Another strategy that has been proposed to increase the proportion of lipid in algal cells is to limit the flow of newly assimilated carbon into other cellular pathways. Many diatoms, including

C. cryptica, can produce a significant amount of a storage carbohydrate called chrysolaminarin, a P-(1 ^3)-linked glucan. Although some data were available on the chemistry of this compound, the biochemical pathways involved in the synthesis of chrysolaminarin were not known. The synthesis of most storage polysaccharides involves the condensation of nucleoside diphosphate sugars; for example, starch is formed in plants from ADPglucose, and UDPglucose is used to form sucrose in plants and glycogen in mammalian cells. These reactions are catalyzed by nucleoside diphosphate sugar pyrophosphorylases, such as UDPglucose pyrophosphorylase (UGPase), which catalyzes the following reaction:

glucose-1-phosphate + UTP ^ UDPglucose + PPi

Roessler first looked for nucleoside diphosphate sugars pyrophosphorylases in cell-free extracts of C. cryptica, and identified significant amounts of UGPase activity. The enzyme activity was characterized to optimize in vitro assay conditions. The enzyme was activated in the presence of Mn2+ and Mg2+ but was not affected by 3-phosphoglycerate or inorganic phosphate; these chemicals are known to affect the activity of ADPglucose pyrophosphorylase in higher plants. Incubation of cell-free extracts with UDP [14C]glucose resulted in the incorporation of the labeled carbon into a P-(1^3)-glucan polymer, presumably chrysolaminarin, supporting the role of UGPase in chrysolaminarin synthesis in diatoms. Subsequent studies identified a second enzyme, UDPglucose:P-(1^3)-glucan-p-glucosyltransferase (also known as chrysolaminarin synthase), which catalyzes the synthesis of glucan using UDPglucose as substrate. The specific activity of both enzymes was examined in C. cryptica cells under Si-replete and Si-depleted conditions. The activity of UDPglucose pyrophosphorylase was similar under both conditions; however, the activity of chrysolaminarin synthase decreased by 31% in Si-deficient cells, suggesting that the partitioning of newly assimilated carbon into lipid may be partly due to decreased synthesis or inhibition of the chrysolaminarin synthase enzyme (Roessler 1987; 1988a).

Further research on UGPase in C. cryptica was put on hiatus for several years while the emphasis was on ACCase (discussed earlier) and on the development of genetic engineering protocols for microalgae (discussed in Section II. B.3.). However, the development of a successful genetic transformation system for C. cryptica, as well as advances in techniques that allow the down — regulation of particular genes (i. e., antisense RNA, ribozymes) generated a renewed interest in UGPase. NREL researcher Eric Jarvis spent 6 months working at Ribozyme Pharmaceuticals, Inc., a biotechnology company in Boulder, Colorado, learning about these new methods. Antisense RNA is a method in which a cell is transformed with a synthetic gene that produces an RNA sequence complimentary to a specific messenger RNA (mRNA). Although the exact mechanism is not clear, the antisense RNA prevents translation from its complimentary mRNA, effectively lowering the level of that particular protein in the cell. Ribozymes are also RNA molecules produced by synthetic genes that can bind to, and cleave, very specific RNA sequences. Ribozymes can be designed to degrade specific mRNA molecules, effectively decreasing expression of a specific gene.

In C. cryptica, chrysolaminarin can make up 20%-30% of the cell dry weight, and thus chrysolaminarin synthesis pathways presumably compete for newly fixed carbon with the pathways for lipid biosynthesis. Dr. Jarvis and Dr. Roessler proposed that inhibiting chrysolaminarin production by inhibiting one or more genes in the carbohydrate synthesis pathway could result in the flow of more carbon into lipid production. Based on the earlier studies on chrysolaminarin synthesis, Dr. Jarvis initiated an effort to isolate the UGPase gene from C. cryptica DNA. A fragment of the C. cryptica UGPase gene was first produced by the PCR using degenerate oligonucleotide primers based on conserved sequences from known UGPase genes from potato, human, yeast, and Dictyostelium. This fragment was cloned and sequenced; the derived amino acid sequence showed 37% identity with the corresponding sequence from potato UGPase, confirming that a C. cryptica UGPase gene fragment had been cloned. The cloned PCR product was then used as a probe to isolate a genomic DNA clone containing the entire C. cryptica UGPase gene from a lambda library. One clone contained a DNA segment with a single long open reading frame, the 5′ end of which showed homology to known UGPase genes. Surprisingly, the 3′ end of this DNA showed homology to known genes coding for the enzyme phosphoglucomutase (PGMase). In chrysolaminarin synthesis, PGMase catalyzes the following reaction:

glucose-6-phosphate ^ glucose-1-phosphate

The glucose-1-P produced in this reaction is the substrate for UGPase in the production of UDPglucose, an immediate precursor of chrysolaminarin, as described earlier. Although PGMase and UGPase are thought to catalyze successive steps in the chrysolaminarin biosynthesis pathway, this was the first report of a naturally occurring fusion of these two genes in any organism. The C. cryptica UGPase/PGMase gene, designated uppl, contained 3,640 bps, including 3 introns, and coded for a protein composed of 1,056 amino acids, with a molecular weight of 114.4 kd.

To confirm that the protein coded for by uppl actually catalyzes both the UGPase and PGMase reactions, the protein was isolated from extracts of C. cryptica by sequential column chromatography (ion exchange, hydroxylapatite, and gel filtration). The two enzyme activities co-eluted throughout the purification procedure, and all fractions containing UGPase/PGMase activity contained a 114 kd protein as determined by SDS-PAGE. These results supported the presence of both enzyme activities in C. cryptica on a single multifunctional protein. A patent submitted by NREL on this unique gene was allowed in October 1996. The research at NREL involving attempts to manipulate uppl gene expression to affect carbon partitioning in C. cryptica will be discussed in Section II. B.3. of this report.