The overall goal of the studies on the biochemistry and molecular biology of lipid synthesis in microalgae was to increase the understanding of the lipid biosynthetic pathways and to identify enzymes that influence the rate of lipid accumulation and lipid quality. This information would be used to genetically manipulate the biosynthetic pathways for improvement in lipid production rates and to manipulate the nature of the lipids produced (i. e., the degree of fatty acid saturation and chain length) to optimize the production of biodiesel.

The development of a genetic transformation system for diatoms allowed NREL researchers to begin testing ways to manipulate microalgal biochemical pathways. The first target enzyme was ACCase. Previous studies at NREL had shown that increased lipid production in diatoms induced by Si starvation was accompanied by an increase in the activity of the ACCase enzyme. Therefore, it was logical to ask whether the activity of the enzyme could be increased in the cells by adding additional copies of the gene encoding ACCase (acc1), and, if so, would increased activity of the protein stimulate the production of lipids in the algal cells?

A full-length copy of the C. cryptica acc1 gene had been cloned and characterized at NREL (see Section II. B.2.f). The plasmid containing this sequence was designated pACC1 (Figure II. B.8). Before the algal transformation system, attempts were made to express the algal gene in a bacterial system to ensure that the cloned gene encoded a functional ACCase enzyme and to test for the effects of overexpression. For expression of the C. cryptica acc1 gene in E. coli, the introns were removed and the 5′ terminus was replaced with the 5′ end of the E. coli P — galactosidase gene, which included the inducible promoter region. This fusion gene was introduced into E. coli. The transformed cells were analyzed for the production of algal ACCase protein by probing blots of (Sodium Dodecyl Sulfate, SDS) polyacrylamide gels with an anti — ACCase antibody or with avidin conjugated to alkaline phosphatase. (Avidin binds to the biotin moity in the functional ACCase protein.) The bacterial cells produced full-length algal ACCase, as well as a large number of shorter polypeptides recognized by the anti-ACCase antibody. Introducting the gene into other E. coli strains deficient in protease activity also produced these shortened peptides; therefore, they were presumed to be the result of truncated transcription or translation. The full-length ACCase protein was properly biotinylated in the transformed bacteria, but not as efficiently as in the E. coli native biotin-binding ACCase subunit. No effects were observed on lipid biosynthesis in the transformed E. coli strain. Attempts were also made to introduce the C. cryptica acc1 gene into yeast, as expression in a eukaryotic system would more likely mimic the effects in algae, but these experiments were unsuccessful.

The next step was to introduce additional copies of the acc1 gene in diatoms, with the goal of increasing the activity of the ACCase enzyme and then assaying the effects of ACCase overexpression on lipid accumulation. The plasmid containing the full-length acc1 gene (pACC1) does not contain a selectable marker for transformation. Studies in other laboratories showed that nonselectable plasmids can be introduced into cells via cotransformation with a plasmid containing a selectable marker gene such as nptII. Although the exact mechanism for

this phenomenon is not clear, it is believed that during a given transformation procedure, a particular subpopulation of the cells becomes “transformation competent”. These cells may then take up multiple copies of DNA molecules present in the reaction. Introduction of pACC 1 into the diatoms was mediated by microprojectile bombardment as described in a previous section, but with pACCNPT5.1 and pACC 1 precipitated onto the tungsten beads in equimolar amounts. Transformed cells were selected based on their induced resistance to G418 and then screened for additional copies of the acc1 wild-type gene using PCR and Southern analysis. Between 20% and 80% of the G418-resistant colonies contained acc1 sequences in the cotransformation experiments. To facilitate the selection of transformants containing extra copies of the acc1 gene, a plasmid was also constructed that contained both acc1 and nptII, designated pACCNPT4; transformants generated using pACCNPT4 and selected for G418-resistance almost always contained the acc1 gene as well.

Transformed cells containing additional C. cryptica acc1 gene sequences were isolated in C. cryptica T13L, C. cryptica CYCLO1, and N. saprophila NAVIC1. Southern analysis indicated that the foreign DNA inserted into host genome, often in one or more random sites, and often in the form of tandem repeats. Several strains that contained one or more full-length sequences of the inserted acc1 gene were analyzed further to test for ACCase overexpression. The CYCLO T13L transformants showed two to three fold higher ACCase activity than wild-type cells, and there was a corresponding increase in acc1 gene transcript (mRNA) levels. However, preliminary analyses of the lipid composition of the cultures overexpressing acc1 did not indicate a detectable increase in lipid levels. These results suggest that the lipid biosynthesis pathways may be subject to feedback inhibition, so that increased activity of the ACCase enzyme is compensated for by other pathways within the cells. It was hoped that expression of C. cryptica T13L acc1 gene in other algal strains might overcome this inhibition. Numerous N. saprophila transformants were generated that contained full-length copies of the C. cryptica acc1 gene; although acc1 mRNA was detected using the RPA, the recombinant ACCase protein was not detected in any of the N. saprophila strains tested. Whether this result was due to inefficient translation of the mRNA, or degradation of the foreign protein due to improper biotinylation or targeting, is not known. Transformants were also generated in a second strain of C. cryptica, CYCLO1, but the program was discontinued before these strains could be analyzed fully.

NREL researcher Eric Jarvis took another approach to genetically manipulating algal pathways for increased lipid production. Previous research had resulted in the cloning and characterization of the uppl gene from C. cryptica (described in Section II. B.2.h.). This gene codes for a fusion protein containing the activities for UDPglucose pyrophosphorylase and phosphoglucomutase, two key enzymes in the production of chrysolaminarin. It was postulated that decreasing expression of the uppl gene could result in a decrease in the proportion of newly assimilated carbon into the carbohydrate synthesis pathways, and consequently increase the flow of carbon to lipids.

Two techniques that are becoming widely used for gene inactivation are ribozymes and antisense RNA. Dr. 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 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.

Several ribozymes sequences designed to cleave uppl RNA were constructed based on computer predictions of the secondary structure of the target RNA. The ribozyme constructs were shown to specifically cleave the target RNA in vitro. The ribozyme sequences were then inserted into the pACCNPT10 vector in the untranslated acc1 sequence between the nptII stop codon and the acc1 termination sequence (see Figure II. B.8). C. cryptica T13L was transformed with these vectors as described earlier and transformants were selected based on acquired resistance to G418. Extracts were made of the transformed strains and analyzed for UGPase activity. Unfortunately, insertion of the ribozyme sequences did not result in detectable decreases in UGPase expression. Although these initial experiments were unsuccessful, gene inactivation technologies acquired during this project seemed a promising approach for manipulation of algal lipid synthesis pathways. At the time project funding was terminated, work was in progress to continue with the ribozyme experiments and to test antisense RNA constructs as an additional method for inactivating algal pathways.