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org/synthbio Review

Application of Adaptive Laboratory Evolution in Lipid and

Terpenoid Production in Yeast and Microalgae
Yu-Lei Jia, Jin Li, Fang-Tong Nong, Chun-Xiao Yan, Wang Ma, Xiao-Feng Zhu, Li-Hui Zhang,*
and Xiao-Man Sun*

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ABSTRACT: Due to the complexity of metabolic and regulatory networks in microorganisms, it is difficult to obtain robust
phenotypes through artificial rational design and genetic perturbation. Adaptive laboratory evolution (ALE) engineering plays an
important role in the construction of stable microbial cell factories by simulating the natural evolution process and rapidly obtaining
strains with stable traits through screening. This review summarizes the application of ALE technology in microbial breeding,
describes the commonly used methods for ALE, and highlights the important applications of ALE technology in the production of
lipids and terpenoids in yeast and microalgae. Overall, ALE technology provides a powerful tool for the construction of microbial cell
factories, and it has been widely used in improving the level of target product synthesis, expanding the range of substrate utilization,
and enhancing the tolerance of chassis cells. In addition, in order to improve the production of target compounds, ALE also employs
environmental or nutritional stress strategies corresponding to the characteristics of different terpenoids, lipids, and strains.
KEYWORDS: adaptive laboratory evolution, Yarrowia lipolytica, microalgae, lipid, terpenoid

1. INTRODUCTION by obtaining industrial microorganisms with greater stress

Under stress conditions, microbial cells can adapt to optimal resistance and substrate utilization. For example, an Aspergillus
niger strain with improved cellulase production and a
growth by regulating their metabolism networks to obtain
Saccharomyces cerevisiae strain for second-generation ethanol
beneficial phenotypes, which is known as “adaptive evolu-
production with higher tolerance to high temperature and
tion”.1 Adaptive laboratory evolution (ALE) is considered to
hydrolysate-derived inhibitors was created by ALE experi-
be a deliberate strategy for strain improvement, which enables
ment.3,4 ALE depends on beneficial spontaneous mutations
microorganisms to evolve under designed conditions. It
under selection conditions.5 By exposing the strain to the same
confers new phenotypic characteristics on microorganisms by
selection conditions for successive generations, beneficial
providing pressure in the microbial population that favors
mutations can be maintained, while other random mutations
mutant growth.2 The strain produced by the ALE experiment
occur at a lower frequency. In recent years, the application of
is more tolerant to certain abiotic stress environments or may genome sequencing technology has greatly promoted the
evolve new phenotypes. development of microbial ALE research.6 Genome sequencing
ALE has become a popular and widely used tool for technology can rapidly analyze the genes of evolved strains or
analyzing the molecular evolution mechanisms of micro-
organisms under controlled laboratory conditions, and even
controlling the direction of microbial evolution (Figure 1). Received: March 27, 2023
Laboratory evolution of microorganisms can serve as a simple Published: April 21, 2023
model system to help us understand the evolutionary process
of natural selection, how diversity is maintained, and to gain
insight into more specific evolutionary phenomena. In
addition, ALE can also be used in biotechnology applications

© 2023 American Chemical Society https://doi.org/10.1021/acssynbio.3c00179

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Figure 1. Workflow of microbial adaptive laboratory evolution.

Figure 2. Application of adaptive laboratory evolution in microbial breeding.

