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1.
Efficient use of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials. Current studies on xylose uptake for various microorganisms have been hampered by the lack of a facile assay for xylose transport. In this work, a rapid in vivo, label-free method for measuring xylose transport in Escherichia coli was developed by taking advantage of the Bacillus pumilus xylosidase (XynB), which cleaved a commercially available xylose analog, p-nitrophenyl-β-d-xylopyranoside (pNPX), to release a chromogenic group, p-nitrophenol (pNP). XynB was expressed alone or in conjunction with a Zymomonas mobilis glucose facilitator protein (Glf) capable of transporting xylose. This XynB-mediated transport assay was demonstrated in test tubes and 96-well plates with submicromolar concentrations of pNPX. Kinetic inhibition experiments validated that pNPX and xylose were competitive substrates for the transport process, and the addition of glucose (20 g/L) in the culture medium clearly diminished the transmembrane transport of pNPX and, thus, mimicked its inhibitory action on xylose uptake. This method should be useful for engineering of the xylose transport process in E. coli, and similar assay schemes can be extended to other microorganisms. 相似文献
2.
Gregory G. Y. Guirimand Takahiro Bamba Mami Matsuda Kentaro Inokuma Kenta Morita Yuki Kitada Yuma Kobayashi Takahiro Yukawa Kengo Sasaki Chiaki Ogino Tomohisa Hasunuma Akihiko Kondo 《Biotechnology journal》2019,14(9)
Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d ‐xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro‐organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499‐SsXR‐AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a β‐d ‐glucosidase (Aspergillus aculeatus β‐glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5‐fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499‐XR‐ScMAL11‐BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae. 相似文献
3.
Aims: To characterize the duel activities of a glycosyl hydrolase family 3 β‐glucosidase/xylosidase from rumen bacterial metagenome and to investigate the capabilities of its β‐d ‐xylosidase activities for saccharification of hemicellulosic xylans. Methods and Results: A β‐glucosidase/xylosidase gene RuBGX1 was cloned from yak (Bos grunniens) rumen using the metagenomic technology. Recombinant RuBGX1, expressed in Escherichia coli, demonstrated high hydrolytic activities on both p‐nitrophenyl‐β‐d ‐glucopyranoside (pNP‐Glc) and p‐nitrophenyl‐β‐d ‐xylopyranoside (pNP‐Xyl) substrates. Analysis of the kinetic properties indicated that RuBGX1 had a lower affinity for pNP‐Glc substrate as the Km was 0·164 mmol l?1 for pNP‐Glc and 0·03 mmol l?1 for pNP‐Xyl at pH 6·0 and 50°C, respectively. The capabilities of RuBGX1 β‐xylosidase for hydrolysis of xylooligosaccharide substrates were further investigated using an endoxylanase‐coupled assay. Hydrolysis time courses illustrated that a significant increase (about 50%) in the reducing sugars, including xylobiose, xylotriose and xylotetraose, was achieved by supplementing endoxylanase with RuBGX1. Enzymatic product analysis using high‐performance anion‐exchange chromatography‐pulsed amperometric detection showed that RuBGX1 could release xyloses from intermediate xylooligosaccharides produced by endoxylanase. Conclusions: The RuBGX1 shows β‐glucosidase activity in hydrolysis of cello‐oligosaccharides; meanwhile, it has β‐xylosidase activity and functions synergistically with endoxylanase to promote the degradation of hemicellulosic xylans. Significance and Impact of the study: This was the first to report the β‐xylosidase activity of family 3 β‐glucosidase/xylosidase functioned in the degradation of hemicellulosic xylans. The bifunctional β‐glucosidase/xylosidase property of RuBGX1 can be used in simultaneous saccharification of cellulose and xylan into fermentable glucose and xylose. 相似文献
4.
