Adaptive Laboratory Evolution Strategy Research Articles

Simultaneous improvement of fructophilicity and ethanol tolerance of Saccharomyces cerevisiae strains through a single Adaptive Laboratory Evolution Strategy

Saccharomyces cerevisiae is the main yeast used in the winemaking industry. Its innate glucophilicity provokes a discrepancy in glucose and fructose consumption during alcoholic fermentation of grape must, which, combined with the inhibitory effect of ethanol accumulated in the fermentation broth, might lead to stuck or sluggish fermentations. In the present study, we realized an Adaptive Laboratory Evolution strategy, where an alcoholic fermentation of a 20 g/L fructose broth was followed by cell selection in a high ethanol concentration environment, employed in two different S. cerevisiae strains named CFB and BLR. The evolved populations originated from each strain after 100 generations of evolution exhibited diverse fermentative abilities. One evolved population, originated from CFB strain, fermented a synthetic broth of 100 g/L glucose and 100 g/L fructose to dryness in 170 h, whereas the parental strain did not complete the fermentation even after 1000 h of incubation. The parameters of growth of the parental and evolved populations of the present study, as well as of the ethanol tolerant populations acquired in a previous study, when grown in a synthetic broth of 100 g/L glucose and 100 g/L fructose, were calculated through a kinetic model, and were compared to each other in order to identify the effect of evolution on the biochemical behavior of the strains. Finally, in a 200 g/L fructose synthetic broth fermentation, only the evolved population derived from CFB strain showed improved fermentative behavior than its parental strain.

Boosting hydrogen production in Rhodospirillum rubrum by syngas-driven photoheterotrophic adaptive evolution

Rhodospirillum rubrum is a photosynthetic purple non-sulphur bacterium with great potential to be used for complex waste valorisation in biotechnological applications due to its metabolic versatility. This study investigates the production of hydrogen (H2) and polyhydroxyalkanoates (PHA) by R. rubrum from syngas under photoheterotrophic conditions. An adaptive laboratory evolution strategy (ALE) has been carried out to improve the yield of the process. After 200 generations, two evolved strains were selected that showed reduced lag phase and enhanced poly-3-hydroxybutyrate (PHB) and H2 synthesis compared to the parental strain. Genomic analysis of the photo-adapted (PA) variants showed four genes with single point mutations, including the photosynthesis gene expression regulator PpsR. The proteome of the variants suggested that the adapted variants overproduced H2 due to a more efficient CO oxidation through the CO-dehydrogenase enzyme complex and confirmed that energy acquisition was enhanced through overexpression of the photosynthetic system and metal cofactors essential for pigment biosynthesis.

Efficient Biosynthesis of Salidroside via Artificial in Vivo enhanced UDP-Glucose System Using Cheap Sucrose as Substrate.

Salidroside, a valuable phenylethanoid glycoside, is obtained from plants belonging to the Rhodiola genus, known for its diverse biological properties. At present, salidroside is still far from large-scale industrial production due to its lower titer and higher process cost. In this study, we have for the first time increased salidroside production by enhancing UDP-glucose supply in situ. We constructed an in vivo UDP-glucose regeneration system that works in conjunction with UDP-glucose transferase from Rhodiola innovatively to improve UDP-glucose availability. And a coculture was formed in order to enable de novo salidroside synthesis. Confronted with the influence of tyrosol on strain growth, an adaptive laboratory evolution strategy was implemented to enhance the strain's tolerance. Similarly, salidroside production was optimized through refinement of the fermentation medium, the inoculation ratio of the two microbes, and the inoculation size. The final salidroside titer reached 3.8 g/L. This was the highest titer achieved at the shake flask level in the existing reports. And this marked the first successful synthesis of salidroside in an in situ enhanced UDP-glucose system using sucrose. The cost was reduced by 93% due to the use of inexpensive substrates. This accomplishment laid a robust foundation for further investigations into the synthesis of other notable glycosides and natural compounds.