populations, thus elucidating the molecular evolution mecha- work, saves time and effort, and reduces researcher fatigue,
nisms of strain, and also providing new targets and directions which can lead to mistakes and premature stoppage of
for further directed genetic modification of microorganisms.7 experiments where adaptive mutations are still possible.12
Maximizing the production of metabolites of interest is a Previous reviews related to ALE have described the overall
major challenge for industrial production. Artificially evolved strategy of adaptive evolution experiments in enhancing
microbial cells can be directly used for biotechnological microbial phenotypes,13,14 as well as its application in industrial
processes. However, evolved microbial strains may compro- biotechnology,15−17 summarized the systems and tools related
mise such engineering approaches due to the production of to the microbial ALE workflow,18−20 and discussed in detail
adverse mutations. This requires researchers to combine more the application of advanced computer-aided design,8 automa-
innovative approaches and advanced technology with ALE,
tion technology,9 and high-throughput screening technology10
including advanced computer-aided design,8 automation
in ALE experiments. This review will update the application of
technology,9 high-throughput screening technology,10 and
biosensors11 to decouple beneficial and unsound mutations, ALE in microbial breeding to promote the diffusion of
thereby reducing the potential deficits of laboratory evolu- microbial ALE technology, as well as describe the commonly
tionary experiments. For example, automation allows for used methods for ALE. More specially, this review will focus
complex culture procedures that cannot be reasonably on the application of ALE technology in lipid and terpenoid
performed manually. It can improve the monitoring of the production in yeast and microalgae. We summarize several
physiological properties of evolutionary cultures, enabling findings that have emerged in the literature in recent years,
researchers to better understand of evolutionary dynamics. provide an in-depth analysis of their collective contribution,
Furthermore, it can eliminate repetitive manual ALE passage and highlight several important case studies. In addition,
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perspectives on current limitations and future advancements of the O-acetyl-L-homoserine sulfhydrylase Cgl0653, which
ALE are also discussed. catalyzes the formation of an L-methionine analogue from
methanol, and the methanol-induced membrane-bound trans-
2. APPLICATION OF ALE IN MICROBIAL BREEDING porter Cgl0833 were shown to be critical for methanol
ALE has become a powerful tool for microbiological research tolerance.29 Previously engineered methanol-dependent strains
and is now widely used to screen for microbial strains with can only digest methanol in the presence of a specific
specific phenotypes, production performance, and stable traits. cosubstrate.30 Therefore, these strains cannot be evolved to
According to the demand, the application of ALE in microbial grow on methanol alone. Based on this, Har et al.31 improved
breeding includes the following aspects, broadening the the growth of methylotrophic Escherichia coli on the media
substrate utilization profile, improving tolerance to stress, with methanol and threonine as cosubstrates through the ALE
and enhancing phenotypes (Figure 2). method. Eventually, the evolved methylotrophic E. coli
2.1. Broadening the Substrate Utilization Profile. synthesized all amino acids from methanol-derived carbon
Lignocellulosic biomass is a promising biofuel feedstock, which when grown on methanol and homoserine.
has the advantages of being a wide range of sources, 2.2. Improving Tolerance to Stress. Microorganisms
inexpensive, and renewable.21 Yarrowia lipolytica, the most have been widely found in various extreme environments such
popular nontraditional oil-producing yeast, activity was usually as high temperatures, high salinity, or acidic conditions. To
decreased by the inhibitors existing in lignocellulosic hydrolysis adapt to short-term or long-term environmental stresses,
products. Four rounds of adaptive evolution experiments were microorganisms may have to undergo frequent gene
carried out to screen evolved strains by continuously applying replacement.
stress conditions in the continuous batch culture with 2.2.1. Temperature Stress. Thermal niche has been the
increasing concentrations of the inhibitor aromatic aldehyde focus of attention as the temperature is a fundamental
(0, 0.25, 0.5, 0.75, and 1.5 g/L). The results showed that the environmental attribute that affects physiological character-
transformation of aromatic aldehydes was one of the main istics and often determines species distribution.32 Notably,
reasons for the high tolerance of the adapted strain.22 To under normal circumstances, the tolerance of microorganisms
cultivate A. niger mutant strain with enhanced tolerance to to stressful pressure in an independent growth environment is
ferulic acid by adaptive evolution technology. A. niger N402 often accompanied by trade-offs based on the accumulation of
was cultured in conditions containing 25 mmol L−1 d−1 specific mutations acquired; that is, when the strain evolved to
fructose, and then adaptive evolution was achieved by adapt to high temperature, it may be accompanied by a
gradually increasing the ferulic acid concentration from 0.5 reduction in the ability to adapt to low temperature. For
mmol L−1 to 5 mmol L−1. The obtained A. niger mutant not example, among 24 E. coli lineages adapted to low temper-
only showed greater ferulic acid tolerance than their parents atures (20 °C), 15 were found to exhibit reduced adaptability
but also had a higher conversion rate to ferulic acid.23 relative to their ancestors at high temperatures (40 °C).33
Geobacillus sp. WSUCF1 is very efficient in depolymerizing However, this trade-off can often be overcome by mutations
unprocessed lignocellulose. However, it produces low levels of brought about by ALE technology. The evolution experiment
laccase. ALE method was used to improve the lignocellulose of E. coli was carried out 2000 times under heat stress (42.2
deconstruction ability of the strain by inducing the expression °C) by ALE technology, and finally obtained an evolutionary
of laccase, a multicopper oxidase, in Geobacillus sp. WSUCF1.
strain of E. coli that could grow at both high temperatures (45
After 15 rounds of adaptive evolution of the strain under the
°C) and low temperatures (20 °C) was obtained. The whole
selective pressure of unprocessed corn stover, the catalytic
genome sequencing showed that the evolutionary strain mainly
laccase activity of an adaptive evolution strain reached 9.23 U/
improved their tolerance to high and low temperatures through
mL, which was 20-fold higher than that of the unadapted
strain. Moreover, the lignin removal rate of the adaptive strain mutation of two adaptive pathways, which were the mutations
was 22%, which was much higher than the 6% removal rate of in the β subunit gene (rpoB) of RNA polymerase (RNAP) and
the unadapted strain.24 the mutations in the termination factor rho of RNAP.
Methanol has become a promising candidate compound in Subsequently, the single mutations of rpoB and rho genes
biological manufacturing due to its extensive sources and demonstrated that two different heat-adaptive genetic path-
abundant energy.25 Because of its high electronic content, the ways had distinct effects on thermal trade-offs.34 In another
yield of the product may be improved compared with report, Papiran and Hamedi35 conducted the ALE experiment
lignocellulose.26 To realize the biomanufacturing of meth- on Lactococcus lactis by gradually increasing the temperature
anol-based fuels and chemicals, there is an urgent need for and the stirring speed. After 162 consecutive transfers, the
microbial cell factories that can effectively utilize methanol as a optimal temperature and stirring speed of were transferred
carbon source.27 However, methanol is more toxic to cells than from 30 °C and 40 rpm to 37 °C and 200 rpm, respectively,
commonly used feedstocks such as sugar and glycerol. and the oxidation and acid resistance of the evolved strain was
Furthermore, methanol as a solvent can cause oxidative stress, enhanced.
which affects the fluidity of the cell membrane and thus lead to Heat tolerance is a polygenic trait that contributes to cell
cell membrane rupture.28 Therefore, it is particularly important survival and growth at abnormally high temperatures. Htg+ is a
to improve the tolerance of microorganisms to methanol. gene that has been identified as being associated with high-
Corynebacterium glutamicum MX-11 was evolved by the ALE temperature growth.36 Caspeta et al.37 found that many genes
strategy at progressively increasing levels of methanol, resulting related to Htg+ may remain unchanged during the evolutionary
in an evolved strain that not only tolerated high concentrations experiment by a one-step temperature jump strategy from 30
of methanol, from an initial concentration of only 4 g/L to 15 to 39.5 °C, immediately followed by 300 generations. Similarly,
g/L, but also showed an increase in growth and methanol ALE was performed on the S. cerevisiae strain by a stepwise
utilization. Transcriptome analysis showed that mutations in temperature increase strategy that allowed it to grow at 42 °C.
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This strategy resulted in the efficient accumulation of higher terminal growth than their ancestors at pH 4.6, and all
mutations associated with Htg+.38 evolved populations showed a gradual loss of activity of lysine
For microorganisms living in cold regions for long periods, decarboxylase, a major acid stress enzyme.50
cold-adapted microorganisms have evolved specific physio- In another report, Overbeck et al.51 constructed MutS
logical mechanisms to tolerate and respond to low temper- deletion mutants of Lactobacillus casei 12A and ATCC 334 by
atures, one of which is related to guanine-cytosine (GC) transiently inactivating the gene encoding the DNA mismatch
content and its variability. Comparative genomic analyses were repair enzyme MutS. Subsequently, the mutant strain was
carried out on typical cold-adapted cryobacterium populations, subjected to a 100 d ALE experiment by a stepwise pH
including 11 type strains and 67 novel strains isolated from reduction strategy, enabling them to grow in a lactic acid
soil, ice, and meltwater of mountain glaciers. The results medium with a pH adjusted to 4.0. It was found that the
showed that the variation of GC3 and the flexibility of DNA ΔmutS mutation was repaired in the L. casei 12A and ATCC
provided the molecular basis for the variation of cold tolerance. 334 ΔmutS mutants. Furthermore, the adapted strain grew
Natural selection is the main reason driving the adaptive faster, had a higher cell density, and produced more lactic acid
evolution of cryobacterium bacteria from moderate to low than the wild-type strain.
temperatures.39 2.2.4. Oxidative Stress. Candida glabrata is a kind of yeast
2.2.2. Osmotic Stress. In E. coli, the sigma factor RpoS of with important industrial applications and resistance to
RNAP is a global regulator that coordinates up to 10% of the oxidative stress is an important feature affecting its robustness
expression of the genome when the bacteria are under stress as a production host. The C. glabrata population gained
conditions such as starvation, acidity, or increased osmotic resistance to oxidative stress by a short-term ALE strategy to
pressure.40 It has been reported that when E. coli lacking RpoS increase the H2O2 concentration from 80 mM to 350 mM. The
evolved adaptively in a medium supplemented with NaCl, evolved strain detoxified H2O2 more rapidly than the wild-type
strains lacking RpoS were found to show less change in and can grow at high concentrations of oxidants. In addition, it
increased fitness and changes in transcriptional patterns was found that the regeneration of NADPH, the regulation of
compared to strains with RpoS function. Furthermore, in a membrane composition, the remodeling of the cell wall, and/
wild-type genetic background, RpoS is required for the or changes of global regulation were all involved in the
transcription of the genes OtsB and OtsA, which encode two tolerance of C. glabrata to H2O2.52 In another report, ALE
enzymes necessary for the synthesis of the osmoprotectant experiments were carried out on Corynebacterium glutamicum
trehalose. The evolved IS10 insertion remodeled the by gradually increasing H2O2 concentrations. The adaptively
expression of OtsB and OtsA from RpoS-dependent to RpoS- evolved strain acquired tolerance to a concentration of 10 mM
independent, thus partially restoring the wild-type response to H2O2. Transcriptome analysis revealed that the pca genes
osmotic stress.41 encoding enzymes of the β-ketoadipate pathway were up-
S. cerevisiae is a good model system for studying salt stress regulated more than 3-fold. Subsequently, the pca gene cluster
tolerance because it contains several highly conserved was expressed in the wild-type Corynebacterium glutamicum
pathways to mediate salt stress responses. S. cerevisiae was strain. It was found that the wild-type strain could not grow at
subjected to 300 generations in a medium containing 0.5 M the concentration of 2 mM H2O2, while the strains expressing
NaCl under continuous salt stress, the growth rate of the the pca gene cluster recovered their growth.53
evolved strain was faster than that of the original strain under 2.3. Enhancing Phenotypes. Enhanced phenotypes
high salt conditions.42 Lipid accumulation in Schizochytrium sp. include enhanced growth phenotypes and enhanced metabolic
can be induced by stress conditions, but this stress induction phenotypes. Due to the high complexity of carbon and energy
usually reduces cell growth and leads to oxidative damage, metabolism in microbial cells, it is difficult in most cases to
ultimately reducing lipid production. Sun et al.43 obtained a achieve control of the yield of target products produced by
Schizochytrium sp. strain with high tolerance to oxidative stress microorganisms during fermentation through rational design
through ALE under high salinity stress (30 g/L NaCl), which methods such as metabolic engineering. In contrast, ALE
exhibited higher total antioxidant capacity and lower levels of technology does not require an in-depth understanding of the
reactive oxygen species compared to the initial strain. complex microbial physiology and can help to obtain a high-
2.2.3. Acid Stress. E. coli has been shown to grow under yield strain of the target product by improving the tolerance of
mild acid stress. To maintain intracellular pH homeostasis, the strain to stress, optimizing the metabolite production
E. coli has developed a variety of strategies, including pathway, and increasing the metabolic flux. Currently, ALE
cytoplasmic buffering,44 proton consumption systems,45 technology has been widely used to optimize the growth of
regulation of cellular metabolism,46 and physiological microorganisms.54,55 The E. coli K-12 MG1655 strain was
responses.47 The ALE experiments were performed on E. coli subjected to 81 d of ALE under the optimal culture conditions,
K-12 MG1655 in a medium at pH 5.5. After 800 generations, and the fitness of the evolved strain was increased by 1.6-fold
the growth rates of the 6 independent populations in evolution compared to the starting strain. Whole-genome sequencing
at pH 5.5 was 18.0% higher than their starting strain, while was showed that the rapid growth phenotype was mainly caused by
comparable to that of the starting strain at pH 7.0. Whole- the mutations of the global transcription gene rpoB, the
genome sequencing found that five of the six sequenced clones metabolic gene pyrE, rph, and the DNA structural gene hns and
had rpoC mutations.48 The rpoC gene encodes a subunit of tdk.54 In addition, many researchers have carried out work in
RNAP that is considered to be a global regulator of gene yeast and microalgae to enhance growth using ALE
expression.49 In a laboratory evolution study of 24 strains from technology, and have obtained strains with better traits.56,57
4 populations of E. coli, generation was first carried out at an It was worth noting that ALE technology enhances microbial
initial pH of 4.8, and at generation 730, the pH was reduced to growth while often leading to an increase in other beneficial
4.6 with hydrochloric acid to continue the subculture. At traits such as biomass or product yield. After 84 d of ALE
generation 2000, all populations were found to have reached experiments were performed on Chlamydomonas reinhardtii,
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Figure 3. Adaptive laboratory evolution (ALE). Comparison between ALE (right side) and traditional metabolic engineering (left side) in the
design, build, test, and learn cycle.12 When phenotypes can be associated with selection without engineering, ALE can be used to completely
replace the design and build steps (a). ALE can be performed by batch culture in a flasks. During each adaptation cycle, nutrients are not limited
and certain growth parameters of microorganisms may fluctuate significantly (b). Alternatively, ALE can be performed by continuous culture in a
chemostat. Cell density and culture conditions can be kept constant during microbial growth (c).

