Development of flow cytometric procedures for the efficient isolation of improved lipid accumulation mutants in a Chlorella sp. microalga
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The successful development of microalgae-based biofuel production will rely on improvements in the amount and rate of fuel molecule precursor accumulation. Mutagenesis and selection is an attractive approach to improve fuel molecule productivity. Previous screening methods have been laborious, the numbers of mutants isolated have been small, and overall performance of mutants may have been compromised by the presence of deleterious secondary mutations generated by random mutagenesis that affect other cellular processes and growth. We report an improved method of isolating high triacylglycerol (TAG) accumulating mutants of a Chlorella sp., KAS603, using flow cytometric-based selection. In addition to selection for high TAG accumulating strains, the method requires that growth of mutants be competitive with other cells in the population. Not only is growth competitive, but there is improved performance in TAG accumulation with repeated selection, suggesting purification from deleterious secondary mutations. The procedure resulted in the isolation of mutants with far higher efficiency (thousands of fold) that outperformed wild type substantially better (1.8–2.5-fold) than with previous methods. This opens the door to new approaches to the characterization of genes involved in TAG accumulation and other cellular processes.
KeywordsChlorella Triacylglycerols Mutant Flow cytometry Selection Biofuels
As research and technology for microalgae-based biofuels develops, it is clear that lowering the cost of production is the single most important factor for commercial viability. There are many factors associated with large-scale production, ranging from the biology to engineering of culture systems, nutrient regime in the growth medium, harvesting and extraction processes, etc., and different aspects contribute differently to the overall cost. Analyses indicate that the two biggest components towards reducing cost would be improved lipid content per cell (for lipid-based fuel precursor molecules) and improved algae growth and biomass accumulation (Pienkos et al. 2011). Although many native algae species have attractive growth and lipid accumulation characteristics, their performance under outdoor cultivation conditions can be less relative to laboratory conditions. In addition, even though native species may accumulate adequate amounts of lipid or triacylglycerol (TAG), the rate of accumulation may hinder economical production. Microalgae species that accumulate fuel precursors in abundance, at a rapid rate, will be most desirable for production systems.
There are two general approaches to improve lipid accumulation ability: selection of better performing strains and directed genetic manipulation. The latter is the “rational” approach, in which specific target genes suspected to be important are manipulated to improve fuel precursor production characteristics. Its major advantage is that the gene to be manipulated is known. Its disadvantages are that (1) it may not be clear until the manipulation was complete whether the gene was a good target, (2) the approach may not encompass genes that have an unanticipated beneficial effect (e.g., transcription factors that regulate pathways may be difficult to identify), and (3) because the manipulation leads to a genetically modified organism, regulatory issues come into play for production. Selection approaches have proven valuable in isolating environmental strains with improved production characteristics (Aquatic Species Program Closeout Report 1998; Mutanda et al. 2011). Selection approaches can be applied to wild-type strains (Montero et al. 2011) or be combined with mutagenesis as a means of genetic improvement with the advantage that no foreknowledge of the genes involved is needed. This enables identification of both anticipated and unanticipated genes responsible for improved phenotypes, and mutagenized and/or selected organisms are not classified as genetically modified. The drawbacks of mutagenesis and selection approaches are that (1) a specific selection method must be available for the desired phenotype; (2) in the case of random mutagenesis, multiple secondary mutations are generated in the genome, some of which could be detrimental to growth or productivity; and (3) identification of the locus of the mutation responsible for the desired phenotype can be challenging given the fact that it may only result from a point mutation in a background of numerous point mutations in other genes. However, with modern techniques of inexpensive high-throughput sequencing available, reasonable strategies can be devised to identify the responsible mutation.
Previous work has demonstrated the utility of selecting algal mutants for improved productivity. In chlorophyte algae, a phenotypic screen for inhibited starch accumulation has resulted in isolation of “starchless” mutants (Ball et al. 1991; Delrue et al. 1992; Plumed et al. 1996; Zabawinski et al. 2001). Further characterization of such a mutant in Chlamydomonas reinhardtii demonstrated substantially improved TAG accumulation ability under nitrogen limitation, with a 2-fold faster rate and 2-fold higher accumulation than wild type (Wang et al. 2009). These approaches have relied on direct plating of mutagenized cells followed by screening of large numbers (tens of thousands) of mutants to identify the desired phenotype, which although effective, is laborious. Another phenotype isolated by plate screening was the small chlorophyll antenna size mutants of the diatom Cyclotella cryptica (Huesemann et al. 2009). These mutants performed well in the lab, but not so in outdoor conditions (Huesemann et al. 2009). The reason for poor performance was not elucidated, but one possibility could have been the presence of detrimental secondary mutations. More recently, procedures were developed for Nannochloropsis sp. to select for higher lipid accumulating mutants using fluorescence-based staining of lipid droplets coupled with flow cytometric selection of the highest percentage of lipid containing cells (Doan and Obbard 2011, 2012). Substantial improvements in lipid content were obtained, especially after repeated rounds of selection (Doan and Obbard 2012). The process was more efficient and less labor intensive than plate screening and resulted in isolation of 13 mutants out of thousands screened. Two mutants demonstrated a 1.5-fold improvement in fatty acid methyl ester (FAME) content relative to wild type (Doan and Obbard 2012). Nannochloropsis is a member of the stramenopile algae and is a promising production strain (Hu et al. 2008), so the strains that were most improved may have a practical application.
