Successful genetic transformation of microalgal strains with demonstrated potential for biodiesel fuel production was finally accomplished in 1994. Two factors that were critical in the development of the transformation system were:

• the cloning of the acetyl-CoA carboxylase gene from C. cryptica, and thus the availability of ACCase regulatory sequences to drive expression of a foreign gene in the diatoms, and

• the purchase by NREL of a microprojectile accelerator (also known as a particle gun) that can efficiently deliver DNA-coated gold or tungsten beads into walled cells.

Except for the transient expression of luciferase in Chlorella protoplasts, all previous attempts at NREL to transform microalgae had been unsuccessful. Whether the problem was the inability to deliver foreign DNA into the cells through the algal cell wall, or inefficient expression of the foreign gene, is not clear.

As discussed in the previous section of this report, a significant amount of work went into developing homologous selectable markers for microalgae, primarily for Monoraphidium. However, there were some attempts, mainly with diatoms, to use a heterologous antibiotic resistance gene as a selectable marker. The GC content of bacteria and diatoms are relatively similar; thus, codon bias should not prevent expression of a bacterial gene in the diatoms. The antibiotic kanamycin and its more potent analog G418, have been used extensively for genetic transformation in higher plants. These antibiotics function by binding to 30S ribosomes and inhibiting protein synthesis. Resistance to kanamycin or G418 can be induced in cells by expressing the bacterial gene neomycin phosphotransferase (nptII). This enzyme phosphorylates the antibiotic, preventing binding to the ribosome. Previous work at NREL (Galloway 1990) demonstrated that some algal strains are sensitive to kanamycin, suggesting that the kanamycin — G418/nptII system might be the basis of a successful transformation system for microalgae.

Further testing showed that most of the algal strains were sensitive to low concentrations of G418; however, the conditions for complete inhibition of cell growth had to be determined empirically for each strain. The required concentration of the antibiotic depended both on the osmoticum of the plating medium and on the plating density of the cells. For example, C. cryptica T13L grows well on both 10% and 50% ASW. When 2 x 106 cells of T13L were plated on 50% ASW agar plates, the cells were resistant to 50 pg-mL-1 G418. The same number of cells plated onto 10% ASW plus 50 pg-mL-1 G418 showed no growth, yet 3 x 107 cells produced a confluent lawn of colonies under the same conditions.

Early attempts to use the nptII gene as a selectable marker used a plasmid construct that had been used successfully for transformation in higher plants. This plasmid, pCaMVNeo, was obtained from Dr. Michael Fromm at the USDA Plant Gene Expression Center, Albany California.

pCaMVNeo contains the nptII gene driven by the cauliflower mosaic virus 35S ribosomal gene promoter (CaMV35S). Attempts were made to introduce pCaMVNeo into C. crypticaCYCLO1 by electroporation, and later into C. ellipsoidea or CYCLO1 by agitating the cells with glass beads or SiC fibers. No G418-resistant colonies were generated by these methods.

After the acetyl-CoA carboxylase (acc1) gene was cloned from C. cryptica T13L, NREL researcher Paul Roessler decided to try to use the 5′- and 3′-regulatory regions from this gene to drive expression of nptII in T13L. A plasmid (pACCNPT10) was constructed that contained a chimeric gene consisting of the coding region of the nptII gene flanked by 445 bp of the acc1 5′ region (the putative promoter) and 275 bp of acc1 coding region following the nptII stop codon, followed by the acc1 3′ noncoding regions (the putative transcriptional terminator). To increase the chance of encompassing the entire acc1 promoter, a second plasmid, pACCNPT5.1, was constructed that contained 819 bp of upstream sequence. In addition, all but 13 bp of the acc1 coding region was removed from the 5′ end of chimeric gene. Details of the plasmid constructions can be found in Dunahay et al. (1995), and plasmid maps are shown in Figure II. B.8.