the growth of the evolved strain was enhanced. In addition, the beneficial mutant phenotypes.60 With the deepening of
biomass concentration of the end point strain cc4334 was 1.48- research on microbial metabolism and regulation, the
fold higher than that of the starting strain, and the lipid content combination of rational design methods and ALE technology
of this strain under nitrogen-deficient conditions was nearly 1- will achieve greater success in the field of microbial breeding.
fold higher compared to the starting strain.57
Maximizing the yield of target metabolites is an explicit goal 3. ALE PASSAGE EXPERIMENTAL DESIGN
of microbial ALE. The growth of cells during 5-aminolevulinic
acid (5-ALA) production is inhibited by high concentrations of Compared to rational metabolic engineering, which relies on
the substrate glycine. Therefore, to obtain a robust strain for 5- comprehensive knowledge and a time-consuming design-build-
ALA production, ALE was performed using W3110 strain test-learn cycle, ALE performs well in several aspects (Figure
harboring the ALA synthase gene in the selective plate 3a). First, it does not require a reserve of knowledge about the
supplemented with glycine. Subsequently, metabolic fluxes genetic basis of the target phenotype. Second, it can be
were enhanced by overexpression of phosphoenolpyruvate performed based on spontaneous mutagenesis without the use
carboxylase (ppc) and knockout of phosphate acetyltransferase of genetic engineering tools. Third, it can generate large
(pta). The ribulose-1,5-bisphosphate carboxylase/oxygenase mutant libraries and enrich the optimal libraries through
(RuBisCO) and phosphoribulokinase (PRK) were added to repeated passages in a high throughput manner. In general,
confer CO2 assimilation capacity. The biomass and 5-ALA of ALE experiments can be performed either by batch culture in a
the obtained adaptive RL8 strain were increased by 129% and flask or continuous culture in a chemostat (Figure 3b and 3c).
205%, respectively.58 In another report, genome replication Among them, batch culture is the most commonly used
engineering assisted continuous evolution (GREACE) was method for ALE because it is easy to culture on a large scale
used for the continuous evolution of E. coli MG1655 strain, the and does not require expensive equipment. In addition,
lysine yield of the evolved strain was increased by 14.8% common mutations can be observed by using deep well plates
compared to the starting strain.59 Although ALE technology with smaller culture volumes, or by increasing the number of
can help to obtain evolved strains with better traits, they often replicates through automating,61 which not only easily controls
need to be complemented by effective screening methods. The multiple environmental factors but also clearly identifies
use of biosensor-based cell sorting technology in combination evolutionary directions in specific environments. However,
with ALE technology in strain C. glutamicum ATCC 13032 batch culture has certain disadvantages, such as the difficulty of
resulted in an evolved strain with approximately 25% higher maintaining population density, growth rate, nutrient avail-
valine production and a 3−4 fold reduction in metabolic ability, and environmental conditions constant throughout the
byproduct production. This result demonstrates that the experiment,62 which may lead to faulty evolutionary pressures
effective combination of ALE technology with other screening to produce undesired phenotypes and difficulty explaining the
methods is an ideal strategy for the rapid acquisition of association between genotype and phenotype.63
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Table 1. Application of Microbial ALE in Terpenoid Production

