scholarly journals Thermotolerant Yeast Strains Adapted by Laboratory Evolution Show Trade-Off at Ancestral Temperatures and Preadaptation to Other Stresses

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Luis Caspeta ◽  
Jens Nielsen
Keyword(s):  
Ethanol Production ◽  
Production Costs ◽  
Thermotolerant Yeast ◽  
Adaptive Laboratory Evolution ◽  
Genetic Changes ◽  
Laboratory Evolution ◽  
Trade Off ◽  
Thermal Niche ◽  
Production Processes ◽  
Yeast Strains

ABSTRACT A major challenge for the production of ethanol from biomass-derived feedstocks is to develop yeasts that can sustain growth under the variety of inhibitory conditions present in the production process, e.g., high osmolality, high ethanol titers, and/or elevated temperatures (≥40°C). Using adaptive laboratory evolution, we previously isolated seven Saccharomyces cerevisiae strains with improved growth at 40°C. Here, we show that genetic adaptations to high temperature caused a growth trade-off at ancestral temperatures, reduced cellular functions, and improved tolerance of other stresses. Thermotolerant yeast strains showed horizontal displacement of their thermal reaction norms to higher temperatures. Hence, their optimal and maximum growth temperatures increased by about 3°C, whereas they showed a growth trade-off at temperatures below 34°C. Computational analysis of the physical properties of proteins showed that the lethal temperature for yeast is around 49°C, as a large fraction of the yeast proteins denature above this temperature. Our analysis also indicated that the number of functions involved in controlling the growth rate decreased in the thermotolerant strains compared with the number in the ancestral strain. The latter is an advantageous attribute for acquiring thermotolerance and correlates with the reduction of yeast functions associated with loss of respiration capacity. This trait caused glycerol overproduction that was associated with the growth trade-off at ancestral temperatures. In combination with altered sterol composition of cellular membranes, glycerol overproduction was also associated with yeast osmotolerance and improved tolerance of high concentrations of glucose and ethanol. Our study shows that thermal adaptation of yeast is suitable for improving yeast resistance to inhibitory conditions found in industrial ethanol production processes. IMPORTANCE Yeast thermotolerance can significantly reduce the production costs of biomass conversion to ethanol. However, little information is available about the underlying genetic changes and physiological functions required for yeast thermotolerance. We recently revealed the genetic changes of thermotolerance in thermotolerant yeast strains (TTSs) generated through adaptive laboratory evolution. Here, we examined these TTSs’ physiology and computed their proteome stability over the entire thermal niche, as well as their preadaptation to other stresses. Using this approach, we showed that TTSs exhibited evolutionary trade-offs in the ancestral thermal niche, as well as reduced numbers of growth functions and preadaptation to other stresses found in ethanol production processes. This information will be useful for rational engineering of yeast thermotolerance for the production of biofuels and chemicals.

scholarly journals Use of Adaptive Laboratory Evolution To Discover Key Mutations Enabling Rapid Growth of Escherichia coli K-12 MG1655 on Glucose Minimal Medium

2014 ◽  
Vol 81 (1) ◽  
pp. 17-30 ◽  
Author(s):  
Ryan A. LaCroix ◽  
Troy E. Sandberg ◽  
Edward J. O'Brien ◽  
Jose Utrilla ◽  
Ali Ebrahim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rapid Growth ◽  
Scientific Discovery ◽  
Maximum Growth ◽  
Time Frame ◽  
Adaptive Laboratory Evolution ◽  
Genetic Changes ◽  
Laboratory Evolution ◽  
Content Type ◽  
K 12

