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

2015 ◽  
Vol 81 (12) ◽  
pp. 4062-4070 ◽  
Author(s):  
Jingen Li ◽  
Jing Xu ◽  
Pengli Cai ◽  
Bang Wang ◽  
Yanhe Ma ◽  
...  

ABSTRACTLimited uptake is one of the bottlenecks forl-arabinose fermentation from lignocellulosic hydrolysates in engineeredSaccharomyces cerevisiae. This study characterized two novell-arabinose transporters, LAT-1 fromNeurospora crassaand MtLAT-1 fromMyceliophthora thermophila. Although the two proteins share high identity (about 83%), they display different substrate specificities. Sugar transport assays using theS. cerevisiaestrain EBY.VW4000 indicated that LAT-1 accepts a broad substrate spectrum. In contrast, MtLAT-1 appeared much more specific forl-arabinose. Determination of the kinetic properties of both transporters revealed that theKmvalues of LAT-1 and MtLAT-1 forl-arabinose were 58.12 ± 4.06 mM and 29.39 ± 3.60 mM, respectively, with correspondingVmaxvalues 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 byd-glucose duringl-arabinose uptake. Moreover, LAT-1 and MtLAT-1 were expressed in theS. cerevisiaestrain BSW2AP containing anl-arabinose metabolic pathway. Both recombinant strains exhibited much fasterl-arabinose utilization, greater biomass accumulation, and higher ethanol production than the control strain. In conclusion, because of higher maximum velocities and reduced inhibition byd-glucose, the genes for the two characterized transporters are promising targets for improvedl-arabinose utilization and fermentation inS. cerevisiae.

2012 ◽  
Vol 79 (5) ◽  
pp. 1500-1507 ◽  
Author(s):  
Suk-Jin Ha ◽  
Heejin Kim ◽  
Yuping Lin ◽  
Myoung-Uoon Jang ◽  
Jonathan M. Galazka ◽  
...  

ABSTRACTSaccharomyces cerevisiaecannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes fromNeurospora crassa. Here, we report that an engineeredS. cerevisiaestrain expressing the putative hexose transporter geneHXT2.4fromScheffersomyces stipitisandgh1-1can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter whenHXT2.4is overexpressed inS. cerevisiae. However, cellobiose fermentation by the engineered strain expressingHXT2.4andgh1-1was much slower and less efficient than that by an engineered strain that initially expressedcdt-1andgh1-1. The rate of cellobiose fermentation by theHXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolvedS. cerevisiaestrain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higherKmand 4-fold higherVmaxvalues than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed inS. cerevisiaeare suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineeredS. cerevisiaestrains.


2021 ◽  
Vol 29 (1) ◽  
pp. 39-46
Author(s):  
O. M. Moroz ◽  
G. I. Zvir ◽  
S. O. Hnatush

Pollutants of inorganic nature (acids, alkalis, mineral salts of different composition, metals) change the course of biological processes of environmental purification, but their influence on the physiological properties of phototrophic sulfur bacteria has not been studied enough. The usage of nitrite ions as an electron donor of anoxygenic photosynthesis by cells of phototrophic green and purple sulfur bacteria Chlorobium limicola IMV K-8, Thiocapsa sp. Ya-2003 and Lamprocystis sp. Ya-2003, isolated from Yavorivske Lake, under the influence of the most widespread inorganic pollutants – hydro- and dihydrophosphates, sulfates, chlorides and chlorates, has been studied. It is shown that KH2PO4, K2HPO4, Na2SO4, NaCl and KClO3, present in the van Niel medium with 4.2 mM NaNO2 at concentrations that are 0.5, 1.0, 2.0, 3.0, 4.0 times different from the maximum permissible concentrations (MPC), influenced the biomass accumulation and nitrite ions oxidation by phototrophic green and purple sulfur bacteria. In media with hydro- and dihydrophosphate ions at concentrations 4.0 times higher than the MPC, inhibition of bacterial growth was up to 1.7 times lower than in the control. The biomass accumulation by bacteria in media with chloride and chlorate ions at concentrations 3.0–4.0 times higher than MPC was 2.0–2.8 times lower compared to the control. In the medium with Na2SO4 at concentrations 2.0–4.0 times higher than MPC, the biomass was 2.0–4.0 times lower than in the control. Nitrites’ oxidation by all strains in the media with the studied pollutants was slowed down. The residual content of nitrite ions in media with hydro- and dihydrophosphate, chloride and chlorate ions at their concentrations 4.0 times higher than MPC, exceeded the NO2– content in the control variants up to 1.7 times. If in the medium without pollutants the cells of C. limicola IMV K-8, Thiocapsa sp. Ya-2003 and Lamprocystis sp. Ya-2003 strains oxidized 72.7%, 72.2% and 71.4%, respectively, of nitrite ions present in the medium, then in the medium with sulfate ions at concentration 4.0 times higher than the MPC, bacteria oxidized nitrite ions only at 39.6%, 34.4% and 27.0%, respectively. Oxidation of a lower quantity of nitrites by phototrophic bacteria in the media with inorganic pollutants led to the production by them of a lower quantity of nitrates. The content of NO3– in the media with hydro-, dihydrophosphate and chlorate ions at all concentrations was up to 1.9 times lower than in the control. In media with sulfate ions at concentrations 2.0–4.0 times higher than MPC and chloride at concentration 4.0 times higher than MPC, the content of nitrate ions was 2.1–4.3 and 2.0 times, respectively, lower than in the control variants. Inorganic pollutants stimulated the synthesis of intracellular carbohydrates in C. limicola IMV K-8. If the content of intracellular glucose in cells grown in the medium without pollutants was 10.3 mg/g dry cell weight, then in cells grown in media with K2HPO4, KH2PO4, Na2SO4, NaCl and KClO3 at concentrations 4.0 times higher than MPC, its content increased by 12.2%, 10.7%, 51.6%, 17.1% and 35.9%, respectively. The glycogen content in the cells grown in the medium without pollutants was 45.1 mg/g dry cell weight. Hydro- and dihydrophosphate, chloride and chlorate ions at concentrations 4.0 times higher than MPC stimulated glycogen synthesis in cells by 47.5%, 57.6%, 67.4% and 74.6%, respectively. The glycogen content in cells grown in the medium with Na2SO4 at concentrations 3.0 and 4.0 times higher than MPC increased by 102.9% and 107.5%, respectively. Therefore, it is established that pollutants of inorganic nature affect the physiological properties of photosynthetic sulfur bacteria and thus change the course of biological processes of environment purification, in particular, from nitrite ions.