strain ALE strategy results reference
77
Yeast Yarrowia Limonene as selective pressure The titer of limonene increased by 52%
lipolytica
78
Saccharomyces Enzyme inhibitor terbinafine as selective pressure The titer of squalene reaches 193
cerevisiae mg/L
79
S. cerevisiae Oxidative stress with the use of H2O2 as selective pressure The titer was raised 3-fold to 18 mg/g
80
S. cerevisiae Oxidative stress with the use of H2O2 as selective pressure The highest titer of astaxanthin
reached 404.78 mg/L in a 5 L
fermentor
90
S. cerevisiae Periodic exposure to hydrogen peroxide as selective pressure 3-fold higher carotenoid productivity
91
Rhodotorula Oxidative stress with the use of H2O2 as selective pressure 2.3-fold higher carotenoid content
toruloides
82
Microalgae Haematococcus Irradiated with 60Co-γ for 10 generations of ALE under the condition of 6-fold higher astaxanthin content
pluvialis 15% CO2
83
Dunaliella salina Semicontinuous cultures under combining light-emitting diode (LED) 3.3- and 2.3-fold higher β-carotene and
lighting conditions, red LED arrays (75%), and blue LED arrays (25%) lutein content, respectively
89
D. salina Semicontinuous cultures under blue LEDs conditions 19.7% higher β-carotene production
under the blue LEDs conditions
84
Phaeodactylum Semicontinuous cultures under combining light-emitting diode (LED) 2-fold higher fucoxanthin content
tricornutum lighting conditions, red LED arrays (75%), and blue LED arrays (25%)

