Adaptive laboratory evolution—Principles and applications for biotechnology

In the paper

“another study reported the emergence of aneuploidy during glucose-limited growth and amplification of the HXT6 gene with significant divergence between co-evolved populations [94].”

What the sources say

• Disputed

  Materials and Methods (page unavailable) says the three S. cerevisiae lines evolved on galactose minimal medium, not during glucose-limited growth. The Results (page unavailable) report mutations in RAS2 and CYR1, with an additional ERG5 mutation in one strain; our copy does not report aneuploidy or HXT6 amplification. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  cites [94] sourceExperimental — accuracy not yet measured

In the paper

“Whereas certain environments can lead to fast selection of improved phenotypes, others, including the long-term adaptation to environmental pH [59,63], represent complex environments where microbial cells show low adaptive potential in terms of absolute fitness gain.”

What the sources say

• Partly supported

  Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  cites [59] sourceExperimental — accuracy not yet measured
• Not checked yetcites [63] source

In the paper

“Early studies in glucose-limited chemostats revealed that glucose-limited selection led to increased biomass yield and a decreased fermentative capacity [93,98].”

What the sources say

• ?Could not verify

  The Continuous cultivation and Results passages describe nitrogen-limited chemostat selection for growth of an engineered strain, not glucose-limited selection. Searches for glucose-limited chemostats, biomass yield, and fermentative capacity found no passage in our copy reporting the claimed combination of increased yield and decreased fermentative capacity. Page numbers were not available. (LLM, conf=0.74)

  cites [93] sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Discussion describes selection by repetitive shake-flask cultivation on xylose, not glucose-limited chemostat selection. The Batch glucose fermentation section reports a small reduction in ethanol production for the evolved strain, but does not establish decreased fermentative capacity or increased biomass yield following glucose-limited selection. Searches for glucose-limited chemostats and those outcomes found no passage addressing the claim. (LLM, conf=0.76)

  cites [98] sourceExperimental — accuracy not yet measured

In the paper

“As such, ALE studies highlight that duplications and large scale genomic variations, although they can lead to significant issues such as genomic instability [152], play a major role in e.g. yeast evolution; this is also indicated by data on wine yeasts, galactose utilization, comparative genomics and drug resistance [115,153-156].”

What the sources say

• Not checked yetcites [152] source
• ?Could not verify

  Citation [152] is attached to the claim that duplications and large-scale genomic variations can cause genomic instability. The paper discusses deleterious gene-dosage changes and the loss of duplicated copies through recombination (section “The Magnitude of the Problem,” page unavailable), but the passages found do not say that these changes cause genomic instability. The yeast-evolution claim is attached to other citations. (LLM, conf=0.72)

  cites [115] sourceExperimental — accuracy not yet measured
• Not checked yetcites [153] source
• ?Could not verify

  Citation [152] is attached to the qualification about genomic instability, not the later statement about yeast evolution. The Introduction and Discussion discuss the evolution of ER-stress responses in Candida glabrata, but our copy does not address whether duplications or large-scale genomic variations cause genomic instability. Searches for genomic instability, duplication, and copy-number variation found no relevant passage. (LLM, conf=0.75)

  cites [154] sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Background and Results and Discussion discuss salt-responsive gene expression and adaptation in Hortaea werneckii, not duplications or large-scale genomic variation as drivers of yeast evolution. Searches for duplicated genes, genomic instability, and evolution found no passage addressing that claim in our copy; the genomic-instability clause also has a separate citation in the citing sentence. (LLM, conf=0.75)

  cites [155] sourceExperimental — accuracy not yet measured
• ?Could not verify

  Citation [152] is attached to the claim that duplications and large-scale genomic variations can cause genomic instability. The Background discusses T-DNA insertions in Arabidopsis, and the Discussion concerns metabolite-data normalization; neither addresses that claim. Searches for duplications, genomic instability, and yeast evolution found no relevant passage in our copy. (LLM, conf=0.76)

  cites [156] sourceExperimental — accuracy not yet measured

In the paper

“In summary Tables 1 and Table 2, it can be seen that a typical ALE experiment is performed for somewhat between 100 and 2000 generations and usually takes a few weeks up to a few months.”

In the paper

“Moreover, biological catalysis offers several advantages over chemical synthesis, such as stereo-selective production of chemical compounds [9].”

What the sources say

• Not checked yetcites [9] source

In the paper

“Synthetic biology is a new emerging field [10,11] and systems metabolic engineering has proven very successful for the design and implementation of biotechnological production processes [12-14].”

