Fed-batch xylitol production with two recombinant Saccharomyces cerevisiae strains expressing XYL1 at different levels, using glucose as a cosubstrate: A comparison of production parameters and strain stability

Meinander, Nina Q.; Hahn‐Hägerdal, Bärbel

doi:10.1002/(sici)1097-0290(19970520)54:4<391::aid-bit12>3.0.co;2-j

Cited by 46 publications

(31 citation statements)

References 25 publications

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“…This is exemplified by an experiment in which a leu2 strain of S. cerevisiae, transformed with a LEU2-bearing expression vector, was grown in carbon-limited chemostat cultures on a synthetic medium without leucine. After prolonged cultivation, up to 45% of the population consisted of leucine-auxotrophic cells and low concentrations of leucine were detectable in culture supernatants (27). Therefore, even under apparently selective conditions, it is advisable to check plasmid stability, e.g., by performing plate assays.…”

Section: Stability Of Transformed Strains and Cross-feedingmentioning

confidence: 99%

Auxotrophic Yeast Strains in Fundamental and Applied Research

Pronk

2002

Appl Environ Microbiol

265

224

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Section: Stability Of Transformed Strains and Cross-feedingmentioning

confidence: 99%

Auxotrophic Yeast Strains in Fundamental and Applied Research

Pronk

2002

Appl Environ Microbiol

265

224

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“…introduction of XYLA from Piromyces and overexpression of endogenous XKS1 , RPE1 , RKI1 , TAL1 , and TKL1 ) and adaptive evolution in xylose media can generate efficient xylose-utilizing strains [4,8,14]. However, there are two drawbacks for these studies: 1) laboratory haploid strains were chosen as hosts, which are generally considered not as robust as industrial diploid strains when fermenting lignocellulosic biomass hydrolysates [1] and 2) plasmid-based protein expression was employed, which is regarded as not stable as integration-based protein expression [15,16]. Although genome integration of xylose isomerase was pursued by Tanino et al [17], however, a laboratory haploid strain was selected to construct xylose-fermenting yeast.…”

Section: Introductionmentioning

confidence: 99%

Construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiaeby rational design and adaptive evolution

et al. 2013

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BackgroundIt remains a challenge for recombinant S. cerevisiae to convert xylose in lignocellulosic biomass hydrolysates to ethanol. Although industrial diploid strains are more robust compared to laboratory haploid strains, however, industrial diploid S. cerevisiae strains have been less pursued in previous studies. This work aims to construct fast xylose-fermenting yeast using an industrial ethanol-producing diploid S. cerevisiae strain as a host.ResultsFast xylose-fermenting yeast was constructed by genome integration of xylose-utilizing genes and adaptive evolution, including 1) Piromyces XYLA was introduced to enable the host strain to convert xylose to xylulose; 2) endogenous genes (XKS1, RKI1, RPE1, TKL1, and TAL1) were overexpressed to accelerate conversion of xylulose to ethanol; 3) Candida intermedia GXF1, which encodes a xylose transporter, was introduced at the GRE3 locus to improve xylose uptake; 4) aerobic evolution in rich xylose media was carried out to increase growth and xylose consumption rates. The best evolved strain CIBTS0735 consumed 80 g/l glucose and 40 g/l xylose in rich media within 24 hours at an initial OD600 of 1.0 (0.63 g DCW/l) and produced 53 g/l ethanol.ConclusionsBased on the above fermentation performance, we conclude that CIBTS0735 shows great potential for ethanol production from lignocellulosic biomass.

