Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Functional Analysis of Two l-Arabinose Transporters from Filamentous Fungi Reveals Promising Characteristics for Improved Pentose Utilization in Saccharomyces cerevisiae.

ABSTRACT: Limited uptake is one of the bottlenecks for l-arabinose fermentation from lignocellulosic hydrolysates in engineered Saccharomyces cerevisiae. This study characterized two novel l-arabinose transporters, LAT-1 from Neurospora crassa and MtLAT-1 from Myceliophthora thermophila. Although the two proteins share high identity (about 83%), they display different substrate specificities. Sugar transport assays using the S. cerevisiae strain EBY.VW4000 indicated that LAT-1 accepts a broad substrate spectrum. In contrast, MtLAT-1 appeared much more specific for l-arabinose. Determination of the kinetic properties of both transporters revealed that the Km values of LAT-1 and MtLAT-1 for l-arabinose were 58.12 ± 4.06 mM and 29.39 ± 3.60 mM, respectively, with corresponding Vmax values of 116.7 ± 3.0 mmol/h/g dry cell weight (DCW) and 10.29 ± 0.35 mmol/h/g DCW, respectively. In addition, both transporters were found to use a proton-coupled symport mechanism and showed only partial inhibition by d-glucose during l-arabinose uptake. Moreover, LAT-1 and MtLAT-1 were expressed in the S. cerevisiae strain BSW2AP containing an l-arabinose metabolic pathway. Both recombinant strains exhibited much faster l-arabinose utilization, greater biomass accumulation, and higher ethanol production than the control strain. In conclusion, because of higher maximum velocities and reduced inhibition by d-glucose, the genes for the two characterized transporters are promising targets for improved l-arabinose utilization and fermentation in S. cerevisiae.

SUBMITTER: Li J

PROVIDER: S-EPMC4524138 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Improving L-arabinose utilization of pentose fermenting Saccharomyces cerevisiae cells by heterologous expression of L-arabinose transporting sugar transporters.

Project description:BackgroundHydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations.ResultsHere we described the cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations.ConclusionsUsing a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations.

| S-EPMC3216861 | biostudies-literature

Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway.

Project description:BackgroundSustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation.ResultsA new recombinant S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized.ConclusionPentose fermenting recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar.

| S-EPMC2720912 | biostudies-literature

Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae.

Project description:Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.

| S-EPMC3208632 | biostudies-literature

Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae.

Project description:The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

| S-EPMC5591001 | biostudies-literature

Heterologous transporters from anaerobic fungi bolster fluoride tolerance in <i>Saccharomyces cerevisiae</i>.

Project description:Membrane-embedded transporters are crucial for the stability and performance of microbial production strains. Apart from engineering known transporters derived from model systems, it is equally important to identify transporters from nonconventional organisms that confer advantageous traits for biotechnological applications. Here, we transferred genes encoding fluoride exporter (FEX) proteins from three strains of early-branching anaerobic fungi (Neocallimastigomycota) to Saccharomyces cerevisiae. The heterologous transporters are localized to the plasma membrane and complement a fluoride-sensitive yeast strain that is lacking endogenous fluoride transporters up to 10.24 mM fluoride. Furthermore, we show that fusing an amino-terminal leader sequence to FEX proteins in yeast elevates protein yields, yet inadvertently causes a loss of transporter function. Adaptive laboratory evolution of FEX proteins restores fluoride tolerance of these strains, in one case exceeding the solute tolerance observed in wild type S. cerevisiae; however, the underlying molecular mechanisms and cause for the increased tolerance in the evolved strains remain elusive. Our results suggest that microbial cultures can achieve solvent tolerance through different adaptive trajectories, and the study is a promising step towards the identification, production, and biotechnological application of membrane proteins from nonconventional fungi.

| S-EPMC6475669 | biostudies-literature

Transcriptomic profiling of engineered and improved Saccharomyces cerevisiae for xylose utilization

Project description:The aim of present study is to understand the impact of xylose utilization on the Saccharomyces cerevisiae physiology after initial genetic engineering and in a strain with an improved xylose utilization phenotype.

2016-09-30 | GSE72785 | GEO

Effect of C-terminal protein tags on pentitol and L-arabinose transport by Ambrosiozyma monospora Lat1 and Lat2 transporters in Saccharomyces cerevisiae.