Ju-Yong Park Dong Jun Park Bong-Woo Chung Jiho Min 《Biotechnology and Bioprocess Engineering》2013,18(3):528-532
We constructed recombinant Saccharomyces cerevisiae harboring the xylose isomerase (XI) gene isolated from Clostridium phytofermentans to metabolize xylose and use it as a carbon and energy source. In this study, the effect of supplementation using co-substrate such as glucose or galactose on xylose utilization was studied in recombinant S. cerevisiae. Glucose, which is transported with high affinity by the same transport system as is xylose, was not affected by the heterologous expression of XI, thus xylose utilization was not observed in recombinant S. cerevisiae. However, supplemental galactose added to the recombinant S. cerevisiae stimulated xylose utilization as well as the expression of XI protein. Recombinant S. cerevisiae consumed up to 23.48 g/L of xylose when grown in media containing 40 g/L of xylose and supplemented with 20 g/L of galactose. These cells also produced 15.89 g/L of ethanol. Therefore, expression of the bacterial XI in recombinant S. cerevisiae was highly induced by the addition of supplemental galactose as a co-substrate with xylose, and supplemented galactose enabled the yeast strain to grow on xylose and ferment xylose to ethanol. 相似文献
5.
In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae. 相似文献
6.
【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA_70145和KLMA_80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km_SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km_SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km_SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km_SUT2和Km_SUT3可... 相似文献
7.
Marlin John Mert Daniël Coenrad la Grange Shaunita Hellouise Rose Willem Heber van Zyl 《Journal of industrial microbiology & biotechnology》2016,43(4):431-440
Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes. 相似文献
8.
《PloS one》2014,9(9)
The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH. 相似文献
9.
Madalena Salema-Oom Vera Valad?o Pinto Paula Gon?alves Isabel Spencer-Martins 《Applied microbiology》2005,71(9):5044-5049
Maltotriose utilization by Saccharomyces cerevisiae and closely related yeasts is important to industrial processes based on starch hydrolysates, where the trisaccharide is present in significant concentrations and often is not completely consumed. We undertook an integrated study to better understand maltotriose metabolism in a mixture with glucose and maltose. Physiological data obtained for a particularly fast-growing distiller's strain (PYCC 5297) showed that, in contrast to what has been previously reported for other strains, maltotriose is essentially fermented. The respiratory quotient was, however, considerably higher for maltotriose (0.36) than for maltose (0.16) or glucose (0.11). To assess the role of transport in the sequential utilization of maltose and maltotriose, we investigated the presence of genes involved in maltotriose uptake in the type strain of Saccharomyces carlsbergensis (PYCC 4457). To this end, a previously constructed genomic library was used to identify maltotriose transporter genes by functional complementation of a strain devoid of known maltose transporters. One gene, clearly belonging to the MAL transporter family, was repeatedly isolated from the library. Sequence comparison showed that the novel gene (designated MTY1) shares 90% and 54% identity with MAL31 and AGT1, respectively. However, expression of Mty1p restores growth of the S. cerevisiae receptor strain on both maltose and maltotriose, whereas the closely related Mal31p supports growth on maltose only and Agt1p supports growth on a wider range of substrates, including maltose and maltotriose. Interestingly, Mty1p displays higher affinity for maltotriose than for maltose, a new feature among all the α-glucoside transporters described so far. 相似文献
10.
Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae
Mark F. Vickers James D. Young Stephen A. Baldwin Michael J. Ellison Carol E. Cass 《Molecular membrane biology》2013,30(1):73-79
The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins. 相似文献
11.
Background
Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced.Results
Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways.Conclusion
To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype. 相似文献12.
Background
Engineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. Consequently, engineered xylose-fermenting yeast strains first utilize D-glucose before D-xylose can be transported and metabolized.Results
We have used an evolutionary engineering approach that depends on a quadruple hexokinase deletion xylose-fermenting S. cerevisiae strain to select for growth on D-xylose in the presence of high D-glucose concentrations. This resulted in D-glucose-tolerant growth of the yeast of D-xylose. This could be attributed to mutations at N367 in the endogenous chimeric Hxt36 transporter, causing a defect in D-glucose transport while still allowing specific uptake of D-xylose. The Hxt36-N367A variant transports D-xylose with a high rate and improved affinity, enabling the efficient co-consumption of D-glucose and D-xylose.Conclusions
Engineering of yeast endogenous hexose transporters provides an effective strategy to construct glucose-insensitive xylose transporters that are well integrated in the carbon metabolism regulatory network, and that can be used for efficient lignocellulosic bioethanol production.13.