Continuous cultivation can maintain a constant growth rate green industrial production of high-value-added natural
and population density. In addition, it can strictly control compounds.73 Although researchers have optimized the
nutrient supply and environmental conditions.62,63 However, biosynthetic pathways of terpenoids, the titer of a series of
this approach also has some drawbacks. On the one hand, due terpenoids seems to reach the highest limit. As an innovative
to the long-running time, it is difficult to maintain the stability experimental method, ALE has been widely used in the
of metabolic engineering strains although mutations are helpful research of terpenoid biosynthesis (Table 1).
in terms of evolution.64 On the other hand, mutants with 4.1. Yeast. Yeast not only has a natural mevalonic acid
adhesive properties to prevent being washed away may also (MVA) pathway for the production of terpenoids but also can
exhibit biased evolution.65 Most importantly, its setup and effectively use a wide range of substrates to produce acetyl-
operation costs are high, and it is difficult to proceed in CoA precursors.74 In addition, a variety of mature gene editing
parallel. Therefore, in order to reduce costs and enable achieve techniques have been established in yeast.75,76 Therefore, yeast
parallel operations, miniaturization of containers may be an is considered a promising strain for the synthesis of terpenoids.
alternative route.66 However, even using small containers and ALE is an easy-manipulative method to improve the cellular
parallel operations, its cost far exceeds the cost of shake flask tolerance that has been used in various cases to get higher titer
culture. In order to overcome these shortcomings, many of terpenoids. Limonene plays a significant role in many
multiplexed automated culture platforms have been developed physiological processes such as reducing inflammation,
to realize ALE automation and parallelization, and considerable minimizing bacterial growth, and inhibiting cancer develop-
progress has been made.67,68 ment. To find and testify the genes contributing to improved
Successful ALE results depend on population heterogeneity limonene tolerance, Yarrowia lipolytica was first raised in a high
caused by mutations.69 Although ALE can induce mutations in concentration of limonene and then subjected to short-term
parent strains to increase diversity, it is also important to ALE of four 48-h transmissions in the presence of 500 mg/L
maintain the cell state during each cycle, which also affects the limonene, achieving a titer 52% higher than that of the starting
diversity of mutations.12 Considering that with the extension of strain.77 Squalene is a kind of polyunsaturated triterpene,
culture time, the growth rate increases while the increase in which is widely used in the food industry, pharmacology, and
fitness decreases, optimizing the passage method is also crucial. cosmetics due to its antioxidant properties and its role as a free
In addition, the amplification of microbial processes is often a radical scavenger. The adaptive evolution of metabolic
major obstacle in process design because cells are exposed to engineering S. cerevisiae strain was carried out for 1500 h to
changing environmental conditions and pressures during the enhance squalene accumulation under the condition of
amplification process.70,71 gradually increasing concentration of enzyme inhibitor
terbinafine (0.01−300 μg/L), resulting in a squalene titer of
193 mg/L.78
4. APPLICATION OF ALE IN TERPENOID
In addition to improving tolerance to terpenoids and
PRODUCTION
bioinhibitors, ALE also employs environmental or nutritional
Terpenoids are an important secondary metabolite, which stress strategies corresponding to the characteristics of different
plays an important role in food and agriculture fields. terpenoids and strains. Carotenoids are a group of substances
Currently, most terpenoids are mainly derived from plant with antioxidant activity and anticancer properties, including β-
extracts and chemical synthesis. However, plant extraction is carotene, lycopene, and astaxanthin, which are widely used in
limited by factors such as resource availability and climate and the food, medicine, and cosmetics industries. According to the
chemical synthesis has disadvantages such as complex synthetic antioxidant properties of carotenoids, appropriate oxidative
routes, low yields, pollution, and high inputs.72 Therefore, the stress can promote microorganisms to couple the carotenoid
search for new synthetic pathways of terpenoids has become a production process with cell growth, and survive depending on
major focus of researchers. In recent years, microorganisms are the accumulation of antioxidant substances. Thus, when the
increasingly being developed as efficient platforms for the peroxidase-knocked S. cerevisiae was periodically subjected to
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Table 2. Application of Microbial ALE in Lipid Production

