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 (A). Duplicate cultures of plasmid-treated protoplasts were also grown in either the dark (■) or light (□).

(Source: Jarvis and Brown 1991).

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 algaM. 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 nitrogen uptake and utilization in lipid accumulation, and isolating the wild-type NR gene would permit further investigation of these questions.

NR mutants can be isolated based on their resistance to chlorate. Cells with functional NR will take up chlorate along with nitrate and reduce the chlorate to the toxic compound chlorite. Therefore, cells with a mutation in the NR gene will be unable to grow using nitrate as the sole N source, but will be able to grow in the presence of chlorate, as long as urea or ammonium is added as an alternative N source. Using this scheme, several putative NR mutants grew from non-mutagenized cells of MONOR2 and C. cryptica T13L. Biochemical assays suggested that at least two of the MONOR2 mutants contained defects within the NR structural gene.

The next step was to isolate the wild-type gene from MONOR2 for complementation of the NR- minus mutants. A partial cDNA clone of NR from Chlorella vulgaris was obtained from Dr. Andrew Cannons (University of Southern Florida). Southern blot analysis indicated that the Chlorella DNA sequence showed significant homology to a sequence in MONOR2 genomic DNA. Degenerate primers for use in the PCR were designed based on conserved regions in the NR genes from three green algal species and several higher plants. A 700-bp PCR product was generated using MONOR2 genomic DNA as a template and confirmed to represent a fragment of the NR gene by sequence analysis. A MONOR2 genomic DNA library was constructed in a lambda phage vector. Although the library appeared to be representative of the algal genome in that it contained approximately 300,000 separate clones of about 20,000 bp each, repeated screening of the library with the NR gene fragment failed to produce any positive results. Two additional libraries were constructed, but again, screening with the MONOR2 NR sequence did not result in the isolation of a genomic NR sequence. It was concluded that the libraries were probably incomplete; i. e., they did not contain DNA representative of the total algal genome, possibly because of problems associated with the unusual composition of the MONOR2 DNA. This project was put on hold when successful transformation was achieved in C. cryptica, and had not been pursued further when the project was terminated in 1996.

A gene that encodes the enzyme orotidine-5′-phosphate decarboxylase (OPDase) was also targeted for use as a selectable transformation marker. OPDase is a key enzyme in the synthesis of pyrimidines. Organisms with defects in the OPDase gene will only grow if pyrimidines such as uracil are added to the growth medium. OPDase mutants can be selected by growing cells in the presence of the drug 5-fluoroorotic acid (FOA); OPDase converts FOA into a compound that

is toxic to the cells. Therefore, OPDase mutants would grow in the presence of FOA and require uracil; wild-type cells (or mutants transformed with the wild-type OPDase gene) would be susceptible to FOA and would require added uracil in the growth media. NREL researcher Eric Jarvis attempted to develop the OPDase system as a selectable marker for MONOR2. Cells were mutagenized by exposure to UV light, then grown in the presence of uracil and FOA. Putative OPDase mutants were identified as FOA-resistant colonies. Based on growth studies and spectrophotometric measurements of OPDase activity, one isolate of MONOR2 (3180a-1) was identified as a probable OPDase mutant for use as a host strain in the transformation system.

The next step, as for NR, was isolate the wild-type OPDase gene from MONOR2. OPDase had previously been isolated from several species and demonstrated significant sequence conservation between genes from different organisms. Dr. Jarvis made a number of attempts to isolate the OPDase gene from MONOR2 via PCR, using degenerate primers based on conserved OPDase gene sequences. Several PCR products were generated using this approach, but sequence analysis of the cloned DNA fragments resulted in no clones with homology to the OPDase gene. Why this approach did not work for OPDase is unclear, as this same PCR technique had been used to isolate a fragment of NR. A second approach, in which a MONOR2 genomic DNA library was screened for OPDase sequences using heterologous probes, was also unsuccessful.

By 1994, a transformation system had been developed for the diatoms using a chimeric gene as a selectable marker (discussed in the following section); however, there was still interest in producing a selectable marker system that would work for high lipid (although genetically recalcitrant) green algal strains, such as MONOR2. Work began on developing a new selectable marker system that used a mutated version of the acetolactate synthase (ALS) gene as a selectable marker. ALS is an enzyme involved in the synthesis of branched-chain amino acid such as leucine and valine. In plants, this enzyme is inhibited by sulfonylurea and imidazolinone herbicides. Previous work at NREL by Galloway (1990) showed that many microalgae are also sensitive to these herbicides. Eric Jarvis repeated these experiments for MONOR2 and demonstrated that these cells are sensitive to low levels of the sulfonylurea herbicides chlorsulfuron and sulfometural methyl. The approach was to isolate the wild-type gene for ALS from MONOR2, and then to produce a gene that encodes a herbicide-resistant form of the enzyme by site-directed mutagenesis. Degenerate primers were produced based on known ALS sequences and used, this time successfully, to isolate an ALS gene fragment from MONOR2 DNA. This sequence was used to screen the MONOR2 DNA libraries for a full-length ALS sequence, but once again, the screening efforts were unsuccessful.

The feeling among the NREL researchers was that the use of a homologous selectable marker system would still be the best approach for developing genetic transformation systems for some organisms, in particular, those with unusual DNA compositions, and for haploid organisms for which generation of mutants should be relatively straightforward. Despite the promise of M. minutum as a high lipid producer, it may have not been the best organism for these studies because of its highly unusual DNA properties and “tough” cell wall that complicated biochemical extractions and assays. Some of the cloning problems seen with this organism might have been

solved if time had permitted the generation of a cDNA library, or a new genomic DNA library using bacterial host strains optimized for use with highly modified or high GC DNA.