Saloheimo A Rauta J Stasyk OV Sibirny AA Penttilä M Ruohonen L 《Applied microbiology and biotechnology》2007,74(5):1041-1052
In the present study, we modified xylose uptake properties of a recombinant xylose-utilizing yeast Saccharomyces cerevisiae by expression of heterologous and homologous permease-encoding genes. In a mutant yeast strain with the main seven hexose
transporter genes deleted, and engineered for xylose utilization, we screened an expression cDNA library of the filamentous
fungus Trichoderma reesei (Hypocrea jecorina) for enhanced growth on xylose plates. One cDNA clone with significant homology to fungal sugar transporters was obtained,
but when the clone was retransformed into the host, it did not support significant growth on xylose. However, during a long
liquid culture of the strain carrying the cDNA clone, adaptive mutations apparently occurred in the host, which led to growth
on xylose but not on glucose. The new transporter homologue, Trxlt1 thus appears to code for a protein specific for xylose uptake. In addition, xylose-transporting properties of some homologous
hexose transporters were studied. All of them, i.e., Hxt1, Hxt2, Hxt4, and Hxt7 were capable of xylose uptake. Their affinities
for xylose varied, K
m values between 130 and 900 mM were observed. The single-Hxt strains showed a biphasic growth mode on xylose, alike the Trxlt1 harboring strain. The initial, slow growth was followed by a long lag and finally by exponential growth. 相似文献
14.
Hxt-Carrier-Mediated Glucose Efflux upon Exposure of Saccharomyces cerevisiae to Excess Maltose 总被引:2,自引:0,他引:2
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When wild-type Saccharomyces cerevisiae strains pregrown in maltose-limited chemostat cultures were exposed to excess maltose, release of glucose into the external medium was observed. Control experiments confirmed that glucose release was not caused by cell lysis or extracellular maltose hydrolysis. To test the hypothesis that glucose efflux involved plasma membrane glucose transporters, experiments were performed with an S. cerevisiae strain in which all members of the hexose transporter (HXT) gene family had been eliminated and with an isogenic reference strain. Glucose efflux was virtually eliminated in the hexose-transport-deficient strain. This constitutes experimental proof that Hxt transporters facilitate export of glucose from S. cerevisiae cells. After exposure of the hexose-transport-deficient strain to excess maltose, an increase in the intracellular glucose level was observed, while the concentrations of glucose 6-phosphate and ATP remained relatively low. These results demonstrate that glucose efflux can occur as a result of uncoordinated expression of the initial steps of maltose metabolism and the subsequent reactions in glucose dissimilation. This is a relevant phenomenon for selection of maltose-constitutive strains for baking and brewing. 相似文献
15.
Trey K. Sato Tongjun Liu Lucas S. Parreiras Daniel L. Williams Dana J. Wohlbach Benjamin D. Bice Irene M. Ong Rebecca J. Breuer Li Qin Donald Busalacchi Shweta Deshpande Chris Daum Audrey P. Gasch David B. Hodge 《Applied and environmental microbiology》2014,80(2):540-554
The fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverse S. cerevisiae strains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na+, acetate, and p-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts of pCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production. 相似文献
16.
Metabolic engineering for improved fermentation of pentoses by yeasts 总被引:23,自引:0,他引:23
The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g–1 sugar consumed, so commercialization seems feasible for some applications. 相似文献
17.
