II Laboratory Studies
II. B.3.c Development of a Genetic Transformation System for Microalgae Introduction:
During the past 2 decades, manipulation of organisms via genetic engineering has become routine in a number of animal, bacterial, fungal, and plant systems. However, before the research was done at NREL, very little work in this area had been done with microalgae. In fact, the only species for which there was a reproducible transformation system was the single-celled,
genetic transformation methods for microalgae with potential for biodiesel production. Based on the collection and screening efforts of the 1980s, this approach was considered to have the highest potential to produce organisms with high constitutive lipid levels, and to use genetic manipulation to understand the molecular regulation of lipid synthesis in the oleaginous algae.
Studies on the biochemistry and molecular biology of lipid production in C. cryptica had
identified acetyl-CoA carboxylase as a key regulatory enzyme in lipid synthesis (Section
II.B.2.e.). One initial goal was to introduce additional copies of this gene into C. cryptica with
the hope of increasing the activity of the enzyme and the flux of fixed carbon into lipid. Several projects will be discussed in the following section of this report that were directed towards the development and use of genetic transformation systems in oleaginous microalgae. The initial approach was to use use available promoters and marker genes that were reported to function in other eukaryotic systems. Various methods were also tried to get DNA into the cell, initially focusing on enzymatically removing the cell wall or perturbating the cell membrane using electroporation. Unsuccessful experiments represented a “Catch 22” scenario, as negative results could mean either the DNA was not getting into the cells, or the DNA entered but could not be expressed at detectable levels. Subsequent experiments were designed to increase the understanding of the processes involved in DNA uptake and expression and to increase the probability of obtaining transformants by developing methods for detecting rare transformation events within a population of cells.
The projects that will be discussed here include a basic study on the DNA composition of microalgal strains, with implications for the choice of reporter or marker genes used to monitor gene expression in transgenic algae. Other aspects of the research that will be discussed include:
• the use of the luciferase gene to monitor DNA uptake and expression in
Chlorella protoplasts,
• attempts to develop heterologous and homologous genetic markers for algal
transformation,
• the development of methods to introduce DNA into algal cells through the cell
wall, and
• the successful development of a stable genetic transformation system for
diatoms.
Once the methods were available to obtain genetic transformants, efforts were made to use the transformation system to manipulate lipid content in the algae by overexpressing or downregulating key genes. In addition, the transformation system was used to introduce a reporter gene under the control of various regulatory sequences, to better understand the regulation of gene expression in microalgae.
Analysis of Microalgal DNA Composition:
Several oleaginous microalgal strains had been identified as potential candidates for biodiesel fuel production. These organisms became the target of genetic engineering efforts to manipulate the lipid biosynthetic pathways. Before the work on genetic transformation of algae at NREL, very little information was available on the molecular biology of these organisms. One of the first steps was to develop techniques to isolate and purify DNA from these organisms. A desirable protocol would disrupt the cell wall using methods gentle enough to prevent shearing of
the genomic DNA. This was not trivial for some species, such as Monoraphidium, which has a
very resistant wall that contains sporopollenin. A method that worked for most species tested (described in Jarvis et al. 1992) was developed based on a protocol used to isolate yeast DNA (Hoffman and Winston 1987). The cells were suspended in buffer that contained 2% Triton X- 100 and 1% SDS, then added to a tube that contained glass beads and an equal volume of phenol:cholorform:isoamyl alcohol (PCI). The cells were agitated for 1 minute using a vortex mixer. The DNA in the aqueous phase was purified by re-extraction with PCI, ethanol precipitation, and treated with RNase A. For some species, the DNA had to be purified further by using precipitation with hexadecyltrimethylammonium bromide (CTAB; Murray and Thompson 1980) to remove contaminating carbohydrates or by purifying the DNA on CsCl gradients. This procedure produced DNA that digested well with many common restriction endonucleases, but even highly purified DNA would not digest well with all restriction enzymes. NREL researcher Eric Jarvis theorized that poor digestion of the DNA by some enzymes could be attributable to characteristics of the DNA. All DNA is composed of four nucleosides; deoxycytidine, deoxyguanosine, deoxythymidine, and deoxyadenosine, (abbreviated dC, dG, dT, dA); in double stranded DNA, dC is always paired with dG, and dT with dA. The percentage of each nucleoside (often represented as %GC) is variable between species. Restriction enzymes cut DNA at specific nucleotide sequences, generally recognizing 4-6 bp motifs. Therefore, the frequency of cutting by a particular enzyme will be affected by the total nucleotide composition of the DNA (i.e., an enzyme that recognizes CCGG would cut infrequently in an organism with a low %GC). The GC content is also reflected in the codon usage by each organism, as DNA with a high GC content would show a bias toward codons ending with G or C in the variable third position. DNA can also contain unusual modified nucleosides, including 5-
hydroxymethyldeoxycytidine (hm5dC) and 5-hydroxymethyldeoxyuridine (hm5dU), although the
biological significance is unclear. Another common modification is the presence of methylated
nucleosides, in particular 5-methyldeoxycytidine (m5dC) and 6-methyldeoxyadenosine (m6dA).
