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Adaptive laboratory evolution -- principles and applications for biotechnology.

Dragosits M, Mattanovich D - Microb. Cell Fact. (2013)

Bottom Line: Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications.Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations.However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria. martin.dragosits@boku.ac.at

ABSTRACT
Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering.

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Adaptive laboratory evolution (ALE). ALE can be performed in the laboratory by (a) sequential serial passages in shake flasks, where nutrients will not be limited and certain growth parameters can heavily fluctuate. (b) Alternatively, chemostat cultures can be applied, where one nutritional component is typically limited and cell density can be much higher than in shake flasks. Additionally, cell density and environmental conditions can be kept constant and more complex cultivation strategies can be implemented. (c) The increase of fitness during laboratory evolution experiments is fast in the first stage but generally slows down during prolonged selection, whereas the number of mutations is steadily increasing; however network complexity leads to a decreasing beneficial effect of additional mutations. [36,67]. (d) Mutations that are usually identified in ALE studies. Single nucleotide polymorphisms (SNPs), smaller insertions and deletions (indels) and larger deletions and insertions contribute to genetic and gene regulatory changes and fitness changes during the selection for improved phenotypes.
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Figure 1: Adaptive laboratory evolution (ALE). ALE can be performed in the laboratory by (a) sequential serial passages in shake flasks, where nutrients will not be limited and certain growth parameters can heavily fluctuate. (b) Alternatively, chemostat cultures can be applied, where one nutritional component is typically limited and cell density can be much higher than in shake flasks. Additionally, cell density and environmental conditions can be kept constant and more complex cultivation strategies can be implemented. (c) The increase of fitness during laboratory evolution experiments is fast in the first stage but generally slows down during prolonged selection, whereas the number of mutations is steadily increasing; however network complexity leads to a decreasing beneficial effect of additional mutations. [36,67]. (d) Mutations that are usually identified in ALE studies. Single nucleotide polymorphisms (SNPs), smaller insertions and deletions (indels) and larger deletions and insertions contribute to genetic and gene regulatory changes and fitness changes during the selection for improved phenotypes.

Mentions: Generally, ALE experiments with microorganisms are easy to establish and the common methods, which are used, are shown in Figure 1a and Figure 1b. Batch cultivation in shake flasks can be performed to propagate microbial cells in parallel serial cultures. At regular intervals (usually on a daily basis), an aliquot of the culture is transferred to a new flask with fresh medium for an additional round of growth. Clearly, this easy setup has the advantages of cheap equipment and the ease of massive parallel cultures. By replacing shake flasks with deep well plates with even smaller culture volumes, hundreds of microbial cultures can be grown in parallel [44]. Several environmental factors can be easily controlled, including temperature and spatial culture homogeneity (by constant mixing of the culture). Nevertheless, batch cultivation has certain shortcomings, including varying population density, fluctuating growth rate, nutrient supply and fluctuating environmental conditions such as environmental pH and dissolved oxygen (partial oxygen pressure, pO2). In many cases, these factors may not be of great importance for the experimental setup but the simplicity of the setup can prevent the implementation of complex environments for microbial selection experiments. As an alternative to serial batch growth, continuous (chemostat) cultures in bioreactor vessels (Figure 1b) are applied [45-50]. The advantages of chemostat cultivation are constant growth rates and population densities. Furthermore, it is possible to tightly control nutrient supply and environmental conditions such as pH and oxygenation. A major drawback is the costs of operation. Even with small vessels and parallel operation, as they are available from many manufacturers, the costs are by far exceeding the costs of shake flask cultivations. A major difference between prolonged selective growth in batch and chemostat cultures is, however, the growth in nutrient-sufficient versus potentially nutrient-limited conditions. Principally, feedback control mechanisms, such as in the case of a pH-auxostat [51] allow nutrient-sufficient growth in chemostat cultures at the maximum specific growth rate; however, nutrient-limited growth at a growth rate below the maximum is predominantly applied in continuous cultures. 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]. Thus, microbial cells are predominantly grown in exponential cultures. In contrast, in continuous cultures, the growth rate is kept constant (or in certain experimental setups stepwise / continuously increasing) by the limitation of a major growth nutrient, such as glucose, nitrogen or phosphate. In this context, cells selected under growth limiting conditions can show growth trade-offs in non-limiting conditions and vice versa[53]. If the selection aims at phenotypes that do not rely on increased growth rates or biomass yield but other traits such as e.g. a yes / no survival phenotype after gene knockout or antibiotic production, solid media or combinations of solid and liquid media can be applied in order to select for proper phenotypes [54,55].