ABSTRACTAdaptive laboratory evolution (ALE) has emerged as an effective tool for scientific discovery and addressing biotechnological needs. Much of ALE's utility is derived from reproducibly obtained fitness increases. Identifying causal genetic changes and their combinatorial effects is challenging and time-consuming. Understanding how these genetic changes enable increased fitness can be difficult. A series of approaches that address these challenges was developed and demonstrated usingEscherichia coliK-12 MG1655 on glucose minimal media at 37°C. By keepingE. coliin constant substrate excess and exponential growth, fitness increases up to 1.6-fold were obtained compared to the wild type. These increases are comparable to previously reported maximum growth rates in similar conditions but were obtained over a shorter time frame. Across the eight replicate ALE experiments performed, causal mutations were identified using three approaches: identifying mutations in the same gene/region across replicate experiments, sequencing strains before and after computationally determined fitness jumps, and allelic replacement coupled with targeted ALE of reconstructed strains. Three genetic regions were most often mutated: the global transcription generpoB, an 82-bp deletion between the metabolicpyrEgene andrph, and an IS element between the DNA structural genehnsandtdk. Model-derived classification of gene expression revealed a number of processes important for increased growth that were missed using a gene classification system alone. The methods described here represent a powerful combination of technologies to increase the speed and efficiency of ALE studies. The identified mutations can be examined as genetic parts for increasing growth rate in a desired strain and for understanding rapid growth phenotypes.

scholarly journals Laboratory Evolution to Alternating Substrate Environments Yields Distinct Phenotypic and Genetic Adaptive Strategies

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Troy E. Sandberg ◽  
Colton J. Lloyd ◽  
Bernhard O. Palsson ◽  
Adam M. Feist
Keyword(s):  
Escherichia Coli ◽  
Adaptive Strategies ◽  
Carbon Substrate ◽  
Natural Occurrence ◽  
Natural Setting ◽  
Data Types ◽  
Adaptive Laboratory Evolution ◽  
Genetic Changes ◽  
Laboratory Evolution ◽  
Content Type

ABSTRACT Adaptive laboratory evolution (ALE) experiments are often designed to maintain a static culturing environment to minimize confounding variables that could influence the adaptive process, but dynamic nutrient conditions occur frequently in natural and bioprocessing settings. To study the nature of carbon substrate fitness tradeoffs, we evolved batch cultures of Escherichia coli via serial propagation into tubes alternating between glucose and either xylose, glycerol, or acetate. Genome sequencing of evolved cultures revealed several genetic changes preferentially selected for under dynamic conditions and different adaptation strategies depending on the substrates being switched between; in some environments, a persistent “generalist” strain developed, while in another, two “specialist” subpopulations arose that alternated dominance. Diauxic lag phenotype varied across the generalists and specialists, in one case being completely abolished, while gene expression data distinguished the transcriptional strategies implemented by strains in pursuit of growth optimality. Genome-scale metabolic modeling techniques were then used to help explain the inherent substrate differences giving rise to the observed distinct adaptive strategies. This study gives insight into the population dynamics of adaptation in an alternating environment and into the underlying metabolic and genetic mechanisms. Furthermore, ALE-generated optimized strains have phenotypes with potential industrial bioprocessing applications. IMPORTANCE Evolution and natural selection inexorably lead to an organism's improved fitness in a given environment, whether in a laboratory or natural setting. However, despite the frequent natural occurrence of complex and dynamic growth environments, laboratory evolution experiments typically maintain simple, static culturing environments so as to reduce selection pressure complexity. In this study, we investigated the adaptive strategies underlying evolution to fluctuating environments by evolving Escherichia coli to conditions of frequently switching growth substrate. Characterization of evolved strains via a number of different data types revealed the various genetic and phenotypic changes implemented in pursuit of growth optimality and how these differed across the different growth substrates and switching protocols. This work not only helps to establish general principles of adaptation to complex environments but also suggests strategies for experimental design to achieve desired evolutionary outcomes.

scholarly journals Adaptive Evolution of Industrial Brewer’s Yeast Strains towards a Snowflake Phenotype

Fermentation ◽  
2020 ◽  
Vol 6 (1) ◽  
pp. 20 ◽  
Author(s):  
Yeseren Kayacan ◽  
Thijs Van Mieghem ◽  
Filip Delvaux ◽  
Freddy R. Delvaux ◽  
Ronnie Willaert
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Aggregation ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Sedimentation Behavior ◽  
Yeast Strains ◽  
Industrial Strains ◽  
Genetic Interference ◽  
Cost Efficient ◽  
Industrial Brewer’S Yeast