What the sources say

• Not checked yetcites [12] source
• Not checked yetcites [13] source
• Not checked yetcites [14] source

In the paper

“As indicated in Figure 1a, in serial batch cultures for ALE, the transfer often occurs before the stationary phase is reached. Clearly, this is a prerequisite to avoid stationary phase adaptation [52].”

What the sources say

• Not checked yetcites [52] source

In the paper

“Microbial cells offer important advantages for ALE studies: (a) most microbial cells have simple nutrient requirements, (b) they can be easily cultivated in the laboratory and (c) microbial cells generally grow very fast and can be cultivated for several hundred generations within several weeks or months”

In the paper

“however, particularly in the last 25 years, there has been an ever increasing number of such experiments with Escherichia coli and Saccharomyces cerevisiae being the most prominent organisms under investigation [26-28].”

What the sources say

• Not checked yetcites [26] source
• Not checked yetcites [27] source
• Not checked yetcites [28] source

In the paper

“This single parallel E. coli adaptation experiment is already exceeding 50000 generations [33-35].”

What the sources say

• Not checked yetcites [33] source
• Not checked yetcites [34] source
• Not checked yetcites [35] source

In the paper

“In this context, we cannot clearly predict the scale at which improved phenotypes emerge during laboratory evolution, although recent literature indicates that such predictions are possible in certain growth contexts such as carbon source adaptation [65].”

What the sources say

• Not checked yetcites [65] source

In the paper

“Whereas the fitness increase is usually fast within the first 100 to 500 generations, it slows considerably down during the course of ALE [36].”

What the sources say

• Not checked yetcites [36] source

In the paper

“In this context, cells selected under growth limiting conditions can show growth trade-offs in non-limiting conditions and vice versa[53].”

What the sources say

• Not checked yetcites [53] source

In the paper

“Hence, adaptive laboratory evolution was already performed about a hundred years ago by William Dallinger [22] and during the middle of the last century [24,25];”

What the sources say

• Not checked yetcites [22] source
• Not checked yetcites [24] source
• Not checked yetcites [25] source

In the paper

“The initial choice of conditions, including media composition and limited versus non-limited growth rate, will greatly influence whether the artificially selected microbial host will be suitable for its dedicated application.”

In the paper

“During adaptive evolution, certain trait values change and are associated with increased (Darwinian) fitness [20]; as such, an improved phenotype or property is often equal to increased fitness.”

What the sources say

• Not checked yetcites [20] source

In the paper

“In certain cases ALE can lead to an even higher increase of up to 1000% as recently shown for the iron reduction rate of Geobacter sulfurreducens[123], although this increase was only achieved after 24 months of selection”

What the sources say

• Not checked yetcites [123] source

In the paper

“Deletions accounted for approximately 29% of the observed genomic changes, followed by insertions (7%) and IS movements (3%) [15].”

What the sources say

• Not checked yetcites [15] source

In the paper

“Nevertheless, the role of HGT in natural evolution remains a controversial topic [133].”

What the sources say

• Not checked yetcites [133] source

In the paper

“Interestingly, it was shown that high mutation rates and mutator strains can emerge naturally during laboratory evolution and contribute to the formation of genetic novelty [33,73,139-141].”

What the sources say

• Not checked yetcites [33] source
• Not checked yetcites [73] source
• Not checked yetcites [139] source
• Not checked yetcites [140] source
• Not checked yetcites [141] source

In the paper

“As summarized in a recent review, SNPs (61%, with G:C to T:A mutations being disproportionally frequent) account for the majority of observed genetic changes [15].”

What the sources say

• Not checked yetcites [15] source

In the paper

“This so-called toolbox model suggests that prokaryotic metabolic pathways emerge through horizontal gene transfer (HGT) and are rapidly acquired and lost based on nutrient availability [132].”

What the sources say

• Not checked yetcites [132] source

In the paper

“they are a prerequisite for the development of novel molecular functions [129-131].”

What the sources say

• Not checked yetcites [129] source
• Not checked yetcites [130] source
• Not checked yetcites [131] source

In the paper

“During the first few thousand generations, glucose-evolved cultures developed larger cell size and a lower final cell density as compared with the ancestor [34].”

What the sources say

• Not checked yetcites [34] source

In the paper

“Furthermore, it was shown that mutational robustness tends to increase with mutation rate and decline with population size [40].”

What the sources say

• Not checked yetcites [40] source

In the paper

“In this study, the biomass yield increased up to 40% and acetate overflow was reduced, whereas the metabolic rate increased and the evolved cells showed a lowered or total loss of motility.”

What the sources say

• Not checked yetcites [69] source

In the paper

“Microbial metabolic networks often provide appropriate functions at a minimal size [134] and furthermore, their complexity seems to be correlated with environmental factors [135].”