strain ALE strategy results reference
94
Yeast Yarrowia lipolytica Selection of the floated cells after the centrifugation 23% higher lipid content
and isolation of the high lipid content cells by
measuring the fluorescent level
95
Y. lipolytica Growth under nitrogen and magnesium limited 30% higher lipid content
conditions followed by selection under carbon-
limited conditions
97
Saccharomyces Gradually decreasing ethanol concentration and High levels of free fatty acids were produced
cerevisiae gradually increasing glucose concentration
91
Rhodotorula Oxidative stress with the use of H2O2 as selective 40% higher lipid content
toruloidesis pressure
106
Rhodosporidium Sugar cane bagasse hemicellulosic hydrolysate as More than 108%, 175%, and 118% higher lipid content,
toruloides selective pressure concentration, and productivity, respectively
107
Ogataea Methanol as selective pressure 2.8-fold higher free fatty acids
polymorpha
43
Microalgae Schizochytrium sp. High salinity as selective pressure 32.7% and 53.3% higher biomass and lipid yield,
respectively
99
Schizochytrium sp. High levels of oxygen as selective pressure 24.1%, 19.1%, and 8.7% higher lipid content, biomass,
and DHA percentage, respectively
98
Parachlorella sp. High salinity as selective pressure 233% increase of the fatty acid productivity
103
Scenedesmus sp. Stepwise increasing of multistresses such as salinity, The highest lipid content of 44.1% was obtained
light intensity, and temperature
104
Crypthecodinium Sethoxydim and sesamol as selective pressure 100% and 130% increase of the lipid and DHA
cohnii productivity, respectively
108
Chlamydomonas Cultivation under nitrogen-depletion and repletion 50% and 175% increase of the lipid content in the wild
reinhardtii conditions and selection of high-lipid content cells strain and low-starch mutant, respectively, under
with FACS analysis nitrogen starvation conditions
109
Aurantiochytrium Sugar cane bagasse hydrolysate as selective pressure 30.7% increase of the DHA content
sp.