Synergistic application of atmospheric and room temperature plasma mutagenesis and adaptive laboratory evolution improves the tolerance of Escherichia coli to L-cysteine.

L-Cysteine production through fermentation stands as a promising technology. However, excessive accumulation of L-cysteine poses a challenge due to the potential to inflict damage on cellular DNA. In this study, we employed a synergistic approach encompassing atmospheric and room temperature plasma mutagenesis (ARTP) and adaptive laboratory evolution (ALE) to improve L-cysteine tolerance in Escherichia coli. ARTP-treated populations obtained substantial enhancement in L-cysteine tolerance by ALE. Whole-genome sequencing, transcription analysis, and reverse engineering, revealed the pivotal role of an effective export mechanism mediated by gene eamB in augmenting L-cysteine resistance. The isolated tolerant strain, 60AP03/pTrc-cysEf , achieved a 2.2-fold increase in L-cysteine titer by overexpressing the critical gene cysEf during batch fermentation, underscoring its enormous potential for L-cysteine production. The production evaluations, supplemented with L-serine, further demonstrated the stability and superiority of tolerant strains in L-cysteine production. Overall, our work highlighted the substantial impact of the combined ARTP and ALE strategy in increasing the tolerance of E. coli to L-cysteine, providing valuable insights into improving L-cysteine overproduction, and further emphasized the potential of biotechnology in industrial production.

Advances in using adaptive laboratory evolution technology for engineering of photosynthetic cyanobacteria

Cyanobacteria are the only prokaryotes capable of oxygenic photosynthesis, which have potential to serve as "autotrophic cell factories". However, the synthesis of biofuels and chemicals using cyanobacteria as chassis are suffered from poor stress tolerance and low yield, resulting in low economic feasibility for industrial production. Thus, it's urgent to construct new cyanobacterial chassis by means of synthetic biology. In recent years, adaptive laboratory evolution (ALE) has made great achievements in chassis engineering, including optimizing growth rate, increasing tolerance, enhancing substrate utilization and increasing product yield. ALE has also made some progress in improving the tolerance of cyanobacteria to high light intensity, heavy metal ions, high concentrations of salt and organic solvents. However, the engineering efficiency of ALE strategy in cyanobacteria is generally low, and the molecular mechanisms underpinning the tolerance to various stresses have not been fully elucidated. To this end, this review summarizes the ALE-associated technical strategies and their applications in cyanobacteria chassis engineering, following by discussing how to construct larger ALE mutation library, increase mutation frequency of strains and shorten evolution time. Moreover, exploration of the construction principles and strategies for constructing multi-stress tolerant cyanobacteria, and efficient analysis the mutant libraries of evolved strains as well as construction of strains with high yield and strong robustness are discussed, with the aim to facilitate the engineering of cyanobacteria chassis and the application of engineered cyanobacteria in the future.

Physiological and transcriptome analyses of Kluyveromyces marxianus reveal adaptive traits in stress response.

Kluyveromyces marxianus is a non-conventional yeast with outstanding physiological characteristics and a high potential for lignocellulosic ethanol production. However, achieving high ethanol productivity requires overcoming several biotechnological challenges due to the cellular inhibition caused by the inhibitors present in the medium. In this work, K. marxianus SLP1 was adapted to increase its tolerance to a mix of inhibitory compounds using the adaptive laboratory evolution strategy to study the adaptation and stress response mechanisms used by this non-Saccharomyces yeast. The fermentative and physiological parameters demonstrated that the adapted K. marxianus P8 had a better response against the synergistic effects of multiple inhibitors because it reduced the lag phase from 12 to 4 h, increasing the biomass by 40% and improving the volumetric ethanol productivity 16-fold than the parental K. marxianus SLP1. To reveal the effect of adaptation process in P8, transcriptome analysis was carried out; the result showed that the basal gene expression in P8 changed, suggesting the biological capability of K. marxianus to activate the adaptative prediction mechanism. Similarly, we carried out physiologic and transcriptome analyses to reveal the mechanisms involved in the stress response triggered by furfural, the most potent inhibitor in K. marxianus. Stress response studies demonstrated that P8 had a better physiologic response than SLP1, since key genes related to furfural transformation (ALD4 and ALD6) and stress response (STL1) were upregulated. Our study demonstrates the rapid adaptability of K. marxianus to stressful environments, making this yeast a promising candidate to produce lignocellulosic ethanol. KEY POINTS: • K. marxianus was adapted to increase its tolerance to a mix of inhibitory compounds • The basal gene expression of K. marxianus changed after the adaptation process • Adapted K. marxianus showed a better physiological response to stress by inhibitors • Transcriptome analyses revealed key genes involved in the stress response.

