量表达ACS1对乙醇胁迫的耐受能力强于过量表达ACS2。【结论】增加胞内乙酰辅酶A的含量可以显著增加甲羟戊酸途径碳代谢流量,并增强S.cerevisiae对发酵过程主要副产物乙醇的耐受能力。     

Phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol): sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) activity was characterized from the mitochondrial fraction of Saccharomyces cerevisiae. The pH optimum for the reaction was 7.0. Maximum activity was dependent on manganese (0.1 mM), magnesium (0.3 mM), or cobalt (1 mM) ions and the nonionic detergent Triton X-100 (1 mM). The apparent Km values for CDP-diacylglycerol and glycerol-3-phosphate were 33 and 27 microM, respectively. Optimal activity was at 30 degrees C with an energy of activation of 5.4 kcal/mol (1 cal = 4.1868 J). Phosphatidylglycerophosphate synthase activity was thermally labile above 40 degrees C. p-Chloromecuriphenylsulfonic acid, N-ethylmaleimide, and mercurous ions inhibited activity. Phosphatidylglycerophosphate synthase activity was partially solubilized from the mitochondrial fraction with 1% Triton X-100.     

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the α subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC₅₀) of the enzyme activity was measured to be 400 μM, whereas the IC₅₀ value was 15 μM for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly → Ser) in the α subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.