Work on the key role of ACCase in lipid biosynthesis in other plant and animal systems, suggested that this enzyme might be a viable target for genetic manipulation in order to increase lipid production in microalgae. This notion was further supported by the work at SERI that showed changes in ACCase activity in Si-starved C. cryptica cells.

The next step was to isolate the ACCase gene from a microalgal species. Although the ACCase gene had been isolated from yeast, rats, and the bacteria E. coli, the gene had not previously been isolated from any photosynthetic organism. In 1990 and 1991, Dr. Roessler took a sabbatical from SERI to work with Dr. John Ohlrogge at Michigan State University. Dr. Ohlrogge studies lipid biosynthetic pathways in higher plants. This work was partially funded by a Plant Biology Postdoctoral Fellowship to Paul Roessler from the National Science Foundation. The goal of this collaboration was to clone and characterize the ACCase gene from C. cryptica. To accomplish this task, the purified ACCase protein was first cleaved with cyanogen bromide (CNBr); the peptides generated were separated by SDS-PAGE, purified, and several of these peptides were analyzed to determine their amino acid sequence. (This work was done in collaboration with Calgene, a plant biotechnology company in Davis, California.)

The amino acid sequences were used to design degenerate oligonucleotide primers that were used in a polymerase chain reaction (PCR) to amplify an ACCase gene fragment from C. cryptica’s total DNA. A 32P-labeled RNA transcript was produced from the ACCase DNA and used to screen a genomic library of C. cryptica DNA. A 14 kb cloned fragment that hybridized to the ACCase probe was cleaved into smaller fragments that were subcloned, sequenced, and analyzed for the presence of open reading frames (ORFs) and non coding intron sequences. This analysis showed that the ACCase gene from C. cryptica contains approximately 6.3 kbp of coding sequence, separated by a 447 bp intron close to the 5′ end, and a 73 bp intron just upstream from the biotin binding site. The protein predicted by this nucleotide sequence would contain 2,089 amino acids and have a molecular weight of 230 kDa. This is somewhat larger than the molecular weight of 185 kDa estimated by SDS-PAGE, discussed earlier. This discrepancy could be accounted for by inaccuracies inherent in using SDS-PAGE to estimate protein size, particularly for large proteins, and the probability of a signal sequence on the ACCase enzyme for targeting the protein to the chloroplast. Post-translational cleavage of the signal would result in a mature protein smaller than predicted from the primary DNA sequence.

The deduced amino acid sequence of the ACCase from C. cryptica was compared with known sequences from yeast and rat (Figure II. B.5). The algal sequence showed approximately 50% identity with other sequences in the biotin carboxylase domain (at the amino terminus of the protein) and in the carboxyl transferase domain (at the carboxyl terminus of the sequence). However, the central portion of enzyme showed only about 30% identity with the yeast and rat enzymes, with most of the similarity in this region occurring in the biotin binding domain. This suggests that the central region of the protein probably functions primarily as a linker or spacer that moves the carboxylated biotin residue closer to the carboxyl transferase domain. The isolation of the ACCase gene from C. cryptica was an important step for the ASP; significantly

in that this was the first time a full-length sequence for an ACCase gene had been isolated from a photosynthetic organism. NREL was granted a patent on this gene in 1996, and there has been interest from at least one major plant biotechnology company in using this gene to manipulate oils and lipids in higher plants (Roessler and Ohlrogge 1993; Roessler et al. 1994).

The availability of the purified ACCase protein and the cloned ACCase gene allowed NREL researchers to study the effects of Si deficiency on ACCase gene expression. Southern blots, in which a fragment of the cloned ACCase gene was used as a probe to analyze C. cryptica DNA, indicated that there is probably only a single copy of the ACCase gene in C. cryptica. ACCase gene fragments were used to monitor mRNA levels in Si-deficient cells using the ribonuclease protection assay (RPA). ACCase mRNA levels increased 2.5-fold between 2 and 6 hours after the beginning of Si-deprivation as compared to Si-replete cells, but then decreased to the control level after 23 hours. Thus, Si concentration appears to affect ACCase gene expression at the level of gene transcription, possibly as a result of increased promoter activity and/or by altering the rates of mRNA degradation. ACCase activity was also measured in cell lysates from Si — starved cultures; enzyme activity increased steadily over 23 hours to a final level 4.5-fold higher than that of Si-replete cultures. The increased level of ACCase activity was correlated with an increase in the amount of ACCase protein, as determined by Western blotting using anti-ACCase polyclonal antibodies. Although Si deficiency caused the levels of ACCase mRNA and protein to increase, the kinetics of the two processes were different.

These results supported the hypothesis that diatoms could respond to Si deprivation by altering the activity of enzymes involved in lipid biosynthesis to partition more fixed carbon into storage lipids. If the activity of ACCase could be increased using mutation or genetic manipulation, it might be possible to produce a strain with constitutively high levels of TAG synthesis. This was a major premise of the genetic engineering experiments discussed in Section II. B.3.