2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Chengqiang Wang ◽  
Yu Shen ◽  
Yanyan Zhang ◽  
Fan Suo ◽  
Jin Hou ◽  
...  

The L-arabinose utilization pathway was established inSaccharomyces cerevisiae, by expressing the codon-optimizedaraA,araB, andaraDgenes ofLactobacillus plantarum. After overexpressing theTAL1,TKL1,RPE1,RKI1, andGAL2genes and adaptive evolution, the L-arabinose utilization of the recombinant strain became efficient. The resulting strain displayed a maximum specific growth rate of 0.075 h−1, a maximum specific L-arabinose consumption rate of 0.61 g h−1 g−1dry cell weight, and a promising ethanol yield of 0.43 g g−1from L-arabinose fermentation.


2013 ◽  
Vol 80 (5) ◽  
pp. 1660-1669 ◽  
Author(s):  
Falk Matthäus ◽  
Markus Ketelhot ◽  
Michael Gatter ◽  
Gerold Barth

ABSTRACTThe codon-optimized genescrtBandcrtIofPantoea ananatiswere expressed inYarrowia lipolyticaunder the control of theTEF1promoter ofY. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis inY. lipolytica,GGS1andHMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in aY. lipolyticastrain withPOX1toPOX6andGUT2deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity ofY. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g−1(dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host.


2015 ◽  
Vol 14 (6) ◽  
pp. 564-577 ◽  
Author(s):  
Geovani López ◽  
Héctor Quezada ◽  
Mariana Duhne ◽  
James González ◽  
Mijail Lezama ◽  
...  

ABSTRACTProduction of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeastSaccharomyces cerevisiaehas two paralogous genes,LEU4andLEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. Theleu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of theleu4Δ mutant with a centromeric plasmid carryingLEU4restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formedin vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine–α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.


Microbiology ◽  
2000 ◽  
Vol 81 (2) ◽  
pp. 373-382 ◽  
Author(s):  
FRANCOISE VEZINHET ◽  
M. ROGER ◽  
MONIQUE PELLECUER ◽  
P. GALZY

Summary: The biochemical modifications of two yeast strains, a/α and α/α, have been studied during incubation in a sporulation medium. The increases in dry cell weight, protein, carbohydrate and lipid contents, as well as the variation in respiration rate are quite similar for the two strains. Mating type gene control of sporulation is discussed.


2011 ◽  
Vol 77 (10) ◽  
pp. 3311-3319 ◽  
Author(s):  
Eric Young ◽  
Ashley Poucher ◽  
Austin Comer ◽  
Alexandra Bailey ◽  
Hal Alper

ABSTRACTMolecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism inSaccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinantSaccharomyces cerevisiaehost unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes ofScheffersomyces stipitisand open reading frames from the yeastsYarrowia lipolyticaandDebaryomyces hansenii.Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised ofD. hanseniiXylHPandS. stipitisXUT1andXUT3demonstrated moderate transport efficiency and higher xylose preferences.


2001 ◽  
Vol 67 (3) ◽  
pp. 1280-1283 ◽  
Author(s):  
Young-Mi Shin ◽  
Tae-Ho Kwon ◽  
Kyung-Suk Kim ◽  
Keon-Sang Chae ◽  
Dae-Hyuk Kim ◽  
...  

ABSTRACT We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 μg per gram (dry cell weight) of the recombinant yeast but was 210 μg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.


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