The maximum productivity observed for this species (12.0 g dry weighHm-2^d-1 ) occurred during continuous culture at 60% full sunlight under N-sufficient conditions. Doubling the light intensity lowered the productivity to 6.1 g dry weighHm-2^d-1. The chemical composition of N — sufficient cells (as an average percentage of total cell dry weight) was 64.2% protein, 12.6% carbohydrate, and 23.1% lipid. After 7 days of growth under N-deficient conditions, the composition was 26.8% protein, 59.7% carbohydrate, and 13.7% lipid. Therefore, this alga accumulates carbohydrates rather than lipids in response to nutrient deficiency, limiting its usefulness as a lipid production strain.

M. salina:

This alga reportedly contained high levels of lipids when grown under N-deficient conditions. The highest productivity (13.9 g dry weighHm-2^d-1) was observed under N-sufficient conditions at a light intensity of 50% full sunlight, although detailed experiments with regards to the effects of light intensity on productivity were not conducted. There was little difference in the lipid

3Reporting of productivities in g dry weight-m"2 — d"1 derives from the goal of mass culturing the algae in shallow open ponds. The objective would be to maximize biomass produced per area of pond. However, it is often difficult to compare results between experiments when the data are reported in this manner, as factors such as culture depth and vessel design would significantly affect productivity of the cultures.

content of cells grown under N-sufficient and N-deficient conditions (20.7% and 22.1%, respectively).

T. sueica:

The highest productivity observed for this strain was 19.1 g dry weight^m-2^d-1, which occurred in N-sufficient batch cultures grown under a light intensity of 60% full sunlight. N deficiency resulted in a large increase in carbohydrate content (from a mean value of 10.7% to a mean value of 47.1%). On the other hand, protein content was reduced substantially (from 67.6% to 28.3%), and the lipid content decreased from 23.1% to 14.6% in response to N deficiency.

Isochrysis sp.(Tahitian strain T-ISO):

This strain is commonly used as a feed organism in aquaculture production systems. A productivity of 11.5 g dry weight^m-2^d-1 was typical for batch cultures of this species, which was approximately 33% higher than the value recorded during semi-continuous growth (dilution of 0.15 L/d). Productivity was lowered during N-deficient growth to 5.5-7.6 g dry weight^m-2^d-1. This strain accumulated carbohydrate in response to N deficiency (from a mean value of 23.1% to 56.9%). Lipid content also increased slightly (from 28.5% to 33.4%), whereas protein content was reduced from 44.9% to 27.3%. The higher lipid content of N-deficient cells did not translate to higher lipid productivities, however, because of the lower overall productivity of the stressed cultures.

B. braunii:

Some very limited experiments were conducted with this species, which is known to accumulate hydrocarbons. A culture grown under a light intensity of 60% full sunlight had a productivity of only 3.4 g dry weight^m-2^d-1. The lipid content of these cells was 29% of the cellular dry weight; the N status of the cells was not reported, but it is assumed that the cells were grown under N-sufficient conditions.

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.

Applied Biology

і A unique collection of oil-producing microalgae.

The ASP studied a fairly specific aspect of algae—their ability to produce natural oils. Researchers not only concerned themselves with finding algae that produced a lot of oil, but also with algae that grow under severe conditions—extremes of temperature, pH and salinity. At the outset of the program, no collections existed that either emphasized or characterized algae in terms of these constraints. Early on, researchers set out to build such a collection. Algae were collected from sites in the west, the northwest and the southeastern regions of the continental U. S., as well as Hawaii. At its peak, the collection contained over 3,000 strains of organisms. After screening, isolation and characterization efforts, the collection was eventually winnowed down to around 300 species, mostly green algae and diatoms. The collection, now housed at the University of Hawaii, is still available to researchers. This collection is an untapped resource, both in terms of the unique organisms available and the mostly untapped genetic resource they represent. It is our sincere hope that future researchers will make use of the collection not only as a source of new products for energy production, but for many as yet undiscovered new products and genes for industry and medicine.

і Shedding light on the physiology and biochemistry of algae.

Prior to this program, little work had been done to improve oil production in algal organisms. Much of the program’s research focused attention on the elusive “lipid trigger.” (Lipids are another generic name for TAGs, the primary storage form of natural oils.) This “trigger” refers to the observation that, under environmental stress, many microalgae appeared to flip a switch to turn on production of TAGs. Nutrient deficiency was the major factor studied. Our work with nitrogen-deficiency in algae and silicon deficiency in diatoms did not turn up any overwhelming evidence in support of this trigger theory. The common thread among the studies showing increased oil production under stress seems to be the observed cessation of cell division. While the rate of production of all cell components is lower under nutrient starvation, oil production seems to remain higher, leading to an accumulation of oil in the cells. The increased oil content of the algae does not to lead to increased overall productivity of oil. In fact, overall rates of oil production are lower during periods of nutrient deficiency. Higher levels of oil in the cells are more than offset by lower rates of cell growth.

і Breakthroughs in molecular biology and genetic engineering.

Plant biotechnology is a field that is only now coming into its own. Within the field of plant biotechnology, algae research is one of the least trodden territories. The slower rate of advance in this field makes each step forward in our research all the more remarkable. Our work on the molecular biology and genetics of algae is thus marked with significant scientific discoveries. The program was the first to isolate the enzyme Acetyl CoA Carboxylase (ACCase) from a diatom. This enzyme was found to catalyze a key metabolic step in the synthesis of oils in algae. The gene that encodes for the production of ACCase was eventually isolated and cloned. This was the first report of the cloning of the full sequence of the ACCase gene in any photosynthetic organism. With this gene in hand, researchers went on to develop the first successful transformation system for diatoms—the tools and genetic components for expressing a foreign gene. The ACCase gene and the transformation system for diatoms have both been patented. In the closing days of the program, researchers initiated the first experiments in metabolic engineering as a means of increasing oil production. Researchers demonstrated an ability to make algae over­express the ACCase gene, a major milestone for the research, with the hope that increasing the level of ACCase activity in the cells would lead to higher oil production. These early experiments did not, however, demonstrate increased oil production in the cells.

The collection, screening, and characterization of microalgal strains represent a major endeavor of ASP researchers during the 1980s. More than 3,000 algal strains were collected from sites within the continental U. S. (Figure II. A.5.) and Hawaii or obtained from other culture collections. This was the first major collection of microalgae that emphasized strains suitable for cultivation in saline waters at high (or variable) temperatures, and with the potential for oil production. The establishment of the SERI Culture Collection as a genetic resource was a major accomplishment of the ASP; unfortunately a large proportion of the collection was lost due to funding cutbacks. However, the approximately 300 strains remaining in the collection will be transferred to the University of Hawaii, and should be available to interested researchers.