The chlorophytes are an evolutionarily distinct class of microalgae with promising production characteristics (Hu et al. 2008). We have worked with Chlorella sp. KAS603, a proprietary species isolated by Kuehnle AgroSystems (Honolulu, Hawaii, USA) with attractive growth, environmental flexibility, and TAG accumulation characteristics. This species accumulates TAG rapidly in response to combined nitrogen and phosphorous depletion. In this report, we systematically develop a mutagenesis and selection scheme using flow cytometric sorting to isolate mutants with improved TAG accumulation characteristics. The data suggest that although epigenetic variation occurs, stable phenotypes are consistent with genetic alterations. We demonstrate a much more efficient recovery of high lipid accumulation mutants with higher TAG levels than with previously reported approaches. These results also open the door towards new approaches to characterize genes involved in TAG accumulation or other cellular processes.
Materials and methods
Algal strains and culture conditions
The green alga Chlorella sp. KAS603 (a proprietary strain from Kuehnle AgroSystems) was grown in 125-mL stoppered Erlenmeyer flasks containing 50 mL 14G growth medium (General Atomics 2012) at 25 °C under 500 μmol photons m−2 s−1 continuous light. Cells were inoculated into 14G growth medium starting with 2 × 106 cells mL−1. Individual sorted cells were grown in 24-well plates before further cultivation in 125 mL Erlenmeyer flasks. To induce TAG accumulation, cells were placed in induction medium (14 L) lacking nitrate and phosphate (General Atomics 2012). Cell growth was measured by counting cell number using a hemocytometer. This species is robust and massively scalable and has been used to produce over one million liters of culture in open ponds for biofuel purposes (A. Kuehnle, personal communication).
UV mutagenesis was performed according to Ball et al. (1991). Briefly, exponentially grown cells were harvested, washed, and suspended in 14G medium at 5 × 106 cells mL−1. Cells were placed in a 60-mm petri dish covered with two layers of a Kimwipe and irradiated using a UV cross linker (UVP CX-2000) at the maximum intensity (1 joule cm−2) at a distance of 7 cm for 30 sec. Irradiated cells were allowed to recover in the dark for 24 h. Cells were then either inoculated into liquid, or plated on agar plates, and placed at 25 °C under 500 μmol photons m−2 s−1 continuous light for 5–7 days or until colonies were visible. Fifty percent survival was considered as optimal irradiance for mutant generation. For double mutagenesis, two singly mutagenized cell lines were subjected to the same treatment.
Chemically mutagenized cells were treated with ethyl methansulfonate (EMS, Sigma); 5 × 108 cells mL−1 were incubated in 0.28 M EMS in phosphate buffer, pH 7 in the dark at 25 °C for various time intervals with constant shaking. The reaction was stopped by adding 5 % sodium thiosulfate at 15, 30, 45, 60, 75, and 90 min. Samples were washed twice with phosphate buffer and resuspended into the medium. Mutagenized cells were plated onto agar plates to determine viability and also grown in liquid medium for sorting by flow cytometry. Untreated cells were used as a control. We selected 45 min of mutagenesis, which resulted in 50 % survival.
Characterization of triacylglycerol content by BODIPY fluorescence using imaging flow cytometry
TAG accumulation was monitored using BODIPY fluorescence as proxy using an Amnis ImageStream imaging flow cytometer. At different times during the course of an experiment, 1 × 107 cells were removed from a culture and pelleted and stored at −20 °C. For analysis, pellets were thawed, resuspended in potassium phosphate buffer (0.1 M, pH 7), and stained with 2.6 μg mL−1 BODIPY (4,4-difluoro-3a,4a-diaza-s-indacene) (Invitrogen, USA) by incubating for 20 min at room temperature. After 20 min, samples were run through the ImageStream. Typically, data were collected on 10,000–20,000 cells. Data were analyzed using IDEAS™ software for the ImageStream and plotted as chlorophyll vs. BODIPY fluorescence.