Altering autotrophic carbon metabolism of Nitzschia closterium to mixotrophic mode for high-value product improvement

An adaptive laboratory evolution (ALE) strategy was designed to evolve autotrophic Nitzschia closterium to mixotrophic growth for high productivity of essential amino acid (EAA), eicosapentaenoic acid (EPA) and fucoxanthin. The N. closterium growth was limited under glucose initially, but a red light emitting diode was innovatively applied to modify carbon metabolism and obtain mixotrophic strain of N. closterium GM. The N. closterium GM biomass concentration was improved by 65.07% comparing with wild type, but exhibited weak photosynthesis and strong glucose metabolism. At carbon metabolism levels, ALE promoted NADPH oxidase activity and induced protein degradation to lipid biosynthesis by elevating acetyl-CoA and pyruvate contents. It also improved carbon flux to TCA cycle, and elevated contents of glucose-6-phosphate, fructose-6-phosphate, glyceraldehyde-3-phosphate for providing sufficient ATP and NADPH. Productivities of EPA, EAA and fucoxanthin were increased by 41.0%, 18.8% and 20.4%, respectively. This ALE strategy was promising in microalgal production of high-value products.

Growth fitness, heme uptake and genomic variants in mutants of oxygen-tolerant Lacticaseibacillus casei and Lactiplantibacillus plantarum strains

Adaptive Laboratory Evolution (ALE) is a powerful tool to improve the fitness of industrially relevant microorganisms, because it circumvents some of the problems related to the use of genetically modified strains. In this study, we used an ALE strategy involving serial batch cultivations in aerobic and respiratory conditions to generate spontaneous mutants from the respiration-competent strain Lacticaseibacillus casei N87. Genotypic changes in selected mutants were investigated using whole genome sequencing (WGS). The O2-tolerant Lactiplantibacillus plantarum C17 and its mutant C17-m58 (obtained from a previous ALE study) were included in heme uptake experiments and in WGS and variant calling analyses. Several Lcb. casei N87 mutants cultivated under aerobic and respiratory conditions showed improved biomass production, O2 uptake and oxidative stress tolerance compared to the parental strain. Mutants of Lcb. casei and Lpb. plantarum differed from the parental strains in the ability to use heme and menaquinone. High heme concentrations (> 10 mg/L), however, were toxic for all strains. Single nucleotide modifications (SNPs) were detected in some genes encoding for proteins and transcriptional regulators involved in carbon metabolism, oxidative stress, redox balance, and cell wall properties, but their role in the evolved phenotypes needs further investigations. We conclude that prolonged adaptation to aerobic and respiratory life-style may be used as natural strategy to generate strains with improved O2-consuming ability and oxidative stress tolerance, two important features to develop robust cultures and to reduce oxidative processes in foods.