Flocculation or cell aggregation is a well-appreciated characteristic of industrial brewer’s strains, since it allows removal of the cells from the beer in a cost-efficient and environmentally-friendly manner. However, many industrial strains are non-flocculent and genetic interference to increase the flocculation characteristics are not appreciated by the consumers. We applied adaptive laboratory evolution (ALE) to three non-flocculent, industrial Saccharomyces cerevisiae brewer’s strains using small continuous bioreactors (ministats) to obtain an aggregative phenotype, i.e., the “snowflake” phenotype. These aggregates could increase yeast sedimentation considerably. We evaluated the performance of these evolved strains and their produced flavor during lab scale beer fermentations. The small aggregates did not result in a premature sedimentation during the fermentation and did not result in major flavor changes of the produced beer. These results show that ALE could be used to increase the sedimentation behavior of non-flocculent brewer’s strains.

scholarly journals Harnessing the Power of Mutagenesis and Adaptive Laboratory Evolution for High Lipid Production by Oleaginous Microalgae and Yeasts

2020 ◽  
Vol 12 (12) ◽  
pp. 5125
Author(s):  
Neha Arora ◽  
Hong-Wei Yen ◽  
George P. Philippidis
Keyword(s):  
Genetic Engineering ◽  
Lipid Production ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Production Costs ◽  
Screening Methods ◽  
Scale Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Oleaginous Microalgae

Oleaginous microalgae and yeasts represent promising candidates for large-scale production of lipids, which can be utilized for production of drop-in biofuels, nutraceuticals, pigments, and cosmetics. However, low lipid productivity and costly downstream processing continue to hamper the commercial deployment of oleaginous microorganisms. Strain improvement can play an essential role in the development of such industrial microorganisms by increasing lipid production and hence reducing production costs. The main means of strain improvement are random mutagenesis, adaptive laboratory evolution (ALE), and rational genetic engineering. Among these, random mutagenesis and ALE are straight forward, low-cost, and do not require thorough knowledge of the microorganism’s genetic composition. This paper reviews available mutagenesis and ALE techniques and screening methods to effectively select for oleaginous microalgae and yeasts with enhanced lipid yield and understand the alterations caused to metabolic pathways, which could subsequently serve as the basis for further targeted genetic engineering.

scholarly journals Engineering improved ethylene production: Leveraging systems Biology and adaptive laboratory evolution

2021 ◽  
Author(s):  
Sophie Vaud ◽  
Nicole Pearcy ◽  
Marko Hanževački ◽  
Alexander M.W. Van Hagen ◽  
Salah Abdelrazig ◽  
...  
Keyword(s):  
Systems Biology ◽  
Ethylene Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution

scholarly journals Exergy efficiency of thermochemical syngas-to-ethanol production plants

2021 ◽  
Vol 3 (5) ◽  
Author(s):  
Marcos Paulo Gabriel da Costa e Silva ◽  
Júlio Cesar de Carvalho Miranda
Keyword(s):  
Ethanol Production ◽  
Process Improvement ◽  
Second Generation ◽  
Exergy Efficiency ◽  
Exergy Destruction ◽  
Exothermic Reactions ◽  
Production Processes ◽  
Different Temperatures ◽  
Exergy Analyses ◽  
Second Generation Ethanol

Abstract This work presents exergy analyses applied in four different conceptual second-generation ethanol production processes through a thermochemical route using catalysts based on Molybdenum (P-1), Copper (P-2), and Rhodium (P-3 and P-4), aiming to assess their exergetic efficiencies. The results show that the conceptual processes have satisfactory exergy efficiencies in both cases, when compared among themselves and when compared with other processes reported in literature. The processes’ efficiency for P-1, P-2, P-3 and P-4 were, respectively, 52.4%, 41.4%, 43.7% and 48.9%. The reactors were the sections in which exergy destruction was more significant, due to the exothermic reactions and mixing points (where streams with different temperatures were mixed). Such results show the potential of thermochemical ethanol production, besides opening the possibilities of process improvement. Graphic abstract

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