The degree of methylation has been associated with levels of gene expression. In addition, some microorganisms use DNA methylation as a defense mechanism, in that methylated DNA sequences are often not recognized by endonucleases from invading pathogens. Although the presence of methylated nucleosides is characteristic for some species, the degree of methylation can vary on a short time scale with changing environmental conditions. In contrast, the %GC and presence of modified nucleosides are characteristic for a particular organism. These characteristics only on an evolutionary time scale.
DNA was isolated from microalgae strains, including 10 species from 5 classes. The nucleoside composition was analyzed by reverse-phase HPLC and by digestion with restriction endonucleases. The results of the HPLC analysis are summarized in Table I.B.4-1. Although the diatoms showed a GC content typical for most eukaryotes (42%-48% GC), the GC content of the
green algae (excepting Stichococcus) was significantly higher. In particular, Monoraphidium
DNA contains 71% GC. The table also shows the presence of m5dC in the algal DNA. All
species tested contained some level of this modified base, although once again Monoraphidium
stands out with approximately 11% m5dC. The only other unusual feature was the presence of
12% hm5dU in the dinoflagellate C. cohnii (data not shown); dinoflagellates were not considered
to be good candidates for biodiesel fuel production, so this observation was not explored further. These data provided a good background for developing genetic transformation systems for these organisms. As mentioned above, the GC content of an organism can be reflected in the codon
usage, suggesting that an organism with a high GC content such as Monoraphidium may not
successfully express heterologous marker genes. This was found to be true for the green alga Chlamydomonas; successful transformation of this organism was achieved only by the use of homologous selectable markers (discussed in more detail later). Also, GC content should be considered when designing synthetic DNA probes based on protein sequences, i.e., for isolation of algal genes by PCR. In addition, DNA methylation can affect the ability to construct DNA libraries and to clone algal DNA, and may require the use of bacterial host strains that are insensitive to DNA methylation.
Table II.B.1. DNA Nucleoside Composition of Several Microalgal Strains (Modified from Dunahay, et al, 1992, p .333 and Jarvis et al, 1992)
Algal species M5dC 5GC Chlorophyceae Chlamydomonas reinhardtii 0.16 61.6 Chlorella ellipsoidea 1.48 51.6 Monoraphidium minutum 11.2 70.9 Bacillariophyceae Cyclotella cryptica 1.95 43.2 Navicula saprophila 0.20 46.2 Nitzschia pusilla 0.78 45.4
Phaeodactylum tricornutum 0.14 48.0 Charophyceae Stichococcus sp. 0.30 44.8 Prasinophyceae Tetraselmis suecica 3.32 57.5 Dinophyceae Crypthecodinium cohnii 1.54 43.7
Transient Expression of Luciferase in Chlorella ellipsoidea:
The first step in transformating any organism is getting the foreign DNA inside the cell. For organisms with a cell wall, methods must be devised to either remove or permeabilize the wall, or to get DNA into the cell through the intact wall. Bacterial cell walls do not seem to represent a significant barrier to DNA uptake, and can be induced to take up foreign DNA simply by being washed in low osmotic medium and glycerol, followed by a brief heat shock. Cell walls can be removed enzymatically from yeast cells to form spheroplasts, or from plant cells to form protoplasts. These wall-less cells can be induced to take up DNA by chemically permeabilizing the cell membrane with polyethylene glycol and/or calcium. Alternatively, DNA can enter yeast spheroplasts or plant protoplasts via electroporation, a method in which a rapid, high voltage electric pulse is used to produce transient pores in a cell membrane.
