Introduction:

During the past 2 decades, manipulation of organisms via genetic engineering has become routine in a number of animal, bacterial, fungal, and plant systems. However, before the research was done at NREL, very little work in this area had been done with microalgae. In fact, the only species for which there was a reproducible transformation system was the single-celled, flagellated green alga C. reinhardtii, which is studied extensively in laboratories as a model photosynthetic cell. The focus of the research in the ASP during the early 1990s was to develop

genetic transformation methods for microalgae with potential for biodiesel production. Based on the collection and screening efforts of the 1980s, this approach was considered to have the highest potential to produce organisms with high constitutive lipid levels, and to use genetic manipulation to understand the molecular regulation of lipid synthesis in the oleaginous algae. Studies on the biochemistry and molecular biology of lipid production in C. cryptica had identified acetyl-CoA carboxylase as a key regulatory enzyme in lipid synthesis (Section II. B.2.e.). One initial goal was to introduce additional copies of this gene into C. cryptica with the hope of increasing the activity of the enzyme and the flux of fixed carbon into lipid.

Several projects will be discussed in the following section of this report that were directed towards the development and use of genetic transformation systems in oleaginous microalgae. The initial approach was to use use available promoters and marker genes that were reported to function in other eukaryotic systems. Various methods were also tried to get DNA into the cell, initially focusing on enzymatically removing the cell wall or perturbating the cell membrane using electroporation. Unsuccessful experiments represented a “Catch 22” scenario, as negative results could mean either the DNA was not getting into the cells, or the DNA entered but could not be expressed at detectable levels. Subsequent experiments were designed to increase the understanding of the processes involved in DNA uptake and expression and to increase the probability of obtaining transformants by developing methods for detecting rare transformation events within a population of cells.

The projects that will be discussed here include a basic study on the DNA composition of microalgal strains, with implications for the choice of reporter or marker genes used to monitor gene expression in transgenic algae. Other aspects of the research that will be discussed include:

• the use of the luciferase gene to monitor DNA uptake and expression in Chlorella protoplasts,

• attempts to develop heterologous and homologous genetic markers for algal transformation,

• the development of methods to introduce DNA into algal cells through the cell wall, and

• the successful development of a stable genetic transformation system for diatoms.

Once the methods were available to obtain genetic transformants, efforts were made to use the transformation system to manipulate lipid content in the algae by overexpressing or downregulating key genes. In addition, the transformation system was used to introduce a reporter gene under the control of various regulatory sequences, to better understand the regulation of gene expression in microalgae.