The isolation of strains of Saccharomyces cerevisiae showing altered plasmid stability characteristics by means of selective continuous culture

O’Kennedy, Ronan; Patching, John W

doi:10.1016/s0168-1656(99)00047-4

Cited by 3 publications

(3 citation statements)

References 33 publications

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“…After 460 generations, the population consisted of at least two subpopulations with distinct phenotypes and thus exhibited the heterogeneity (or polymorphism) that is often observed during evolution experiments (11,18,23,29,34). The phenotype of the smaller class I subpopulation, representing one-third of the clones isolated, was rather similar to that of parental strain TMB3001 on glucose but was significantly improved on xylose.…”

Section: Discussionmentioning

confidence: 94%

“…Since chemostat-evolved, asexual populations are typically heterogeneous (18,23,34), an aliquot of the population after 460 generations was plated on xylose minimal medium. Under anaerobic conditions the number of CFU was 54% Ϯ 4% of the number of CFU found on aerobic YPD medium plates, thus providing the first evidence that there was population heterogeneity.…”

Section: Resultsmentioning

confidence: 99%

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Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

Sonderegger

Sauer

2003

Appl Environ Microbiol

236

169

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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.Over the last decade, metabolic engineering has become a standard procedure for strain improvement and has been very successful when simple cellular traits have been targeted (19,26). Although the genomics age and the associated genomewide analytical technologies provide further impetus for rational approaches, metabolic engineering of more complex cellular systems or cellular systems that are not fully understood remains a challenge (4). Along with random, combinatorial approaches, such as directed evolution in contemporary protein engineering (3), evolutionary approaches are becoming increasingly important for augmenting metabolic engineering of complex phenotypes (21,22,24,36). In certain cases, however, even seemingly simple metabolic systems resist straightforward rational engineering.

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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

Sonderegger

Sauer

2003

Appl Environ Microbiol

236

169

View full text Add to dashboard Cite

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“…A critical factor for successful selection of segregationally stable host-vector combinations is the selection pressure applied. While the above positive selections for antibiotic resistant cells were successful, a similar experiment that used a negative selection for plasmid-bearing clones of S. cerevisiae with an auxotrophic marker did not enrich for more stable clones over a period of 420 generations [126]. Although a large variety of clones with altered recombinant plasmid stability evolved over time, it appeared to be mainly a result of non-specific periodic selection.…”

Section: Plasmid Stabilitymentioning

confidence: 99%