What the sources say

• Not checked yetcites [134] source
• Not checked yetcites [135] source

In the paper

“however, such mutational adaptations might be counterproductive for some applications of microbial cells in biotechnology.”

In the paper

“Many ALE studies analyzed the impact and molecular adaptation towards both stresses (Table 1 and Table 2), leading to major findings regarding the emergence of specialists, generalists, the underlying mutations and the applicability of evolved cells for industrial microbiology.”

In the paper

“For example, an experimental E. coli culture could evolve the ability to aerobically utilize citrate [37], or the ability to use the non-natural carbon source 1,2-propanediol [71]; also, a strain deficient in glutathione biosynthesis was able to develop compensatory mutations to restore this function [147].”

What the sources say

• Not checked yetcites [37] source
• Not checked yetcites [71] source
• Not checked yetcites [147] source

In the paper

“In a 1997 study, E. coli was evolved in chemostat cultures in order to select for improved growth on glycerol. The authors reported phenotypic changes in terms of colony morphology accompanied with increased growth rate and biomass yield and decreased acetate formation [50].”

What the sources say

• Not checked yetcites [50] source

In the paper

“It is clear that a higher mutation rate can only be beneficial to a certain extent as it also results in a genetic load [142].”

What the sources say

• Not checked yetcites [142] source

In the paper

“In nature as well as in biotechnological processes, microorganisms are challenged with changing environmental conditions, where they experience fluctuations in temperature, pH, oxygenation, atmospheric or hydrostatic pressure but also water and nutrient availability; thus, the selective pressures can be divided in two main categories: nutrient availability and environmental stress [17].”

What the sources say

• Not checked yetcites [17] source

In the paper

“Only recently, the cumulative number of cell divisions (CCD) was proposed as a more appropriate denominator to evaluate microbial evolution [126].”

What the sources say

• Not checked yetcites [126] source

In the paper

“Glucose import, sensing and regulation represent major targets during glucose-limited chemostat growth, as a further study also identified frequent mutations in HXT genes and glucose sensors and regulators such as RGT1 and MIG2[53].”

What the sources say

• Not checked yetcites [53] source

In the paper

“Adaptive changes towards ethanol tolerance were linked to the up-regulation of oxyR and nrdR, indicating major changes in the cellular respiratory system but also changes in amino acid metabolism [85].”

What the sources say

• Not checked yetcites [85] source

In the paper

“Transcript analysis showed up-regulation of trehalose and glycogen metabolism, whereas no mutations were identified in galactose or storage carbohydrate related genes. Interestingly, the authors rather identified mutations in RAS/PKA signaling as a source for increased fitness on galactose medium [106].”

What the sources say

• Not checked yetcites [106] source

In the paper

“The authors did not identify expression changes in HXT genes, although other studies on improved xylose and arabinose utilization indicated that HXT genes may also be involved in increased xylose uptake rates [99,104].”

What the sources say

• Not checked yetcites [99] source
• Not checked yetcites [104] source

In the paper

“Similarly, Zelle and co-workers established growth on glucose by genetic- and evolutionary engineering in a pyruvate carboxylase-negative S. cerevisiae strain. The authors identified a point mutation in the recombinant sfcA gene, which shifted the co-factor preference from NADH to NADPH [107].”

What the sources say

• Not checked yetcites [107] source

In the paper

“Mutations that lead to altered expression patterns accumulated predominantly in lacI and lacO1 regions of the lac operon [76]. Altogether, these mutations were responsible for a reduced (diauxic) lag phase and increased maximum growth rates in lactose growth medium.”

What the sources say

• Not checked yetcites [76] source

In the paper

“Growth rates and glucose uptake rates increased up to 3.6 and up to 2.6 fold in evolved ∆pgi strains as compared with the ancestor and were accompanied with different phenotypes regarding acetate and formate production.”

What the sources say

• Not checked yetcites [70] source

In the paper

“Decreased activity levels of enzymes related to glycolysis but also correlating changes in the expression levels of genes such as ENO1, ENO2, TDH1 and PYK1 and down-regulation of the stress response (MSN2/4) were observed.”

What the sources say

• Not checked yetcites [98] source

In the paper

“Strikingly, the authors also noticed that malT regulatory mutations, although common in aerobic conditions, were absent during oxygen-limited chemostat growth, where mutations in the glucose permease ptsG were common [48].”

What the sources say

• Not checked yetcites [48] source

In the paper

“but most importantly the rpoS mutation alone did not positively influence the growth rate in the ancestral ∆pgi strain; rpoS mutations were epistatically linked to additional mutations.”