Adaptive laboratory evolution and shuffling of Escherichia coli to enhance its tolerance and production of astaxanthin

BackgroundAstaxanthin is one of the strongest antioxidants in nature and has been widely used in aquaculture, food, cosmetic and pharmaceutical industries. Numerous stresses caused in the process of a large scale-culture, such as high acetate concentration, high osmolarity, high level of reactive oxygen species, high glucose concentration and acid environment, etc., limit cell growth to reach the real high cell density, thereby affecting astaxanthin production.ResultsWe developed an adaptive laboratory evolution (ALE) strategy to enhance the production of chemicals by improving strain tolerance against industrial fermentation conditions. This ALE strategy resulted in 18.5% and 53.7% increases in cell growth and astaxanthin production in fed-batch fermentation, respectively. Whole-genome resequencing showed that 65 mutations with amino acid substitution were identified in 61 genes of the shuffled strain Escherichia coli AST-4AS. CRISPR interference (CRISPRi) and activation (CRISPRa) revealed that the shuffled strain with higher astaxanthin production may be associated with the mutations of some stress response protein genes, some fatty acid biosynthetic genes and rppH. Repression of yadC, ygfI and rcsC, activation of rnb, envZ and recC further improved the production of astaxanthin in the shuffled strain E. coli AST-4AS. Simultaneous deletion of yadC and overexpression of rnb increased the production of astaxanthin by 32% in the shuffled strain E. coli AST-4AS.ConclusionThis ALE strategy will be powerful in engineering microorganisms for the high-level production of chemicals.

A Two-Stage Adaptive Laboratory Evolution Strategy to Enhance Docosahexaenoic Acid Synthesis in Oleaginous Thraustochytrid.

Thraustochytrid is a promising algal oil resource with the potential to meet the demand for docosahexaenoic acid (DHA). However, oils with high DHA content produced by genetic modified thraustochytrids are not accepted by the food and pharmaceutical industries in many countries. Therefore, in order to obtain non-transgenic strains with high DHA content, a two-stage adaptive laboratory evolution (ALE) strategy was applied to the thraustochytrid Aurantiochytrium sp. Heavy-ion irradiation technique was first used before the ALE to increase the genetic diversity of strains, and then two-step ALE: low temperature based ALE and ACCase inhibitor quizalofop-p-ethyl based ALE were employed in enhancing the DHA production. Using this strategy, the end-point strain E-81 with a DHA content 51% higher than that of the parental strain was obtained. The performance of E-81 strain was further analyzed by component analysis and quantitative real-time PCR. The results showed that the enhanced in lipid content was due to the up-regulated expression of key enzymes in lipid accumulation, while the increase in DHA content was due to the increased transcriptional levels of polyunsaturated fatty acid synthase. This study demonstrated a non-genetic approach to enhance lipid and DHA content in non-model industrial oleaginous strains.

Simultaneous Improvement of Limonene Production and Tolerance in Yarrowia lipolytica through Tolerance Engineering and Evolutionary Engineering.

Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast Yarrowia lipolytica that were up-regulated when exposed to limonene. When overexpressed, 8 of the gene candidates improved tolerance of this yeast to exogenously added limonene. To determine whether overexpression of these genes could also improve limonene production, we individually coexpressed the tolerance-enhancing genes with a limonene synthase gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene production in Y.lipolytica. Particularly, overexpressing YALI0F19492p led to an 8-fold improvement in product titer. Furthermore, through short-term adaptive laboratory evolution strategy, in combination with morphological and cytoplasmic membrane integrity analysis, we shed light on the underlying mechanism of limonene cytotoxicity to Y.lipolytica. This study demonstrated an effective strategy for improving limonene tolerance of Y.lipolytica and limonene titer in the host strain through the combinatorial use of tolerance engineering and evolutionary engineering.

Adaptive laboratory evolution induced novel mutations in Zymomonas mobilis ATCC ZW658: a potential platform for co-utilization of glucose and xylose.