H2O2 shock, the yield of β-carotene could be increased 3-fold Light conditions are another key factor affecting the
to 18 mg/g DCW in a short-term evolution.79 Similarly, to biosynthesis of terpenoids in microalgae.87 It has been
increase astaxanthin production, a combined strategy consist- reported that as light intensity increases, microalgae will
ing of physical mutagenesis by atmospheric and room produce more light-protective pigments such as carotenoids to
temperature plasma (ARTP) and adaptive evolution driven adapt to the increased light intensity.86 In addition, the
by H2O2 was established. Through multiple rounds of cross wavelength of light also affects the photosynthesis of
ARTP and H2O2 treatment, the astaxanthin titer of the flask microalgae. In general, microalgae show higher pigment
was 65.9 mg/L, which was nearly 4-fold higher than that of the absorption in blue and red light compared to other colors.88
initial S. cerevisiae strain. Subsequently, the highest reported To improve the β-carotene production of D. salina, the blue-
light adapted D. salina (ALE-D. salina) method was developed
titer of astaxanthin (404.78 mg/L) in S. cerevisiae was achieved
through ALE and combined with the blue-red LED wave-
in a 5 L fermenter by further optimizing the fermentation
length-shifting system (B-R system) applied to ALE-D. The β-
conditions.80 carotene concentration of the evolved strain reached 33.94
4.2. Microalgae. Microalgae have efficient terpenoid μM, an increase of 19.7% compared to the wild-type D. salina
biosynthetic pathways, including the eukaryotic mevalonic without ALE treatment under the B-R system.89
acid (MVA) pathway and the prokaryotic methyl-D-erythritol
4-phosphate (MEP) pathway. In addition, it can fix inorganic
5. APPLICATION OF ALE IN LIPID PRODUCTION
carbon through photosynthesis to promote its growth and
biosynthesis of terpenoids.81 Therefore, microalgae are Microbial lipids are synthesized by oleaginous microorganisms
considered a rich source of natural terpenoids. It was reported capable of accumulating energy in the form of lipids. When
that the production of astaxanthin, β-carotene, lutein, and carbon sources exist but key nutrients such as nitrogen sources
fucoxanthin was successfully increased after the ALE experi- are deficient, cell growth is interrupted while cells transition
ment was performed on Haematococcus pluvialis, Dunaliella from growth to oil production. Subsequently, when the carbon
salina, and Phaeodactylum tricornutum.82−84 source is exhausted, the lipids accumulated by the cells are
used as intracellular carbon sources to maintain cell growth.92
Microalgae can absorb energy from sunlight and then fix
ALE is considered to be a cost-effective strategy to improve
inorganic carbon (CO2) as a source of carbon.85 Therefore,
microbial oil production, aiming to cultivate microbial strains
CO2 and light are two important factors affecting the growth with cost-saving and high oil production (Table 2).
and terpenoids biosynthesis of microalgae. Generally, high 5.1. Yeast. Oleaginous yeasts, especially S. cerevisiae and
concentrations of CO2 (5−20%) are conducive to photosyn- Y. lipolytica, are excellent microbial chassis cells for lipid
thesis in microalgae,86 and terpenoid production is always synthesis due to their mature genetic manipulation tools and
accompanied by an increase in photosynthetic efficiency. It was unique metabolic characteristics.93 To enhance lipid accumu-
reported that when the high astaxanthin-producing strain lation in the Y. lipolytica strain, Liu et al.94 developed an ALE
H. pluvialis was irradiated with 60Co-γ for 10 generations of strategy associated with the floating cell enrichment process.
ALE under the condition of 15% CO2, it resulted in a 6-fold Each adaptation cycle consisted of culturing the cells in a high
increase in astaxanthin content.82 glucose content medium suitable for lipogenesis and then
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centrifuging and recovering the floating cells as inoculum for A multifactor synergy is also a common tool in ALE. A novel
the next selection cycle, a process that was repeated for a total synergistic two-factor ALE strategy based on concomitant low
of five rounds. Finally, the lipid content of the adapted strain temperature and high salinity has been applied to increase the
was increased by 23% compared to the original strain. In productivity of Schizochytrium sp. After 30 adaptation cycles,
another report, Daskalaki et al.95 proposed an ALE method the maximum cell dry weight of the evolved strain was 126.4 g/
that relies on Y. lipolytica in alternating environments that L and the DHA yield was 38.12 g/L, 27.42% and 57.52%
promote growth, encourage stored lipid synthesis, and reward higher than the parental strain, respectively.102 In another
high-energy cells. Yeast was grown under nitrogen and report, multiple tolerance was successfully induced in an
magnesium-limited conditions and then selected under adaptive evolutionary strain of Scenedesmus sp. SPP by stepwise
carbon-limited conditions. The evolved population obtained increasing physicochemical factors such as salinity, light
after 77 generations were able to accumulate 44% w/w of intensity, and temperature, resulting in an evolved strain with
lipids, which was 30% more than the initial strain. a lipid content of up to 44.1%.103
Medium-chain fatty acids (MCFA) are valuable components In addition, chemical regulators play a role in the regulation
for the production of biofuels and oil chemicals. However, the of cell metabolism and are often used to specifically interfere
efficient synthesis of this fatty acid using microbial biocatalysts with or inhibit the enzyme proteins or signal molecules of
is challenging due to the cytotoxicity of MCFA. In view of this, competing pathways to increase product production. However,
endogenous fatty acid synthase (FAS) and orthologous most chemical regulators have adverse effects on the growth
bacterial type I FAS were designed for MCFA production in and fatty acid accumulation of microalgae. In view of this, a
S. cerevisiae. To improve cell tolerance to toxic MCFA, directed chemical regulator-based ALE was developed and the
evolution of membrane transporter Tpo1 and ALE of strain application of sethoxydim to Crypthecodinium cohnii increased
were carried out, ultimately increasing MCFA production by the expression level of acetyl-CoA carboxylase, resulting in an
1.3-fold and 1.7-fold, respectively.96 increase in lipid and DHA production by approximately 50%
In many research works, major reprogramming of microbial and 90%, respectively. Subsequently, ALE experiments were
performed to improve the sensitivity of the strain to sesamol.
metabolism is often required to ensure high throughput of the
When 1 mM sesamol was added, the evolved strain obtained
product of interest, which often requires the use of ALE
increased lipid and DHA production by approximately 100%
techniques in combination with genetic engineering techni-
and 130%, respectively.104 In another report, Schizochytrium sp.
ques. In one report, metabolic engineering and process design
S31 was domesticated in successive generations using the ALE
of S. cerevisiae, including altered subcellular metabolic trans- method by gradually increasing the domestication concen-
port, fine-tuned NADPH and ATP supply, and reduced carbon tration of the fatty acid synthesis pathway inhibitor
flux to biomass, enabled the production of 33.4 g/L of sethoxydim. After nearly 200 d of continuous domestication
extracellular free fatty acids. Subsequently, pyruvate decarbox- for 65 generations, the tolerance concentration of Schizochy-
ylase was deleted and the engineered S. cerevisiae strain was trium sp. S31 to sethoxydim increased from 200 μmol/L to
cultured on a basic medium containing 0.5% glucose and 2% 500 μmol/L. Compared to the wild-type strain, the biomass
ethanol with successive generations every 48 or 72 h. The and DHA production of the evolved strain were increased by
ethanol concentration was gradually reduced and the glucose 19.74% and 29.38%, respectively, in a 5 L fermenter.105
concentration gradually increased for 45 d generations.
Subsequently, the strain was transferred to a basic medium 6. CONCLUSIONS AND FUTURE PERSPECTIVES
containing 2% glucose as the sole carbon source, and growth
ALE technology has greatly accelerated the efficiency of
was promoted by successive generations every 48 h or 72 for
modifying metabolic pathways and cell tolerance, and it has
50 d generations. After approximately 200 generations, the
been widely used in improving the level of target product
evolved strain achieved growth in glucose. Finally, the synthesis, expanding the range of substrate utilization, and
metabolically engineered S. cerevisiae was converted from enhancing the tolerance of chassis cells. However, the
ethanol fermentation to pure lipogenic metabolism, resulting in construction and screening of cell factories are often limited
a high level of free fatty acid production.97 by the determination of target phenotypes. Therefore,
5.2. Microalgae. Microalgal lipids are rich in polyunsatu- researchers need to continuously improve and optimize
rated fatty acids (PUFAs), including EPA, DHA, and ARA, existing techniques such as the use of biosensors to transform
which can be used as a source of functional foods and difficult-to-detect phenotypes into rapidly measurable pheno-
feedstock for green biofuels. Salt stress is widely used in ALE types.110 In addition, excellent strains obtained by ALE often
to increase the lipid content of microalgae.98 According to the contain a large number of mutation loci, of which a limited
report, the lipid productivity of the evolved strain microalgae number is strongly associated with the target phenotype.
Schizochytrium sp. increased 1.96-fold to 80.14 g/L by Therefore, more efficient and accurate bioinformatics algo-
combining ALE with stress at high salt concentrations.99 rithms need to be developed to reduce the difficulty of genetic
Similarly, P. tricornutum was adaptively evolved through 70% mechanism analysis.
salinity for 252 d, and then the evolved strain was treated with Although ALE technology provides a powerful tool for the
15 mg/L fulvic acid, which finally led to the highest EPA construction of microbial cell factories, there are still many
content of the evolved P. tricornutum of 13.9%.100 Temper- issues to overcome such as the often time-consuming and
ature stress is also a common strategy for increasing the lipid expensive labor costs of the evolution process. Therefore, it can
content of microalgae through ALE. Combined with 70 circles be combined with advanced computer-aided design,8 automa-
of ALE, Schizochytrium sp. adapted to the high-temperature tion technology,9 and high-throughput screening technology10
stimulation from 28 to 34 °C, whose cell growth rate improved in future developments, which will greatly promote the
by 60.55% while lipid and DHA concentration reached 4.31 synthesis of target high-value compounds and advance the
and 4.33-fold the starting strain.101 development of microbial ALE engineering. In addition, the
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ACS Synthetic Biology pubs.acs.org/synthbio Review