What the sources say

• Not checked yetcites [63] source

In the paper

“Altered cell morphology was also observed, as well as higher affinity towards glucose, although no expression change in the major HXT low-affinity glucose transporters was observed [98]”

What the sources say

• Not checked yetcites [98] source

In the paper

“Mutations in acrAB and marC were found independently in these studies; further changes were linked to carbon and nitrogen metabolism via gatY and tnaA[86], enterobactin synthesis [63] or attenuated rpoS activity via hfq mutations [87].”

What the sources say

• Not checked yetcites [63] source
• Not checked yetcites [86] source
• Not checked yetcites [87] source

In the paper

“In xylose adaptation studies, increased growth rates and higher ethanol yields were observed in recombinant yeast. In this case, trehalose and glycogen metabolic processes were significantly down-regulated, nutrient and stress signaling was affected by down-regulation of YAK1 and also accompanied by down-regulation of CWP1 and consequently major cell wall changes [108].”

What the sources say

• Not checked yetcites [108] source

In the paper

“The authors found frequent point mutations in rpoS, udhA (soluble transhydrogenase), pntAB and a prophage deletion. They did not observe these mutations in the glucose-adapted wildtype strain; other regulatory mutations (including rpoABC) occurred in both backgrounds”

What the sources say

• Not checked yetcites [70] source

In the paper

“For other carbon sources, it was shown that selection for improved galactose utilization could be achieved by serial transfer with a 24% increase of μmax within 62 days. Galactose uptake rates and ethanol production were increased.”

What the sources say

• Not checked yetcites [106] source

In the paper

“Furthermore, experiments on pentose utilization highlighted that increased gene copy numbers of the heterologous genes in recombinant S. cerevisiae can emerge during evolutionary engineering and contribute to increased growth in anaerobic conditions [104,109].”

What the sources say

• Not checked yetcites [104] source
• Not checked yetcites [109] source

In the paper

“In the model yeast S. cerevisiae, adaptation towards salt tolerance led to increased cell size and increased ploidy. Additionally, gene expression changes in CTT1 and MSN4 were observed, as well as a high-frequency SNP in the transcriptional regulator MOT2[116].”

What the sources say

• Not checked yetcites [116] source

In the paper

“A recent study showed that cross-protection towards lignocellulosic hydrolysates can be evolved in S. cerevisiae. Interestingly, several mutants showed increased fitness in the presence of inhibitor combinations but reduced fitness when exposed to individual hydrolysate components [112].”

What the sources say

• Not checked yetcites [112] source

In the paper

“Similarly, aerobic chemostat-selected S. cerevisiae cells also showed increased fitness in anaerobic glucose-limited chemostats and in acetate-limited chemostats, but decreased fitness in nitrogen-limited batch cultivations [53].”

What the sources say

• Not checked yetcites [53] source

In the paper

“In fact, a recent study in S. cerevisiae indicated that anticipatory gene regulatory patterns can evolve under cycling salt and oxidative stress conditions on a laboratory time scale within 300 generations and that cross-stress protection against salt and oxidative stress is strikingly asymmetrical, with oxidative stress protecting against salt stress but not vice versa[120].”

What the sources say

• Not checked yetcites [120] source

In the paper

“Thus, evolutionary trade-offs and cross-protection against process-relevant parameters are ubiquitous, but the mode of cultivation and the specificities of the selection pressure largely determine to which extent these phenomena arise.”

In the paper

“Increased copper tolerance was reported to be mediated by a genomic amplification of CUP1, decreased basal levels of the copper transporters CTR2 and CCC2 and decreased activity of antioxidant enzymes [117,128].”

What the sources say

• Not checked yetcites [117] source
• Not checked yetcites [128] source

In the paper

“Importantly, epistatic interactions can be hardly inferred from metabolic modeling studies although significant epistatic connections for strain engineering may be easily identified in ALE studies.”

In the paper

“In another study, increased ethanol tolerance was mediated by mutations in the translational regulator SSD1 and UTH1, a protein of unknown function, and indicated that cell wall stability is a major factor in ethanol tolerance of S. cerevisiae[119].”

What the sources say

• Not checked yetcites [119] source

In the paper

“From these data it can be concluded that: (a) long-term adaptation leads to both large-scale transcriptional de-regulation and optimization of terminal nodes (fine-tuning of the expression levels of individual genes)”

In the paper

“Glycerol-limited growth led to stable mutants in a single E. coli population, where one mutant showed increased growth in glycerol-sufficient batch cultures and a second mutant did not. Furthermore, the mutant that showed increased growth in batch culture also showed increased fitness under general nutrient limitation, heat and osmotic stress [50].”