Based on their research backgrounds, NREL researchers tended to view microalgae as either single cell plants, or pigmented yeasts. In either case, the initial tendency was to try to produce wall-less algal cells as targets for transformation. There had previously been some reports of
protoplast production in green microalgae of the genus Chlorella (Braun and Aach 1975;
Berliner 1977). NREL researcher Eric Jarvis decided to attempt to introduce foreign DNA into Chlorella protoplasts, with the eventual goal of adapting these protocols for other algal strains with biodiesel production potential.
The production of a stably transformed line of cells involves several steps, including introducing the foreign DNA into the target cell, expressing the foreign gene, stabilizating (replicating) the new DNA by the host cell, and survival and proliferation of the genetically altered cells. Transient expression assays can be used to monitor and optimize just the first two of these processes, i.e., DNA entry and expressing a foreign gene in a population of cells, and thus can be useful intermediate steps in developing genetic transformation systems. Transient assays usually involve the introduction of a gene that codes for an enzyme detectable by a simple biochemical assay (often referred to as a reporter gene). Dr. Jarvis decided to use one such gene, the firefly
The alga used for these studies was C. ellipsoidea (strain CCAP 211/1a, obtained from the Culture Collection of Algae and Protozoa, Freshwater Biological Association, United Kingdom). Protoplasts were produced using a protocol adapted from Global and Aach (1985). The cells were grown to early stationary phase, then incubated overnight in 10 mg/mL Cellulysin, a crude commercial preparation of the cellulose-degrading enzyme cellulase. Protoplast production was monitored by sonication of the treated cells in water; generally about 80% of the cells were disrupted by this treatment and were considered to be protoplasts. A plasmid containing the luciferase gene driven by plant regulatory sequences was introduced in the protoplasts by mixing
the cells with the plasmid DNA for 30 minutes in the presence of 50 mM CaCl2 and 13%
polyethylene glycol (mw 4000). The cells were washed and incubated in a regeneration medium overnight. The cells were then harvested and luciferase activity was monitored in crude protein extracts. Luciferase catalyzes the oxidation of luciferin with the production of a photon of light via the following reaction:
luciferase, Mg2+, O2
LUCIFERIN + ATP ---> OXYLUCIFERIN + AMP + CO2 + hv
The light produced can be monitored using a scintillation counter or a luminometer.
The results of these experiments are shown in Figure II.B.6. Luciferase activity was detectable in protoplasts treated with the luciferase plasmid, but not in protoplasts that had not been exposed to plasmid or to polyethylene glycol. Intact cells did not take up the DNA. There was a significant decrease in luciferase expression when carrier DNA was left out of the transformation reaction (“carrier DNA” is usually sheared genomic DNA from calf thymus or salmon sperm that is added to reduce the effects of cellular nucleases on the added plasmid DNA). Monitoring of the luciferase activity over time showed that the activity was maximal at about 24 hours after exposing the protoplasts to the plasmid; expression decreased over time and was virtually undetectable after 80-100 hours. Unfortunately, attempts to regenerate the protoplasts into viable walled cells were unsuccessful.
These results were important as they demonstrated the first successful steps in developing a genetic transformation system for microalga, including the production of viable protoplasts, the introduction of DNA into the protoplasts, and the expression of a foreign gene by the algal cells. This last point was very significant, as homologous genes were required to achieve
transformation in another green alga (Chlamydomonas). The dogma in the field was that
heterologous gene expression in green algae would likely be unsuccessful due to codon biases resulting from high GC contents. The work resulted in a publication (Jarvis and Brown 1991), and was the basis for later studies in which the luciferase gene was used to monitor promoter
activities in Cyclotella (discussed later). However, attempts to adapt this procedure to algal
strains with significance to the biodiesel project were unsuccessful. The composition of microalgal cell walls is highly variable between species and even between isolates of the same species. Some unsuccessful efforts were made to determine the enzymatic conditions for wall degradation for several oleaginous algal strains. However, the conclusion, in the words of the
project manager at the time, was that this was “an endless pit of fruitless endeavor”, and the decision was made to explore other methods of introducing DNA into microalgal cells. In
addition, although low levels of luciferase expression were acheived in Chlorella, the decision
was made to pursue the development of selectable marker systems that would allow the isolation of very rare individual transformants within a population of microalgal cells. This will be discussed in the following section.
