New generation of <i>lox</i>P‐mutated deletion cassettes for the genetic manipulation of yeast natural isolates

Carter, Zorana; Delneri, Daniela

doi:10.1002/yea.1774

Cited by 70 publications

(60 citation statements)

References 35 publications

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“…Gene deletions in EBY.VW4000 were performed using the loxP/cre recombinase system with kanMX and hphNT1 markers (47,48). Further details are found in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Farwick

Bruder

Schadeweg

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

274

347

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All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growthbased screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.xylose transport | major facilitator superfamily | transporter engineering | pentose metabolism | HXT

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“…Gene deletions in EBY.VW4000 were performed using the loxP/cre recombinase system with kanMX and hphNT1 markers (47,48). Further details are found in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Farwick

Bruder

Schadeweg

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

274

347

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“…The plasmids pHD8 and pHD22 were constructed by homologous recombination in yeast from up to 17 single overlapping PCR fragments. The templates used were genomic DNA from S. cerevisiae , the plasmids pUG6 [73], pZC1 [74], p426H-i-opt.XI [24], YEparaAsynth and YEparaDsynth [33] as well as the codon-optimized genes of XKS1 and E.coli araB (method described in [33] - Sloning BioTechnology) and NQM1 and TKL2 (DNA2.0). The assembly of the multi-copy plasmid carrying the XylA gene, was similar to the plasmid pHD8, but the genes flanked by the i1 and i3 regions (see Figure 1a) were substituted by restriction digestion and ligation with one copy of the XylA gene.…”

Section: Methodsmentioning

confidence: 99%

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Demeke

Dietz

et al. 2013

Biotechnol Biofuels

279

213

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BackgroundThe production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production.ResultsAn expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate.ConclusionsAn industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates.

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“…Strains were constructed by standard methods, either by crosses or by transformation (Ausubel, 2001). The deletion alleles were created by replacing the respective open reading frame (ORF) with the KanMX or NatMX (Brachmann et al , 1998) or HphMX4 markers (Carter and Delneri, 2010). The KanMX::GAL1 pr -ORF alleles (where ORF = SAM2 , MUP1 , MUP3 , or INO1 ) was constructed by placing the KanMX marker and the GAL1 promoter upstream of the respective ORF (Longtine et al , 1998).…”

Section: Methodsmentioning

confidence: 99%

Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast

Hickman

Petti

Ho-Shing

et al. 2011

MBoC

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New generation of loxP‐mutated deletion cassettes for the genetic manipulation of yeast natural isolates

Cited by 70 publications

References 35 publications

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast

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