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.     

The wine yeast strain-dependent expression of genes implicated in sulfide production in response to nitrogen availability

Sulfur metabolism in S. cerevisiae is well established, but the mechanisms underlying the formation of sulfide remain obscure. Here we investigated by real time RT-PCR the dependence of expression levels of MET3, MET5/ECM17, MET10, MET16 and MET17 along with SSU1 on nitrogen availability in two wine yeast strains that produce divergent sulfide profiles. MET3 was the most highly expressed of the genes studied in strain PYCC4072, and SSU1 in strain UCD522. Strains behaved differently according to the sampling times, with UCD522 and PYCC4072 showing the highest expression levels at 120h and 72h, respectively. In the presence of 267mg assimilable N/l, the genes were more highly expressed in strain UCD522 than in PYCC4072. MET5/ECM17 and MET17 were only weakly expressed in both strains under any condition tested. MET10 and SSU1 in both strains, but MET16 only in PYCC4072, were consistently up-regulated when sulfide production was inhibited. This study illustrates that strain genotype could be important in determining enzyme activities and therefore the rate of sulfide liberation. This linkage, for some yeast strains, of sulfide production to expression levels of genes associated to sulfate assimilation and sulfur amino acid biosynthesis could be relevant for defining new strategies for genetic improvement of wine yeasts.     

Cloning and functional analysis of the GSH1/MET1 gene complementing cysteine and glutathione auxotrophy of the methylotrophic yeast Hansenula polymorpha

The Hansenula polymorpha GSH1/MET1 gene was cloned by complementation of glutathione-dependent growth of H. polymorpha gsh1 mutant isolated previously as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistant and cadmium ion sensitive clone. The H. polymorpha GSH1 gene was capable of restoring cadmium ion resistance, MNNG sensitivity, normal glutathione level and cell proliferation on minimal media without addition of cysteine or glutathione, when introduced into the gsh1 mutant cells. It was shown that the H. polymorpha GSH1 gene has homology to the Saccharomyces cerevisiae MET1 gene encoding S-adenosyl-L-methionine uroporphyrinogen III transmethylase, responsible for the biosynthesis of sulfite reductase cofactor, sirohaem. The H. polymorpha GSH1/MET1 gene deletion cassette (Hpgsh1/met1::ScLEU2) was constructed and corresponding null mutants were isolated. Crossing data of the point gsh1 and null gsh1/met1 mutants demonstrated that both alleles were located to the same gene. The null gsh1/met1 mutant showed total growth restoration on minimal media supplemented with cysteine or glutathione as a sole sulfur source, but not with inorganic (sulfate, sulfite) or organic (methionine, S-adenosylmethionine) sources of sulfur. Moreover, both the point gsh1 and null gsh1/met1 mutants displayed increased sensitivity to the toxic carbon substrate methanol, formaldehyde, organic peroxide and cadmium ions.     

Development of a glutathione production process from proteinaceous biomass resources using protease-displaying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources.     

Alcoholic fermentation by wild-type <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> versus recombinant strains with an elevated level of intracellular glutathione

The ability of baker’s yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains.     

MET17 and hydrogen sulfide formation in Saccharomyces cerevisiae

Commercial isolates of Saccharomyces cerevisiae differ in the production of hydrogen sulfide (H(2)S) during fermentation, which has been attributed to variation in the ability to incorporate reduced sulfur into organic compounds. We transformed two commercial strains (UCD522 and UCD713) with a plasmid overexpressing the MET17 gene, which encodes the bifunctional O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase), to test the hypothesis that the level of activity of this enzyme limits reduced sulfur incorporation, leading to H(2)S release. Overexpression of MET17 resulted in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H(2)S produced. In contrast, OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced H(2)S formation. Overexpression of OAS/OAH SHLase activity was greater in UCD713 when grown under low-nitrogen conditions, but the impact on reduction of H(2)S was greater under high-nitrogen conditions. Thus, there was not a good correlation between the level of enzyme activity and H(2)S production. We measured cellular levels of cysteine to determine the impact of overexpression of OAS/OAH SHLase activity on sulfur incorporation. While Met17p activity was not correlated with increased cysteine production, conditions that led to elevated cytoplasmic levels of cysteine also reduced H(2)S formation. Our data do not support the simple hypothesis that variation in OAS/OAH SHLase activity is correlated with H(2)S production and release.     

高产谷胱甘肽酵母菌株的选育及其代谢通量分析

利用UV和HNO2及其复合诱变处理S.cerevisiae 的原生质,筛选得到ZnCl2和半胱氨酸抗性菌株S.cerevisiae YZM-14(ZnCl2r,Cysr),其谷胱甘肽(GSH)产量(84.72mg/L)、生物量(7.63g/L)及胞内GSH含量(11.10mg/g)分别是出发菌株的2.79倍、1.63倍和1.71倍,且性状稳定。根据细胞比生长速率和GSH得率变化曲线,将GSH生物合成过程分为三个阶段,第二阶段诱变菌株与出发菌株相比PP途径代谢通量增加8.1 mmol/(g·h),GSH前体合成途径通量增加,且诱变菌株的有机酸分泌通量减少,提高了细胞的碳源利用效率,增大了GSH的生成。     

Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production

The production of hydrogen sulfide (H₂S) during fermentation is a common and significant problem in the global wine industry as it imparts undesirable off-flavors at low concentrations. The yeast Saccharomyces cerevisiae plays a crucial role in the production of volatile sulfur compounds in wine. In this respect, H₂S is a necessary intermediate in the assimilation of sulfur by yeast through the sulfate reduction sequence with the key enzyme being sulfite reductase. In this study, we used a classical mutagenesis method to develop and isolate a series of strains, derived from a commercial diploid wine yeast (PDM), which showed a drastic reduction in H₂S production in both synthetic and grape juice fermentations. Specific mutations in the MET10 and MET5 genes, which encode the catalytic α- and β-subunits of the sulfite reductase enzyme, respectively, were identified in six of the isolated strains. Fermentations with these strains indicated that, in comparison with the parent strain, H₂S production was reduced by 50–99%, depending on the strain. Further analysis of the wines made with the selected strains indicated that basic chemical parameters were similar to the parent strain except for total sulfite production, which was much higher in some of the mutant strains.     

CRISPR/Cas9介导的高产谷胱甘肽原养型酵母工程菌的构建

谷胱甘肽(Glutathione,GSH)是具有多种生理功能的非蛋白质类巯基化合物,已广泛应用于药品、食品等行业,且市场需求量逐年增加。遗传工程育种是提高细胞内GSH含量的重要策略,但在遗传操作过程中使用到的营养缺陷型遗传标记可能会影响菌株的正常生长,且不利于高密度发酵的进行。为回复工程菌株的营养缺陷型,利用g RNA转录表达框和靶基因同源DNA片段直接共转化酵母细胞,由细胞内表达的Ⅱ型CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9)介导的基因组编辑技术将营养缺陷型GSH工程菌株W303-1b/FGP回复为原养型菌株。结果显示,与营养缺陷型菌株相比,原养型菌株生长周期缩短,且可以利用简单的合成培养基进行培养,方便菌株的大规模培养。     

Extracellular glutathione fermentation using engineered Saccharomyces cerevisiae expressing a novel glutathione exporter

A novel extracellular glutathione fermentation method using engineered Saccharomyces cerevisiae was developed by following three steps. First, a platform host strain lacking the glutathione degradation protein and glutathione uptake protein was constructed. This strain improved the extracellular glutathione productivity by up to 3.2-fold compared to the parental strain. Second, the ATP-dependent permease Adp1 was identified as a novel glutathione export ABC protein (Gxa1) in S. cerevisiae based on the homology of the protein sequence with that of the known human glutathione export ABC protein (ABCG2). Overexpression of this GXA1 gene improved the extracellular glutathione production by up to 2.3-fold compared to the platform host strain. Finally, combinatorial overexpression of the GXA1 gene and the genes involved in glutathione synthesis in the platform host strain increased the extracellular glutathione production by up to 17.1-fold compared to the parental strain. Overall, the metabolic engineering of the glutathione synthesis, degradation, and transport increased the total (extracellular + intracellular) glutathione production. The extracellular glutathione fermentation method developed in this study has the potential to overcome the limitations of the present intracellular glutathione fermentation process in yeast.     

Physiological analysis of mutants of Saccharomyces cerevisiae impaired in sulphate assimilation.

The assimilation of sulphate in Saccharomyces cerevisiae, comprising the reduction of sulphate to sulphide and the incorporation of the sulphur atom into a four-carbon chain, requires the integrity of 13 different genes. To date, the functions of nine of these genes are still not clearly established. A set of strains, each bearing a mutation in one MET gene, was studied. Phenotypic studies and enzyme determinations showed that the products of at least five genes are needed for the synthesis of an enzymically active sulphite reductase. These genes are MET1, MET5, MET8, MET10 and MET20. Wild-type strains of S. cerevisiae can use organic metabolites such as homocysteine, cysteine, methionine and S-adenosylmethionine as sulphur sources. They are also able to use inorganic sulphur sources such as sulphate, sulphite, sulphide or thiosulphate. Here we show that both of the two sulphur atoms of thiosulphate are used by S. cerevisiae. Thiosulphate is cleaved into sulphite and sulphide prior to utilization by the sulphate assimilation pathway, as the metabolism of one sulphur atom from thiosulphate requires the presence of an active sulphite reductase.     

Human glutamate cysteine ligase gene regulation through the electrophile response element

Glutathione (GSH) is the primary nonprotein thiol in the cell. It has many important roles in cell function, including regulating redox-dependent signal transduction pathways. The content of GSH within the cell varies with stress. In many cases, a process involving GSH synthesis results in adaptation to subsequent stressors. Sustained increases in GSH content are controlled primarily through induction of two genes, Gclc and Gclm, leading to the synthesis of the rate-limiting enzyme for GSH synthesis, glutamate cysteine ligase. Each of these genes in humans has a number of putative enhancer elements in their promoters. Overall, the most important element in both Gclc and Gclm expression is the electrophile response element. We review the evidence that has led to this conclusion and the implications for the redox-dependent regulation of this critical intracellular antioxidant.     

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L^-1 glutathione with glutathione content and yield of 4.3% and 25.6 mg_glutathione/g_glucose, respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L^-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.     

Subcellular distribution of glutathione and cysteine in cyanobacteria

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria.     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。