scholarly journals Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

2003 ◽  
Vol 69 (4) ◽  
pp. 1990-1998 ◽  
Author(s):  
Marco Sonderegger ◽  
Uwe Sauer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Selection Procedure ◽  
Pichia Stipitis ◽  
Anaerobic Growth ◽  
Evolutionary Engineering ◽  
Xylose Utilization ◽  
Anaerobic Conditions ◽  
Naturally Occurring ◽  
Microaerobic Conditions ◽  
Utilization Pathway

ABSTRACT Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.

scholarly journals Examination of the Anaerobic Growth ofCampylobacter concisusStrains

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Hoyul Lee ◽  
Rena Ma ◽  
Michael C. Grimm ◽  
Stephen M. Riordan ◽  
Ruiting Lan ◽  
...  
Keyword(s):  
Optimal Growth ◽  
Anaerobic Growth ◽  
Mass Spectrometry Analysis ◽  
Atmospheric Conditions ◽  
Anaerobic Conditions ◽  
Gas Generation ◽  
Spectrometry Analysis ◽  
Virulence Proteins ◽  
Oral Bacterium ◽  
Microaerobic Conditions

Campylobacter concisusis an oral bacterium that is associated with intestinal diseases.C. concisuswas previously described as a bacterium that requires H2-enriched microaerobic conditions for growth. The level of H2in the oral cavity is extremely low, suggesting thatC. concisusis unlikely to have a microaerobic growth there. In this study, the anaerobic growth ofC. concisuswas investigated. The growth of fifty-seven oralC. concisusstrains and six entericC. concisusstrains under various atmospheric conditions including anaerobic conditions with and without H2was examined. The atmospheric conditions were generated using commercially available gas-generation systems.C. concisusputative virulence proteins were identified using mass spectrometry analysis. Under anaerobic conditions, 92% of the oralC. concisusstrains (52/57) and all six enteric strains grew without the presence of H2and the presence of H2greatly increasedC. concisusgrowth. An oralC. concisusstrain was found to express a number of putative virulence proteins and the expression levels of these proteins were not affected by H2. The levels of H2appeared to affect the optimal growth ofC. concisus. This study provides useful information in understanding the natural colonization site and pathogenicity ofC. concisus.

scholarly journals Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

2013 ◽  
Vol 79 (9) ◽  
pp. 2962-2967 ◽  
Author(s):  
Zihe Liu ◽  
Tobias Österlund ◽  
Jin Hou ◽  
Dina Petranovic ◽  
Jens Nielsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Folding ◽  
Protein Secretion ◽  
Electron Acceptor ◽  
Anaerobic Growth ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Content Type ◽  
Final Electron ◽  
Final Electron Acceptor

ABSTRACTIn this study, we focus on production of heterologous α-amylase in the yeastSaccharomyces cerevisiaeunder anaerobic conditions. We compare the metabolic fluxes and transcriptional regulation under aerobic and anaerobic conditions, with the objective of identifying the final electron acceptor for protein folding under anaerobic conditions. We find that yeast produces more amylase under anaerobic conditions than under aerobic conditions, and we propose a model for electron transfer under anaerobic conditions. According to our model, during protein folding the electrons from the endoplasmic reticulum are transferred to fumarate as the final electron acceptor. This model is supported by findings that the addition of fumarate under anaerobic (but not aerobic) conditions improves cell growth, specifically in the α-amylase-producing strain, in which it is not used as a carbon source. Our results provide a model for the molecular mechanism of anaerobic protein secretion using fumarate as the final electron acceptor, which may allow for further engineering of yeast for improved protein secretion under anaerobic growth conditions.

scholarly journals Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase

2010 ◽  
Vol 76 (23) ◽  
pp. 7796-7802 ◽  
Author(s):  
David Runquist ◽  
B�rbel Hahn-H�gerdal ◽  
Maurizio Bettiga
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Parent Strain ◽  
Selection Process ◽  
Xylose Reductase ◽  
Dry Weight ◽  
Batch Cultivation ◽  
Genetically Engineered ◽  
Ethanol Productivity ◽  
Anaerobic Growth ◽  
Xylose Utilization

ABSTRACT Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V max of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased ∼40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] � h versus 0.007 g/g [DW] � h), and the anaerobic growth rate was increased from ∼0 h−1 to 0.08 h−1. The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.

scholarly journals Minimize the Xylitol Production in Saccharomyces cerevisiae by Balancing the Xylose Redox Metabolic Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Yixuan Zhu ◽  
Jingtao Zhang ◽  
Lang Zhu ◽  
Zefang Jia ◽  
Qi Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Budding Yeast ◽  
Ectopic Expression ◽  
Redox Balance ◽  
Inducible Promoter ◽  
Pentose Phosphate ◽  
Xylose Utilization ◽  
Xylose Metabolism ◽  
Utilization Pathway