Project description:Functional expression in heterologous hosts is often less successful for integral membrane proteins than for soluble proteins. Here, two Ambrosiozyma monospora transporters were successfully expressed in Saccharomyces cerevisiae as tagged proteins. Growth of A. monospora on l-arabinose instead of glucose caused transport activities of l-arabinose, l-arabitol, and ribitol, measured using l-[1-(3)H]arabinose, l-[(14)C]arabitol, and [(14)C]ribitol of demonstrated purity. A. monospora LAT1 and LAT2 genes were cloned earlier by using their ability to improve the growth of genetically engineered Saccharomyces cerevisiae on l-arabinose. However, the l-arabinose and pentitol transport activities of S. cerevisiae carrying LAT1 or LAT2 are only slightly greater than those of control strains. S. cerevisiae carrying the LAT1 or LAT2 gene fused in frame to the genes for green fluorescent protein (GFP) or red fluorescent protein (mCherry) or adenylate kinase (AK) exhibited large (>3-fold for LAT1; >20-fold for LAT2) increases in transport activities. Lat1-mCherry transported l-arabinose with high affinity (Km ? 0.03 mM) and l-arabitol and ribitol with very low affinity (Km ? 75 mM). The Lat2-GFP, Lat2-mCherry, and Lat2-AK fusion proteins could not transport l-arabinose but were high-affinity pentitol transporters (Kms ? 0.2 mM). The l-arabinose and pentitol transport activities of A. monospora could not be completely explained by any combination of the observed properties of tagged Lat1 and Lat2, suggesting either that tagging and expression in a foreign membrane alters the transport kinetics of Lat1 and/or Lat2 or that A. monospora contains at least one more l-arabinose transporter.

| S-EPMC3993308 | biostudies-literature

Augmenting Pentose Utilization and Ethanol Production of Native <i>Saccharomyces cerevisiae</i> LN Using Medium Engineering and Response Surface Methodology.

Project description:Economics of ethanol production from lignocellulosic biomass depends on complete utilization of constituent carbohydrates and efficient fermentation of mixed sugars present in biomass hydrolysates. Saccharomyces cerevisiae, the commercial strain for ethanol production uses only glucose while pentoses remain unused. Recombinant strains capable of utilizing pentoses have been engineered but with limited success. Recently, presence of endogenous pentose assimilation pathway in S. cerevisiae was reported. On the contrary, evolutionary engineering of native xylose assimilating strains is promising approach. In this study, a native strain S. cerevisiae LN, isolated from fruit juice, was found to be capable of xylose assimilation and mixed sugar fermentation. Upon supplementation with yeast extract and peptone, glucose (10%) fermentation efficiency was 78% with ~90% sugar consumption. Medium engineering augmented mixed sugars (5% glucose + 5% xylose) fermentation efficiency to ~50 and 1.6% ethanol yield was obtained with concomitant sugar consumption ~60%. Statistical optimization of input variables Glucose (5.36%), Xylose (3.30%), YE (0.36%), and peptone (0.25%) with Response surface methodology led to improved sugar consumption (74.33%) and 2.36% ethanol within 84 h. Specific activities of Xylose Reductase and Xylitol Dehydrogenase exhibited by S. cerevisiae LN were relatively low. Their ratio indicated metabolism diverted toward ethanol than xylitol and other byproducts. Strain was tolerant to concentrations of HMF, furfural and acetic acid commonly encountered in biomass hydrolysates. Thus, genetic setup for xylose assimilation in S. cerevisiae LN is not merely artifact of xylose metabolizing pathway and can be augmented by adaptive evolution. This strain showed potential for commercial exploitation.

| S-EPMC6166573 | biostudies-literature

Comparative transcriptomes reveal novel evolutionary strategies adopted by Saccharomyces cerevisiae with improved xylose utilization capability

Project description:The xylose fermentation capability of an industrainl Saccharomyces cerevisiae strain was enhanced by adaptive evolution. Eight homozygots were generated by tetrads dissection. The underlying molecular basis of the enhanced xylose fermentation capability was analyzed.

2016-04-30 | GSE80822 | GEO

Xylose Utilization Gene in Saccharomyces cerevisiae

Project description:This SuperSeries is composed of the SubSeries listed below.

2010-04-09 | GSE19121 | GEO