What the sources say

• Not checked yetcites [50] source

In the paper

“Another interesting study analyzed to which extent multi-stress resistant S. cerevisiae populations can be evolved. The authors tested various chemostat and batch stress regimes and found that batch selection for freeze-thaw tolerance also selected for increased thermal, oxidative and ethanol stress resistance [113].”

What the sources say

• Not checked yetcites [113] source

In the paper

“Yeast cells that were evolved for efficient galactose utilization [106] subsequently showed trade-offs when grown on glucose as carbon source. This effect was due to antagonistic pleiotropy that was based on mutations of the regulatory protein RAS2[162].”

What the sources say

• Not checked yetcites [106] source
• Not checked yetcites [162] source

In the paper

“Whereas core sub-networks and pathways tend to be well conserved, the up- and downstream connections to other cellular networks vary significantly among different species and lead to species-specific differences of stress resistance in adverse environments [169,170]”

What the sources say

• Not checked yetcites [169]
• Not checked yetcites [170]

In the paper

“Examples for adaptive cross-protection are also available for S. cerevisiae where cobalt resistance also improved tolerance for other metal ions and pulsed cobalt stress exposure during laboratory evolution even resulted in evolutionary cross-protection to thermal and oxidative stress [114].”

What the sources say

• Not checked yetcites [114] source

In the paper

“and n-butanol tolerance is weakly compatible with oxidative stress [63] and leads to trade-offs in hexane and chloramphenicol resistance [86].”

What the sources say

• Not checked yetcites [63] source
• Not checked yetcites [86] source

In the paper

“Moreover, for eukaryotic host organisms such as yeasts, alternating asexual and sexual propagation during selection can be applied in order to decouple beneficial and unsound mutations and thus reduce potential deficits of laboratory evolution experiments [118].”

What the sources say

• Not checked yetcites [118] source

In the paper

“In this context, computational approaches provide important benefits towards rational engineering of microbial cell factories but as it can be foreseen right now, they cannot replace or prevent ‘trial and error’ laboratory experiments for the foreseeable future.”

In the paper

“In contrast to classical approaches it is highly structured and biological networks are split into smaller functional modules [11,183], leading to complex optimization needs.”

What the sources say

• Not checked yetcites [11] source
• Not checked yetcites [183]

In the paper

“Full-scale models of microorganisms are available [185,186] and we can simulate microbial growth.”

What the sources say

• Not checked yetcites [185]
• Not checked yetcites [186]

In the paper

“Towards this end, a recent study with S. cerevisiae and Saccharomyces paradoxus showed that the evolutionary rescue (ER) frequency was positively correlated with stress concentration during 100 generations of evolution in S. cerevisiae but negatively correlated in S. paradoxus[44].”

What the sources say

• Not checked yetcites [44] source

In the paper

“Other studies used ethylmethanesulfonate (EMS) treated Bacillus boroniphilus to select for increased boron resistance [88] or EMS-treated S. cerevisiae in order to achieve increased nutrient utilization and stress resistance [95,113,114].”

What the sources say

• Not checked yetcites [88] source
• Not checked yetcites [95] source
• Not checked yetcites [113] source
• Not checked yetcites [114] source

In the paper

“Similarly, in silico approaches are widely applied in order to address evolutionary questions [40,172].”

What the sources say

• Not checked yetcites [40] source
• Not checked yetcites [172]

In the paper

“Microbial gene regulatory networks are generally very robust towards perturbations and large-scale re-wiring results in a significant number of viable clones with increased evolvability as compared with the wildtype [181].”

What the sources say

• Not checked yetcites [181]

In the paper

“Computational models are well-suited to study evolutionary change on the population level, whereas in silico evolution of artificial cells that include simulated regulatory networks are currently very limited [21].”

What the sources say

• Not checked yetcites [21] source

In the paper

“Certain functions such as overflow metabolism in S. cerevisiae may have evolved in order to provide an evolutionary advantage in specific environments and to protect carbon resources from other competing species [175].”

What the sources say

• Not checked yetcites [175]

In the paper

“For example, laboratory selection can lead to large-scale rewiring of gene regulatory networks, but an approach, that the authors called gTME, global transcription machinery engineering, can also yield similar results [2].”

What the sources say

• Not checked yetcites [2] source

In the paper

“Besides insights into co-evolutionary behavior, this approach can be exploited in order to increase antibiotic production such as in the case of co-cultivation of Streptomyces clavuligerus and Staphylococcus aureus[55].”

What the sources say

• Not checked yetcites [55] source