Six to ten million (6–10 × 106) cells of a particular mutant were harvested, resuspended in a microcentrifuge tube containing 1.2 mL methanol, vortexed vigorously for 1 min, and then centrifuged. If pellets were still green, 1.2 mL methanol was added and heated at 80 °C for a few hours until the green color disappeared (alternatively, pellets were extracted with a higher volume of methanol). Pellets were dried and iodine/KI (5 mM iodine/10 mM KI) solution was added. After 2 min of incubation, cells containing starch changed to a dark-brown or black color while cells containing less starch changed to a pale yellow color.
Mutant screening by flow cytometer sorting
Cells (2–3 × 107 cells mL−1) were harvested and stained with BODIPY as described above. TAG level was measured using BODIPY fluorescence as a proxy (Cooper et al. 2010). Cells were excited with a 488-nm laser, and emission was evaluated at 530/40 nm on a Becton Dickinson Influx sorting flow cytometer. Different isolation parameters were tested (see “Results”), but the only top few percent of cells in terms of BODIPY fluorescence were isolated. We routinely sorted 10,000 cells to be used for plating and 100,000–200,000 cells for growth in the liquid medium. Cells were plated immediately after sorting and cells grown in liquid culture were used for resorting.
Imaging flow cytometry analysis of TAG accumulation
TAG accumulation ability is related to the stage of culture growth
Since exact control over culture status is not possible (the condition of cells at “day x” for any given culture is not exactly the same for another), even a few hours variation at a sensitive time (e.g., between day 2 and day 3) could result in a dramatic change in TAG accumulation ability. For this reason, we used two wild-type cultures in all subsequent mutant screening experiments in an attempt to account for variation. Figure 2d shows the mean and average standard deviation for each time point for two wild-type replicates in eight separate TAG induction experiments. The average percent standard deviation of the mean was 21 % across all time points. These data indicate that although variation occurred, induction levels were consistent enough to make evaluations.
Sorting different subpopulations of cells containing different TAG content
Cell mutagenesis and screening mutants using fluorescence-activated cell sorting
The data in Fig. 3 indicate that the phenotype of high or low lipid accumulation persisted over several generations, and data in Figs. 2 and 3 suggest that epigenetic effects could be involved. Since epigenetic effects are not expected to be stable over the long term, we attempted to make genetic changes to increase phenotypic stability and to potentially further improve lipid accumulation ability. We UV-mutagenized HF isolates and, after 2 days of recovery in liquid medium, subjected cells to TAG induction and sorted cells on the basis of high BODIPY fluorescence. Two approaches were tested: sorting of a subpopulation containing 10,000 cells with the highest 1–3 % fluorescence which were plated immediately after sorting, and sorting of a subpopulation containing 100,000–200,000 cells with the highest 10 % fluorescence which were then grown in liquid culture. Plating efficiency of immediately-sorted cells was extremely low; in a typical experiment, three to five colonies out of 10,000 would be isolated if the top 1 % fluorescent cells were sorted (survival was better if the top 3 % were isolated). This suggests that the highest lipid accumulating cells were marginally viable.
TAG accumulation in doubly mutagenized cells
Changes in TAG accumulation after sequential sorting
Summary of screening results
Summary of mutant screening
No. of screened colonies
No. of TAG 1.8–2.5 × WT
No. of TAG 1–1.5 × WT
Percent high accumulation
UV-irradiated single mutant
UV-irradiated double mutant
Chemical mutagenesis (EMS)
Wild-type sorted cells
The cell sorting approach we applied proved to be a highly efficient method to isolate mutants with better TAG accumulation characteristics. Traditional methods have relied on plating of cells after mutagenesis, followed by screening of tens of thousands of colonies for a particular phenotype. For example, starchless mutants have been isolated at an efficiency of approximately 1:10,000 (Ball et al. 1991; Delrue et al. 1992; Plumed et al. 1996; Zabawinski et al. 2001). More recently, the efficacy of continuous selection in liquid has been demonstrated in Nannochloropsis sp. which resulted in 13 mutants with increased FAME content obtained out of thousands screened (Doan and Obbard 2012). Our results on the singly mutagenized cells indicate an even higher recovery of mutants with improved TAG accumulation ability. High TAG accumulating mutants were isolated in our approach at a frequency of 1:2.7, which is 3,700 times better than typical plate screening with far less effort. Improved recovery over that in the Nannochloropsis sp. study (Doan and Obbard 2012), which used a conceptually similar sorting approach, could be due to the mutagenesis method. We show that the recovery of chemically induced mutants in much poorer than UV-induced mutants (Table 1). In addition to improved efficiency of mutant isolation, the data suggest the possibility of differences in the extent of improvement of lipid content between the two methods. One needs to consider that different organisms are being compared as well as TAG vs. FAME accumulation; however, in comparison to wild type, the chemical mutagenesis-based approach used on Nannochloropsis sp. resulted in 11 mutants with higher FAME content than wild type, but only two with greater than 1.5-fold improvement (Doan and Obbard 2012), compared with 32 mutants obtained by UV mutagenesis in this study with 1.8–2.5-fold higher TAG accumulation than wild type, and a large number of others with lesser improvement (Table 1).