Xylose is the second most abundant sugar in lignocellulose, but it cannot be used as carbon source by budding yeast Saccharomyces cerevisiae. Rational promoter elements engineering approaches were taken for efficient xylose fermentation in budding yeast. Among promoters surveyed, HXT7 exhibited the best performance. The HXT7 promoter is suppressed in the presence of glucose and derepressed by xylose, making it a promising candidate to drive xylose metabolism. However, simple ectopic expression of both key xylose metabolic genes XYL1 and XYL2 by the HXT7 promoter resulted in massive accumulation of the xylose metabolic byproduct xylitol. Through the HXT7-driven expression of a reported redox variant, XYL1-K270R, along with optimized expression of XYL2 and the downstream pentose phosphate pathway genes, a balanced xylose metabolism toward ethanol formation was achieved. Fermented in a culture medium containing 50 g/L xylose as the sole carbon source, xylose is nearly consumed, with less than 3 g/L xylitol, and more than 16 g/L ethanol production. Hence, the combination of an inducible promoter and redox balance of the xylose utilization pathway is an attractive approach to optimizing fuel production from lignocellulose.

scholarly journals Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of l-Arabinose

2007 ◽  
Vol 73 (15) ◽  
pp. 4881-4891 ◽  
Author(s):  
H. Wouter Wisselink ◽  
Maurice J. Toirkens ◽  
M. del Rosario Franco Berriel ◽  
Aaron A. Winkler ◽  
Johannes P. van Dijken ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Alcoholic Fermentation ◽  
Plant Biomass ◽  
Ethanol Yield ◽  
Dry Weight ◽  
Pentose Phosphate ◽  
Anaerobic Growth ◽  
Evolutionary Engineering ◽  
First Case

ABSTRACT For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h−1 g [dry weight]−1) and ethanol production (0.29 g h−1 g [dry weight]−1) and a high ethanol yield (0.43 g g−1) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.

scholarly journals Construction of a Xylan-Fermenting Yeast Strain through Codisplay of Xylanolytic Enzymes on the Surface of Xylose-Utilizing Saccharomyces cerevisiae Cells

2004 ◽  
Vol 70 (9) ◽  
pp. 5407-5414 ◽  
Author(s):  
Satoshi Katahira ◽  
Yasuya Fujita ◽  
Atsuko Mizuike ◽  
Hideki Fukuda ◽  
Akihiko Kondo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Yeast Strain ◽  
High Performance ◽  
Surface Engineering ◽  
Xylose Reductase ◽  
Pichia Stipitis ◽  
Direct Conversion ◽  
Engineering System ◽  
Xylose Utilization

ABSTRACT Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan. We used a cell surface engineering system based on α-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase II (XYNII) from Trichoderma reesei QM9414 and β-xylosidase (XylA) from Aspergillus oryzae NiaD300, on the cell surface. In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XylA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface. These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA. In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S. cerevisiae strain was constructed by codisplaying XYNII and XylA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g. These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.

scholarly journals Ability for Anaerobic Growth Is Not Sufficient for Development of the Petite Phenotype in Saccharomyces kluyveri

2001 ◽  
Vol 183 (8) ◽  
pp. 2485-2489 ◽  
Author(s):  
Kasper Mo/ller ◽  
Lisbeth Olsson ◽  
Jure Piškur
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Positive Characteristic ◽  
Kluyveromyces Lactis ◽  
Sensu Stricto ◽  
Point Of View ◽  
The Other ◽  
Anaerobic Growth ◽  
Anaerobic Conditions ◽  
Saccharomyces Kluyveri ◽  
Petite Mutants

ABSTRACT Saccharomyces cerevisiae is a petite-phenotype-positive (“petite-positive”) yeast, which can successfully grow in the absence of oxygen. On the other hand, Kluyveromyces lactisas well as many other yeasts are petite negative and cannot grow anaerobically. In this paper, we show that Saccharomyces kluyveri can grow under anaerobic conditions, but while it can generate respiration-deficient mutants, it cannot generate true petite mutants. From a phylogenetic point of view, S. kluyveri is apparently more closely related to S. cerevisiae than toK. lactis. These observations suggest that the progenitor of the modern Saccharomyces and Kluyveromycesyeasts, as well as other related genera, was a petite-negative and aerobic yeast. Upon separation of the K. lactis andS. kluyveri-S. cerevisiae lineages, the latter developed the ability to grow anaerobically. However, while the S. kluyveri lineage has remained petite negative, the lineage leading to the modern Saccharomyces sensu stricto and sensu lato yeasts has developed the petite-positive characteristic.

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