A systematic adaptive laboratory evolution strategy was employed to develop a potential Zymomonas mobilis strain with the ability to co-utilize glucose and xylose. Z. mobilis ATCC ZW658, a recombinant xylose fermenting strain, was subjected to adaptive laboratory evolution over a period of 200days under strict selection pressure of increasing concentration of xylose. The evolved strain exhibited 1.65 times increase in the overall specific xylose utilization rate when compared with the parent strain. Furthermore, the strain displayed significantly improved performance in terms of co-fermentation of xylose in the presence of glucose with specific glucose and xylose utilization rate of 1.24gg-1 h-1 and 1.34gg-1 h-1, respectively. Altered phenotypic response of the evolved strain, in terms of improved xylose utilization, co-utilization of mixed sugars, enhanced growth, ethanol production, and reduced xylitol production has been explained by novel mutations, identified using next-generation sequencing, in xylose assimilating, metabolizing, and crucial regulatory pathway genes and key enzyme activity assays.

Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

Abstract Background In industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze–thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain. Results Elite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze–thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield. Conclusions A multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze–thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation. How to cite: Zhang Q, Jin Y, Fang Y, et al. Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high gravity bioethanol fermentation. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.06.003

Impaired oxidative stress and sulfur assimilation contribute to acid tolerance of Corynebacterium glutamicum.

The industrial organism Corynebacterium glutamicum is often subjected to acid stress during large-scale fermentation for the production of bio-based chemicals. The capacity of the cells to thrive in acidic environments is a prerequisite for achieving high product yields. In this study, we obtained an acid-adapted strain using an adaptive laboratory evolution strategy. Physiological characterizations revealed that the adapted strain achieved improved cell viability after acid-stress challenge, with a higher cytoplasmic pHin level, a lower intracellular reactive oxygen species (ROS), and an enhanced morphological integrity of the cells, when compared to those of the original control strain. Transcriptome analysis indicated that several important cellular processes were altered in the adapted strain, including sulfur metabolism, iron transport, and central metabolic pathways. Further research displayed that KatA and Dps cooperatively mediated intracellular ROS scavenging, which was required for resistance to low-pH stress in C. glutamicum. Furthermore, the repression of sulfur assimilation by the McbR regulator also contributed to the improvement of acid-stress tolerance. Moreover, two copper chaperone genes cg1328 and cg3292 were found to be involved in promoting cell survival under acid-stress conditions. Finally, a new recombinant C. glutamicum strain with enhanced acid tolerance was generated by the combined overexpression of katA, dps, mcbR, and cg1328, showing 18.4 ± 2.5% higher biomass yields than the wild-type strain under acid-stress conditions. These findings will provide new insights into the understanding and genetic improvement of acid tolerance in C. glutamicum.

Beyond the Theoretical Yields of Dark-Fermentative Biohydrogen.

Theoretical hydrogen (H2) yield by dark fermentative route is 12mol/mol of glucose. Biological H2 production yields of 3.8mol/mol of glucose by microbes have been reported. Transient gene inactivation in combination with adaptive laboratory evolution strategy has enabled the H2 yield to exceed the stoichiometric production values.

Growth-Coupled Carotenoids Production Using Adaptive Laboratory Evolution.

Adaptive laboratory evolution is a powerful technique for strain development. However, the target phenotypes using this strategy have been limited by the required coupling of the phenotype-of-interest with fitness or survival, and thus adaptive evolution is generally not used to improve product formation. If the desired product confers a benefit to the host, then adaptive evolution can be an effective approach to improve host productivity. In this book chapter, we describe an effective adaptive laboratory evolution strategy for improving product formation of carotenoids, a class of compounds with antioxidant potential, in the yeast Saccharomyces cerevisiae.

An auto-inducible Escherichia coli strain obtained by adaptive laboratory evolution for fatty acid synthesis from ionic liquid-treated bamboo hydrolysate