Yun-Cheng Li Zi-Xi Gou Ze-Shen Liu Yue-Qin Tang Takashi Akamatsu Kenji Kida 《Biotechnology letters》2014,36(10):2011-2021
In the industrial production of bioethanol from lignocellulosic biomass, a strain of Saccharomyces cerevisiae that can ferment xylose in the presence of inhibitors is of utmost importance. The recombinant, industrial-flocculating S. cerevisiae strain NAPX37, which can ferment xylose, was used as the parent to delete the gene encoding p-nitrophenylphosphatase (PHO13) and overexpress the gene encoding transaldolase (TAL1) to evaluate the synergistic effects of these two genes on xylose fermentation in the presence of weak acid inhibitors, including formic, acetic, or levulinic acids. TAL1 over-expression or PHO13 deletion improved xylose fermentation as well as the tolerance of NAPX37 to all three weak acids. The simultaneous deletion of PHO13 and the over-expression of TAL1 had synergistic effects and improved ethanol production and reduction of xylitol accumulation in the absence and presence of weak acid inhibitors. 相似文献
18.
19.
Vito Porcelli Angelo Vozza Valeria Calcagnile Ruggiero Gorgoglione Roberto Arrigoni Flavia Fontanesi Carlo M.T. Marobbio Alessandra Castegna Ferdinando Palmieri Luigi Palmieri 《BBA》2018,1859(11):1249-1258
The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family (MCF) and 58 MCF members are coded by the genome of Arabidopsis thaliana, most of which have been functionally characterized. Here two members of this family, Ymc2p from S. cerevisiae and BOU from Arabidopsis, have been thoroughly characterized. These proteins were overproduced in bacteria and reconstituted into liposomes. Their transport properties and kinetic parameters demonstrate that Ymc2p and BOU transport glutamate, and to a much lesser extent L-homocysteinesulfinate, but not other amino acids and many other tested metabolites. Transport catalyzed by both carriers was saturable, inhibited by mercuric chloride and dependent on the proton gradient across the proteoliposomal membrane. The growth phenotype of S. cerevisiae cells lacking the genes ymc2 and agc1, which encodes the only other S. cerevisiae carrier capable to transport glutamate besides aspartate, was fully complemented by expressing Ymc2p, Agc1p or BOU. Mitochondrial extracts derived from ymc2Δagc1Δ cells, reconstituted into liposomes, exhibited no glutamate transport at variance with wild-type, ymc2Δ and agc1Δ cells, showing that S. cerevisiae cells grown in the presence of acetate do not contain additional mitochondrial transporters for glutamate besides Ymc2p and Agc1p. Furthermore, mitochondria isolated from wild-type, ymc2Δ and agc1Δ strains, but not from the double mutant ymc2Δagc1Δ strain, swell in isosmotic ammonium glutamate showing that glutamate is transported by Ymc2p and Agc1p together with a H+. It is proposed that the function of Ymc2p and BOU is to transport glutamate across the mitochondrial inner membrane and thereby play a role in intermediary metabolism, C1 metabolism and mitochondrial protein synthesis. 相似文献
20.
Strains of Selenomonas ruminantium vary considerably in their capacity to ferment xylooligosaccharides. This ability ranges from strain GA192, which completely
utilized xylose through xylotetraose and was able to ferment considerable quantities of larger oligosaccharides, to strain
HD4, which used only the simple sugars present in the hydrolysate. The ability of S. ruminantium GA192 to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidase activities. The production
of these activities appears to be regulated in response to carbon source used for growth. Both arabinosidase and xylosidase
were induced by growth on xylose or xylooligosaccharides, but no activity was detected in glucose-or arabinose-grown cultures.
A genetic locus from S. ruminantium GA192 was cloned into Escherichia coli JM83 that produced both xylosidase and arabinosidase activities. Analyses of crude extracts from the E. coli clone and S. ruminantium GA192 by using native polyacrylamide gel electrophoresis and methylumbelliferyl substrates indicated that a single protein
was responsible for both activities. The enzyme expressed in E. coli was capable of degrading xylooligosaccharides derived from xylan. DNA sequencing of the locus demonstrated the presence of
an open reading frame that encodes for a protein of 61,174 molecular weight.
Received: 12 January 2001 / Accepted: 5 March 2001 相似文献