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“…Translated to generation time, strain TMB 3001 was stable for more than 40 generations, which is considerably longer than the four to five generations of stability reported for strain 1400 (pLNH32), which carries the same genes on a 2m-derived vector (22). Recombinant xylose-utilizing Saccharomyces strains carrying 2m-based vectors have been stably maintained in batch cultivation (22,24,27,44,48), but in continuous cultivation they tend to be unstable (27,28). The instability of strains carrying 2m-based vectors may result from genetic instability at the plasmid level, i.e., spontaneous loss of the transformed phenotype and the plasmid (19,26,28) or high frequency of recombination, resulting in cells that still carry the selectable marker but have lost the cloned gene (15,28).…”

mentioning

confidence: 99%

“…Recombinant xylose-utilizing Saccharomyces strains carrying 2m-based vectors have been stably maintained in batch cultivation (22,24,27,44,48), but in continuous cultivation they tend to be unstable (27,28). The instability of strains carrying 2m-based vectors may result from genetic instability at the plasmid level, i.e., spontaneous loss of the transformed phenotype and the plasmid (19,26,28) or high frequency of recombination, resulting in cells that still carry the selectable marker but have lost the cloned gene (15,28). Consequently, strains carrying less foreign DNA usually dominate the culture since the high-copy-number plasmids and the high expression of the heterologous genes can reduce the growth rate and glycolytic flux (26)(27)(28)43).…”

mentioning

confidence: 99%

“…The instability of strains carrying 2m-based vectors may result from genetic instability at the plasmid level, i.e., spontaneous loss of the transformed phenotype and the plasmid (19,26,28) or high frequency of recombination, resulting in cells that still carry the selectable marker but have lost the cloned gene (15,28). Consequently, strains carrying less foreign DNA usually dominate the culture since the high-copy-number plasmids and the high expression of the heterologous genes can reduce the growth rate and glycolytic flux (26)(27)(28)43). For the construction of TMB 3001, a single target sequence rather than multiple integrations in ribosomal DNA or transposons was chosen to diminish the burden on the cells caused by expression of foreign DNA.…”

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confidence: 99%

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Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1 , XYL2 , and XKS1 in Mineral Medium Chemostat Cultures

Eliasson

Christensson

Wahlbom

et al. 2000

Appl Environ Microbiol

356

269

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For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. To obtain an economically feasible industrial process for ethanol production from lignocellulose, it is necessary to ferment all sugars present with high yields and productivities (53). The commonly used Saccharomyces cerevisiae has many advantages as an ethanol producer, such as fast sugar consumption, high ethanol yield from hexoses, and high resistance to inhibitory compounds that are present in the hydrolysates. However, a major drawback is that S. cerevisiae cannot utilize the pentose sugar xylose, only its isomer xylulose. In xylose-utilizing yeasts, the conversion from xylose to xylulose is a two-step process catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH) (10), whereas bacteria perform the conversion in one step with xylose isomerase (XI) (23).Xylose fermentation by recombinant S. cerevisiae carrying heterologous XYL1 and XYL2 genes from Pichia stipitis, which encode XR and XDH, respectively, has resulted mainly in xylitol formation (24,44,48). Similarly, if xylA from Thermus thermophilus, which encodes XI, is introduced into S. cerevisiae, then only limited xylose fermentation is observed (47). Limited xylose fermentation by recombinant S. cerevisiae has been ascribed to poor xylose uptake (9, 24, 25), a cofactor imbalance generated by the discrepancy in cofactor usage by XR and XDH (8,24,49), limitations in the pentose phosphate pathway (12,24,38,48), and insufficient induction or activation of ethanologenic enzymes (5,17,20,29). When homologous XKS1, which encodes xylulokinase (XK), was overexpressed in a Saccharomyces sp. strain carrying XYL1 and XYL2, the ethanol yield and the xylose uptake rate increased under oxygenlimited conditions, but xylitol was still a major by-product (22).Although the shortcomings of xylose fermentation by recombinant S. cerevisiae have been investigated in several studies, data from anaerobic fermentations do not exist and quantitative data are sparse. Chemostat cultivations in which growth rate and concentrations of substrates and products are constant enable quantitative determinations of metabolic fluxes. Analysis o...

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Fed-batch xylitol production with two recombinant Saccharomyces cerevisiae strains expressing XYL1 at different levels, using glucose as a cosubstrate: A comparison of production parameters and strain stability

Cited by 46 publications

References 25 publications

Auxotrophic Yeast Strains in Fundamental and Applied Research

Auxotrophic Yeast Strains in Fundamental and Applied Research

Construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiaeby rational design and adaptive evolution

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1 , XYL2 , and XKS1 in Mineral Medium Chemostat Cultures

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