Although intracellular TAG content has been analyzed previously using fluorescent neutral lipid stains coupled with flow cytometry (Doan and Obbard 2012), the application of imaging flow cytometry provides additional benefits. Primarily, distinct stages in the TAG accumulation process could be identified and correlated directly with the appearance of the cells (Fig. 1). The ability to image tens of thousands of cells in a short time provides statistical robustness to the analyses. This is especially important given the spread in TAG content comparing cells in the population (Fig. 1). Such a spread, on the order of up to a hundred fold, has been seen in other microalgae (Doan and Obbard 2012 and unpublished observations). Because these are clonal populations of cells, a probable explanation is that there is epigenetic variation. The results of sorting nonmutagenized cells in Fig. 3 are consistent with this.
The relation between culture stage and TAG accumulation ability, which could also relate to epigenetic variation, is shown in Fig. 2. Previous work on Nannochloropsis sp. showed a similar relationship, but those experiments monitored differences between exponential, mid-stationary, and late stationary phases, with 8- and 12-day gaps between exponential and mid-stationary and mid-stationary and late stationary phases, respectively (Doan and Obbard 2012). The results in Fig. 2 demonstrate substantial differences on the order of individual days for KAS603. In addition, as the cells enter stationary phase, there was an increase in FAME with Nannochloropsis sp. (Doan and Obbard 2012), but a sequential decrease in KAS603 with the exception of day 5 (Fig. 2). The data for day 5 suggest that the response can be complex and that there are lingering effects related to culture stage. Elucidating the mechanisms involved would be beneficial, because the rate of TAG accumulation between days 2 and 3 in this condition is the single highest rate comparing all conditions.
The most efficient approach for selecting mutants resulted from a single round of mutagenesis (Table 1). The lack of improvement in doubly mutagenized cells may likely be a result of accumulation of deleterious secondary mutations; in this case, selection was unable to rescue the high TAG accumulation phenotype (Fig. 6).
Previous work showed that successive rounds of selection of a bulk population of cells improved the overall TAG accumulation response of the bulk population of cells (Doan and Obbard 2012). In our study, we performed successive rounds of selection on individual clones of mutants (Fig. 7). For singly and doubly mutagenized cells, there was a trend of improvement of TAG accumulation ability with successive sorting (Fig. 7). Coupled with the lack of this trend in wild-type cells (Fig. 7), the data indicate that the improvement has a genetic basis. One possible explanation is that under continued cultivation, deleterious secondary mutations are selected against, and a purifying selection occurs.
The flow cytometric sorting approaches described here and elsewhere (Doan and Obbard 2012), which rely on isolation of clones only after repeated selection under competitive growth conditions, offer substantial and practical advantages over plating approaches. One major advantage is the requirement for competitive growth, which will eliminate clones with detrimental mutations affecting growth. In addition, the approach does not preselect for a particular genotype—in theory, any type of mutant that gives rise to a high lipid accumulating phenotype could be isolated by this method. This opens the door to using random mutagenesis not only to isolate useful phenotypes, but as a tool to identify the underlying genetic mechanisms. With high-throughput sequencing approaches, it is entirely feasible to sequence the genome and transcriptome of mutants for comparison with wild type to identify the locus of mutation. If only a few mutants generated by random mutagenesis are compared, then the presence of mutations throughout the genome may make identification of the responsible gene difficult. With larger numbers of mutants, the consistent presence of mutations in the same gene will highlight that gene as a good candidate which could be further tested by manipulation. The approach described in this report enabled isolation of large numbers of high performance mutants, in which multiple classes of mutations could be present. Characterization of multiple mutant classes could lead to the identification of the roles of various metabolic steps in the process of high TAG accumulation.
This work was supported by subcontract 4500024860 from General Atomics.
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