Work by SERI/NREL subcontractors in the early 1980s supported the idea that there is significant genetic variation within algal populations (i. e., Gallagher, Section ILB. Lc.). Therefore, one possible method for producing high lipid algal strains would be selection of natural genetic variants with desired traits, such as high lipid levels or increased tolerance to high salinity or temperature. The limiting factor to this approach has always been the difficulty of selecting for individuals exhibiting a desired trait among a large population of cells. The use of lipophilic dyes such as Nile Blue or Nile Red, coupled with flow cytometry, showed some potential for isolation of high lipid strains of microalgae (see work by Solomon and Cooksey, Sections II. B. 1.e and f). It was not clear, however, that the variations detected in subpopulations of cells were the result of genetic variations that would be passed on to progeny.

An alternative approach is to induce genetic variation in a population of cells using mutagenesis. Again, the ability to select for the desired trait is a limiting factor, but the production of large numbers of mutants by artificial means is a proven method for generating organisms with heritable traits, often the result of a mutation within a single gene. As a prelude to the initiation of mutagenesis and selection experiments with oleaginous microalgae, NREL researcher Ruth Galloway performed a series of experiments designed to understand the factors required to produce mutants in microalgae. These included media requirements for growth, the ability to form colonies on agar plates, sensitivity to herbicides and other growth inhibitors, and the sensitivity of algal strains to mutagens such as UV light or fluorodeoxyuridine (Galloway 1990). Nine algal strains from the SERI Culture Collection were tested, including organisms from three classes: the chlorophyceae (M. minutum MONOR1 and MONOR2), the eustigmatophyceae (Nannochloropsis (NANNP1 and NANNP2), and the bacilliarophyceae (C. cryptica T13L, C.

mulleri CHAET9, Amphora AMPHO17, Nitzschia pusilla NITSC12, and N. saprophila NAVIC1).

Growth of each strain was evaluated qualitatively after spotting the cultures onto media containing nitrogen or carbon sources, or after growing the cells under phototrophic, mixotrophic, or heterotrophic conditions. Cell growth was also evaluated in the presence of a large number of growth inhibitors including various antibiotics and herbicides. Although there was some variability between the algal strains, several generalizations could be made. Most strains could use either NO3- or NH4+ as a nitrogen source. Mixotrophic growth on various carbon sources was more variable, and only AMPHO17, MONOR2, and CYCLOT13L were able to grow heterotrophically, using glucose as a carbon source. The ability to grow heterotrophically would be important for the isolation of photosynthetic mutants.

Predictably, antibiotics that inhibit bacterial cell wall synthesis such as ampicillin and carbenicillin did not inhibit the growth of the algal strains. Antibiotics that inhibit bacterial protein synthesis by binding to the 30S ribosome showed variation in their effects on algal growth. For example, all strains tested grew well on kanamycin and neomycin and showed no growth on erythromycin; while the growth response differed for the strains on spectinomycin and streptomycin. Whether this result was due to differences in 30S (organellar) ribosomal structure between the algal strains, to differences in uptake of the antibiotics by the individual strains, or to other factors that affect sensitivity, is unclear. All strains showed sensitivity to photosynthesis inhibitors diuron, metronidazol, and atrazine, and to the herbicide glyphosate (“RoundUp”), which affects the shikimic acid pathway. However, sensitivity varied between the strains to compounds that affect the enzyme acetolactate synthase and to chemicals that inhibit microtubule synthesis. (The details of these growth experiments can be found in Tables 2, 3, 4, 5, and 7 of Galloway 1990). Many of the growth inhibitors used in this study affect specific proteins in the target organism, and many of these proteins have been well characterized in a number of systems. Isolating the corresponding gene from an algal mutant using heterologous gene probes to characterize the mutation and/or to use the mutant gene as a selectable marker for transformation studies should be relatively easy.

Attempts were also made to generate mutants in the algal strains by exposing the cells to UV light or to fluorodeoxyuridine, followed by plating the cultures on toxic levels of various growth inhibitors. Using UV mutagenesis, streptomycin-resistant mutants were obtained in MONOR2, as well as glyphosate-resistant mutants in both strains ofNannochloropsis, sulfometuron methyl — resistant mutants in NANNP1, and atrazine-diuron-resistant mutants in NAVIC1. In addition, tunicamycin-resistant mutants of NAVIC 1 were produced following treatment with fluorodeoxyuridine. Mutants were not obtained for the other diatoms, CY CLOT 13L, CHAET9, or NITZS12, whether this was due to poor colony formation by these strains, inefficacy of the mutagen, or inappropriately high levels of the selective agent is not known. One interesting point was that the green algal strains, Monoraphidium and Nannochloropsis, produced mutants with traits thought to be due to recessive nuclear gene mutations (i. e., glyphosate resistance or photosynthesis mutants). On the other hand, in Navicula, the only diatom in which mutants were generated, the types of mutations produced were indicative of dominant mutations, i. e.,

atrazine/diuron resistance (resulting from a chloroplast gene mutation) or resistance to tunicamycin, an inhibitor of n-glycosylation. These results indicate that Monoraphidium and Nannochloropsis are probably haploid; the diatoms are diploid. Design of strategies for generation of algal mutants will have to consider the ploidy of the target organism. For example, generating nitrate reductase-deficient mutants for use in a genetic transformation system using homologous selectable markers (described in detail later) should be relatively simple in haploid strains, but would be much more difficult in diploids. For the diatoms, a better approach would be to utilize a dominant gene as a selectable marker, such as a mutant form of the enzyme acetolactate synthase (discussed later), or a heterologous gene such as the neomycin phosphotransferase II (NPTII). The latter gene confers drug resistance by inactivating antibiotics such as kanamycin or geneticin (G418).

In summary, the research performed by Dr. Galloway demonstrated the potential to produce algal mutants with a wide variety of phenotypes, particularly in the green algae, using simple mutagenesis and selection techniques. It would be important to first optimize and understand the growth conditions for the target strains. The conditions to be used for selection (inhibitor specificities and concentrations) should be determined for each strain. However, the generation of mutants will probably be more useful as a tool in developing selectable marker systems, rather than as a method to directly produce high lipid algal strains, primarily because there is no simple way to screen for high-lipid phenotypes. The use of mutagenesis to develop of homologous selectable marker systems for algal transformation will be discussed in detail later.

Mutagenesis and selection was used successfully in another study at NREL to generate mutants in one aspect of lipid synthesis, fatty acid desaturation (Schneider et al. 1995, described in Section II. B.2.g.). In this experiment, UV mutagenized cells of Nannochloropsis were allowed to form colonies, then grown in small-scale liquid cultures. Lipids were extracted from each sample and analyzed by gas chromatography for any significant alteration in the proportion of fatty acids. This project resulted in the identification of a mutant lacking in 20:5 fatty acids, apparently due to a mutation in a 20:4 desaturase. In this case, a simple screen was used to look for changes in a quantitative trait. This result suggests that, with the right method to screen for mutants with the desired properties, mutagenesis could result in microalgae with altered lipid compositions. However, this project was very labor intensive, with hundreds of colonies screened to identify a single mutant.