Adaptive laboratory evolution -- principles and applications for biotechnology.

Dragosits M, Mattanovich D - Microb. Cell Fact. (2013)

Adaptive laboratory evolution (ALE). ALE can be performed in the laboratory by (a) sequential serial passages in shake flasks, where nutrients will not be limited and certain growth parameters can heavily fluctuate. (b) Alternatively, chemostat cultures can be applied, where one nutritional component is typically limited and cell density can be much higher than in shake flasks. Additionally, cell density and environmental conditions can be kept constant and more complex cultivation strategies can be implemented. (c) The increase of fitness during laboratory evolution experiments is fast in the first stage but generally slows down during prolonged selection, whereas the number of mutations is steadily increasing; however network complexity leads to a decreasing beneficial effect of additional mutations. [36,67]. (d) Mutations that are usually identified in ALE studies. Single nucleotide polymorphisms (SNPs), smaller insertions and deletions (indels) and larger deletions and insertions contribute to genetic and gene regulatory changes and fitness changes during the selection for improved phenotypes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716822&req=5

Figure 1: Adaptive laboratory evolution (ALE). ALE can be performed in the laboratory by (a) sequential serial passages in shake flasks, where nutrients will not be limited and certain growth parameters can heavily fluctuate. (b) Alternatively, chemostat cultures can be applied, where one nutritional component is typically limited and cell density can be much higher than in shake flasks. Additionally, cell density and environmental conditions can be kept constant and more complex cultivation strategies can be implemented. (c) The increase of fitness during laboratory evolution experiments is fast in the first stage but generally slows down during prolonged selection, whereas the number of mutations is steadily increasing; however network complexity leads to a decreasing beneficial effect of additional mutations. [36,67]. (d) Mutations that are usually identified in ALE studies. Single nucleotide polymorphisms (SNPs), smaller insertions and deletions (indels) and larger deletions and insertions contribute to genetic and gene regulatory changes and fitness changes during the selection for improved phenotypes.
Mentions: Generally, ALE experiments with microorganisms are easy to establish and the common methods, which are used, are shown in Figure 1a and Figure 1b. Batch cultivation in shake flasks can be performed to propagate microbial cells in parallel serial cultures. At regular intervals (usually on a daily basis), an aliquot of the culture is transferred to a new flask with fresh medium for an additional round of growth. Clearly, this easy setup has the advantages of cheap equipment and the ease of massive parallel cultures. By replacing shake flasks with deep well plates with even smaller culture volumes, hundreds of microbial cultures can be grown in parallel [44]. Several environmental factors can be easily controlled, including temperature and spatial culture homogeneity (by constant mixing of the culture). Nevertheless, batch cultivation has certain shortcomings, including varying population density, fluctuating growth rate, nutrient supply and fluctuating environmental conditions such as environmental pH and dissolved oxygen (partial oxygen pressure, pO2). In many cases, these factors may not be of great importance for the experimental setup but the simplicity of the setup can prevent the implementation of complex environments for microbial selection experiments. As an alternative to serial batch growth, continuous (chemostat) cultures in bioreactor vessels (Figure 1b) are applied [45-50]. The advantages of chemostat cultivation are constant growth rates and population densities. Furthermore, it is possible to tightly control nutrient supply and environmental conditions such as pH and oxygenation. A major drawback is the costs of operation. Even with small vessels and parallel operation, as they are available from many manufacturers, the costs are by far exceeding the costs of shake flask cultivations. A major difference between prolonged selective growth in batch and chemostat cultures is, however, the growth in nutrient-sufficient versus potentially nutrient-limited conditions. Principally, feedback control mechanisms, such as in the case of a pH-auxostat [51] allow nutrient-sufficient growth in chemostat cultures at the maximum specific growth rate; however, nutrient-limited growth at a growth rate below the maximum is predominantly applied in continuous cultures. 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]. Thus, microbial cells are predominantly grown in exponential cultures. In contrast, in continuous cultures, the growth rate is kept constant (or in certain experimental setups stepwise / continuously increasing) by the limitation of a major growth nutrient, such as glucose, nitrogen or phosphate. In this context, cells selected under growth limiting conditions can show growth trade-offs in non-limiting conditions and vice versa[53]. If the selection aims at phenotypes that do not rely on increased growth rates or biomass yield but other traits such as e.g. a yes / no survival phenotype after gene knockout or antibiotic production, solid media or combinations of solid and liquid media can be applied in order to select for proper phenotypes [54,55].

Bottom Line: Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications.Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations.However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria. martin.dragosits@boku.ac.at

ABSTRACT
Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering.

Show MeSH
Related in: MedlinePlus