Figure II.B.6. Transient expression of firefly luciferase in Chlorella ellipsoidea.
A. (Top) - Histogram showing luciferase expression in protoplasts of C. ellipsoidea.
Expression of the luciferase gene is expressed in relative light units (RLU), which are the net photons counted during a 5-min period. See text for explanation.
B. (Bottom) - Kinetics of luciferase expression in C. ellipsoidea protoplasts. Each symbol
represents the result of a single assay. Control cultures were grown in the dark (▲) or light
(∆). Duplicate cultures of plasmid-treated protoplasts were also grown in either the dark (■)
or light (□).
Development of Homologous Selectable Markers for Monoraphidium and Cyclotella:
Transient expression assays can be useful for the rapid assessment of DNA uptake and
expression by cells as demonstrated by the expression of luciferase in Chlorella protoplasts,
described earlier. However, attempts to produce similar results in other algal strains were unsuccessful. The problem with an experiment that produces no signal is that it is impossible to know if this is because the DNA did not get into the cell, or if the DNA entered the cell but was not expressed at detectable levels. In the latter case, poor expression could result from degradation of the foreign DNA, inappropriate regulatory signals, or differences in the codon usage.
One of the most promising organisms with regard to high lipid production and tolerance to
environmental fluxes was the green alga M. minutum (strain MONOR2). However, MONOR2
DNA was shown to be highly unusual in GC content and degree of methylation. As mentioned
elsewhere in this report, successful transformation of the green alga C. reinhardtii, which also
has an elevated GC content, required the use of homologous selectable markers. The literature suggested that this unusual GC content would inhibit the expression of foreign genes, such as bacterial antibiotic resistance genes that had been used successfully as transformation markers in plant and mammalian systems. Based on this information, it was decided to attempt to develop homologous selectable markers for transforming MONOR2 and other strains with programmatic importance. Use of a selectable marker, in contrast to a transient expression assay, would allow the identification of very rare transformation events. Under the appropriate selection conditions, one transformed cell can be detected in a very large population of nontransformed cells, whereas in transient assays, a significant number of cells in a population must be expressing the foreign gene in order to detect the new enzymatic activity. The use of a homologous gene as a marker would greatly increase the chance for successful expression of the introduced gene, as there would be no problems associated with codon bias or foreign regulatory sequences. Although some success was achieved toward the development of a homologous selectable marker system, the emphasis of the research at NREL was shifted after the successful development of a transformation system for diatoms that used a chimeric selectable marker. A significant effort was put into the development of homologous markers, particularly for non-diatom species, from 1989 to 1994, so it is relevant here to summarize the progress made in this area.
The general protocol for developing a homologous selectable transformation system involves several steps. First, a mutation is created or identified in a specific gene. The gene should be essential for growth under “normal” conditions; however, the mutated strains will grow under modified growth conditions. This will allow for positive selection of transformed cells. Then the corresponding wild-type gene is isolated and inserted into a plasmid vector. The wild-type gene is introduced into the mutant cells, and transformants are detected by the ability to grow under the normal, defined growth conditions. In contrast to the transient assay described earlier, use of a selectable marker involves not only DNA entry and expression, but also stabilization of the new DNA in the cell and viability and growth of the newly transformed cells. Genes with good potential for use as selectable markers should not only code for a protein essential for growth
under defined conditions, but should also produce a protein that can be detected by a simple enzymatic assay. In addition, the use of a gene that has been well characterized in other systems will help isolate the gene from the species of interest and simplify the development of enzyme assays and growth conditions for isolating mutants and transformed cells.
Two genes that meet these criteria were targeted for the development of homologous selectable
markers for MONOR2 and for C. cryptica T13L. One codes for the enzyme nitrate reductase
(NR). NR had been used successfully to transform Chlamydomonas (Kindle et al. 1989) and
several species of fungi (Daboussi et al. 1989) and methods were available to isolate NR mutants and selection of transformed strains. In addition, there was some interest at NREL in the role of