Adaptive laboratory evolution (ALE) is a useful metabolic engineering strategy, which allows the selection of the microorganisms with beneficial phenotype through accumulative beneficial mutations among genetic variations occurrencely. Following ALE strategy, a rational constructed Escherichia coli strain DQ101 for fatty acids synthesis was adaptively evolved for 90days with increasing [C4mim]Cl concentration from 1% to 7% (w/v). The evolved strain DQ102 reached a final OD600 of 4.93 at the end of the 24h culture with 7% (w/v) ionic liquid. DQ102/pDQTES with a thioesterase ‘TesA overexpression could produce 1.12g/L fatty acid with a productivity of 0.023g/L-h from ionic liquid-treated bamboo hydrolysate. With another β-hydroxyacyl-ACP dehydratases (fabZ) overexpression, DQ102/pDQTESZ could reach a higher concentration of 2.29g/L with a productivity of 0.048g/L-h. These results indicated that ALE could be implemented as a useful tool for metabolic engineering and production of bio-fuels, as well as commodity and specialty chemicals.

Directional regulation of the metabolic heterogeneity in anaerobic mixed culture to enhance fermentative hydrogen production by adaptive laboratory evolution

In this study, an adaptive laboratory evolution strategy was originally developed to enhance fermentative hydrogen production by directionally regulating the metabolic heterogeneity in anaerobic mixed culture. The results indicated that the co-introduction of 4-methylpyrazole and oxamate could redistribute the metabolic flux to butyrate-type hydrogen fermentation. Subsequently, a synergistic evolutionary pressure, combining exogenous butyrate stress with 4-methylpyrazole and oxamate, was employed to evolve hydrogen-producing mixed culture with continuous fermentation system. The metabolic engineering strategy could directionally regulate the metabolic heterogeneity through efficiently shaping powerful butyrate-type hydrogen-producing community, by which evolved culture acquired a significantly improved hydrogen yield and productivity. Furthermore, compared with original culture, evolved culture possessed much higher activities of pyruvate-ferredoxin oxidoreductase and hydrogenase but a much lower ferredoxin-NAD+ oxidoreductase activity, and these enzymatic evolutionary mechanisms were crucially important for the enhanced hydrogen fermentation.

Enhanced glutathione production by evolutionary engineering of Saccharomyces cerevisiae strains.

Glutathione is an important natural tripeptide mainly used because of its antioxidative properties. Commercial glutathione is microbially synthesized by yeasts and the growing demand requires the development of new production strains. An adaptive laboratory evolution strategy using acrolein as a selection agent was employed to obtain strains with an enhanced glutathione accumulation phenotype accompanied by an acrolein resistance phenotype. Two particularly interesting isolates were obtained: one with a high volumetric productivity for glutathione reaching 8.3 mg(glutathione)/L h, which is twice as high as the volumetric productivity of its parental strain. This strain reached an elevated intracellular glutathione content of 3.9%. A second isolate with an even higher acrolein tolerance exhibited a lower volumetric productivity of 5.8 mg(glutathione)/L h due to a growth phenotype. However, this evolved strain accumulated glutathione in 3.3-fold higher concentration compared to its parental strain and reached a particularly high glutathione content of almost 6%. The presented results demonstrate that acrolein is a powerful selection agent to obtain high glutathione accumulation strains in an adaptive laboratory evolution experiment.

Enhancement of lipid production in low-starch mutants Chlamydomonas reinhardtii by adaptive laboratory evolution

Adaptive laboratory evolution (ALE) is an effective method to improve microalgal strains. The growth phenotypes of three strains (cc4324, cc4326 and cc4334) of green microalgae Chlamydomonas reinhardtii were enhanced by ALE. As a result, endpoint strains exhibited higher growth rates. Upon the utilisation of ALE strategy, the biomass concentrations of the endpoint strains of cc4324, cc4326 and cc4334 became 1.17, 1.33 and 1.48 times of those of the starting strains. The total lipid content of the original strains was increased gradually from 32% to 36.67% in the endpoint strain cc4326 and abruptly increased from 24.27% to 44.67% in the endpoint strain cc4334 by nitrogen starvation. Slight growth impairment was also observed in low-starch mutants exposed to nitrogen starvation stress. However, this impairment was quickly resolved after nitrogen was replenished. These findings demonstrated that the biomass concentration and lipid productivity of low-starch mutants can be enhanced by ALE.