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■ AUTHOR INFORMATION
Corresponding Authors
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of an industrial strain of Saccharomyces cerevisiae for combined
tolerance to inhibitors and temperature. Biotechnol. Biofuels 2013, 6,
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China; orcid.org/0000-0002-2323-2408; properties of adaptive walks in evolving populations of fungus. PLoS
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Xiao-Man Sun − School of Food Science and Pharmaceutical (6) Tenaillon, O.; Barrick, J. E.; Ribeck, N.; Deatherage, D. E.;
Engineering, Nanjing Normal University, Nanjing 210023, Blanchard, J. L.; Dasgupta, A.; Wu, G. C.; Wielgoss, S.; Cruveiller, S.;
Medigue, C.; Schneider, D.; Lenski, R. E. Tempo and mode of
China; orcid.org/0000-0002-6268-4546;
genome evolution in a 50,000-generation experiment. Nature 2016,
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Authors (7) Shendure, J.; Balasubramanian, S.; Church, G. M.; Gilbert, W.;
Rogers, J.; Schloss, J. A.; Waterston, R. H. DNA sequencing at 40,
Yu-Lei Jia − School of Food Science and Pharmaceutical
past, present and future. Nature 2017, 550, 345−353.
Engineering, Nanjing Normal University, Nanjing 210023, (8) Wong, B. G.; Mancuso, C. P.; Kiriakov, S.; Bashor, C. J.; Khalil,
China A. S. Precise, automated control of conditions for high-throughput
Jin Li − School of Food Science and Pharmaceutical growth of yeast and bacteria with eVOLVER. Nat. Biotechnol. 2018,
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China (9) Zhang, J.; Chen, Y.; Fu, L.; Guo, E.; Wang, B.; Dai, L.; Si, T.
Fang-Tong Nong − School of Food Science and Accelerating strain engineering in biofuel research via build and test
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China
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China (12) Sandberg, T. E.; Salazar, M. J.; Weng, L. L.; Palsson, B. O.;
Xiao-Feng Zhu − College of Life Sciences, Sichuan University, Feist, A. M. The emergence of adaptive laboratory evolution as an
Chengdu 610065, China efficient tool for biological discovery and industrial biotechnology.
Complete contact information is available at: Metab. Eng. 2019, 56, 1−16.
(13) Shepelin, D.; Hansen, A. S. L.; Lennen, R.; Luo, H.; Herrgård,
https://pubs.acs.org/10.1021/acssynbio.3c00179
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Author Contributions (14) Williams, T. C.; Pretorius, I. S.; Paulsen, I. T. Synthetic
Yu-Lei Jia: Conceptualization, Methodology, Investigation, evolution of metabolic productivity using biosensors. Trends
Formal analysis, Data curation, Visualization, Writing� Biotechnol. 2016, 34, 371−381.
original draft. Jin Li and Fang-Tong Nong: Investigation, (15) Stella, R. G.; Wiechert, J.; Noack, S.; Frunzke, J. Evolutionary
Formal analysis. Chun-Xiao Yan and Wang Ma: Methodology, engineering of Corynebacterium glutamicum. Biotechnol. J. 2019, 14,
Formal analysis. Xiao-Feng Zhu: Writing�review and editing. No. e1800444.
Li-Hui Zhang and Xiao-Man Sun: Conceptualization, Super- (16) Fernández-Cabezón, L.; Cros, A.; Nikel, P. I. Evolutionary
vision, Methodology, Formal analysis, Visualization, Writing� approaches for engineering industrially relevant phenotypes in
review and editing, Project administration, Resources, Funding bacterial cell factories. Biotechnol. J. 2019, 14, No. e1800439.
acquisition. (17) Bachmann, H.; Pronk, J. T.; Kleerebezem, M.; Teusink, B.
Evolutionary engineering to enhance starter culture performance in
Notes food fermentations. Curr. Opin. Biotechnol. 2015, 32, 1−7.
The authors declare no competing financial interest. (18) Zhu, Z.; Zhang, J.; Ji, X.; Fang, Z.; Wu, Z.; Chen, J.; Du, G.

■ ACKNOWLEDGMENTS
The research was supported by the National Natural Science
Evolutionary engineering of industrial microorganisms-strategies and
applications. Appl. Microbiol. Biotechnol. 2018, 102, 4615−4627.
(19) Morrison, M. S.; Podracky, C. J.; Liu, D. R. The developing
Foundation of China (No. 22038007), the Postgraduate toolkit of continuous directed evolution. Nat. Chem. Biol. 2020, 16,
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Research and Practice Innovation Program of Jiangsu Province
(20) Jang, S.; Kim, M.; Hwang, J.; Jung, G. Y. Tools and systems for
(KYCX22_1610), and Sichuan Science and Technology evolutionary engineering of biomolecules and microorganisms. J. Ind.
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