DNA entry into the algal cells was accomplished using the DuPont/Bio-Rad PDS/1000He microprojectile accelerator. The process, called biolistics, had been used successfully for introducting DNA into walled cells of higher plants, fungi, bacteria, and Chlamydomonas. In this procedure, plasmid DNA is precipitated onto small tungsten or gold particles and accelerated into cells using a burst of helium pressure. Early versions of this device used a gun powder charge to accelerate the particles. Because of prohibitive costs and restrictive licensing agreements, a homemade version of the particle gun was designed and built at NREL. No transformants were generated using this device, but as these experiments were performed before the acc1-nptII chimeric plasmids where constructed, whether the device actually functioned as planned is unclear. Ultimately, a commerical microprojectile accelerator was purchased. This device was optimized for very simple operation and used helium pressure to propel the DNA-coated particles. These properties resulted in greater reproducibility between shots and decreased toxicity caused by gases generated during the explosive charge.

There was some initial skepticism on the part of at least one NREL researcher as to whether microprojectile bombardment would work to introduce DNA into diatoms through the Si frustule. However, the diatoms were transformed using the particle gun and the chimeric vectors in the first try. This turned out to be a simple and reproducible procedure (Figure II. B.9.). For each transformation, algal cells were harvested and spread in an approximate monolayer in the center of an agar plate containing growth medium and 50 pg-mL-1 ampicillin to inhibit bacterial growth. The plates were allowed to dry for 2 hours before bombardment. Just before bombardment, plasmid DNA was precipitated onto 0.5-1.0 pm tungsten particles, which were then propelled into the cells using the microprojectile accelerator. The exact parameters used are described in Dunahay et al. (1995). The cells were incubated for 2 days under nonselective conditions to allow the cells to recover and express the nptII gene. The cells were then washed from the orignal plates and replated onto agar that contained the appropriate concentration of G418. G418-resistant colonies appeared in 7-10 days. These putative transformants were picked

from the plates and tested for continued resistance to G418. The presence of the foreign gene was tested by hybridizing the algal DNA with an nptII gene probe (Southern analysis). The cells were tested for the presence of the and for the NPTII protein by probing with an NPTII-specific antibody (Western blotting), Figures II. B. 10 and II. B. 11.

Both the pACCNPT 10 and pACCNPT 5. 1 plasmids worked well to generate transformants in two strains of C. cryptica (T13L and CYCLO1), as well as in the diatom N. saprophila (NAVIC1). These two species belong to different orders (C. cryptica is a centric diatom, Order Centrales; N. saprophila is a pennate diatom, Order Pennales). Southern analysis indicated that the plasmid DNA was not replicating independently in the cells but had integrated into the host genome, presumably into the nuclear DNA. The chimeric gene integrated into one or more independent sites, often in form of tandem repeats. The nptII DNA remained stably integrated into the host genome for more than 1 year, even when the cells were grown under nonselective conditions.

The successful development of a genetic transformation system for the diatoms was a major achievement for the ASP. This was the first report of genetic transformation of any diatom species, and one of the few reports in which a heterologous gene was used as a selectable marker for stable nuclear transformation of an alga. The use of algal regulatory sequences to drive expression of the bacterial gene in diatoms apparently was a key factor in the successful development of a transformation protocol for these organisms. When the pCaMVNeo plasmid was introduced into diatoms via particle bombardment, no G418-resistant transformants were generated. However, when another plasmid that contains the CaMV35S promoter and the firefly luciferase gene were introduced into the diatoms by cotransformation with pACCNPT5.1, a number of transformants selected based on their resistance to G418 also expressed significant luciferase activity. This result suggests that even though microalgae can in some cases recognize and use foreign promoter sequences, homologous promoters may be necessary to drive expression of foreign selectable markers at levels high enough to overcome the selective pressure. The research that resulted in the development of a genetic tranformation system for diatoms resulted in a publication (Dunahay et al. 1995) that was a finalist for the Provasoli Award for best publication in the Journal of Phycology for that year. In addition, a patent describing this technology was applied for and issued in August 1997. Diatoms represent a very large proportion of the world’s biomass, and are responsible for nearly one-fourth of the net primary production. However, little is known about the biochemistry and molecular biology of these organisms. The availability of a genetic transformation system for diatoms could have a major impact on increasing the understanding of the basic biology of these organisms and should promote their use in biotechnological applications in addition to the intended goal of lipid production. The following section will describe the initial attempts to use the genetic transformation protocol to manipulate levels of storage lipids in C. cryptica.