August 1, 2012.
The definitive version is available at http://dx.doi.org/.
Mortierella alpina , a oleaginous filamentous fungus, is one of industrial fungal strains known for the production of arachidonic acid. It is also of particular interest for hydrocarbon biofuel production since it is able to produce up to 50% of its mass in rich, long-chain polyunsaturated fatty acids [PUFA’s]. In addition to high fatty acid production, M. alpina like many other oleaginous fungi, already have mechanisms for accumulating significant concentrations of hydrophobic compounds making it a naturally equipped candidate to handle potential toxic concentrations of hydrocarbons. To date, three different transformation methods have been established for M. alpina that are capable of sporulation. These are particle bombardment, chemical, and Agrobacterium-mediated transformations. However, many M. alpina strains, such as M. alpina ATCC3222, are poor or absent of sporulation, of which have only protective coats, and the much less penetrable plant cell wall, yet remain of interest for their high lipid production. The goal of this study was to develop an efficient transformation method for those strains, hence allowing researchers to further manipulate these fungi for further improvement of lipid production. Included was optimization of best culture medium for growth and maintenance, optimal conditions for protoplast generation, and replacement of the homologous KusA gene. In molecular principle, homologous recombination is the most efficient method of disrupting, modifying, or replacing a target gene. This integration of exogenous DNA does not come readily to most organisms. Capitalizing from prior research efforts, it is known that deletion of KusA increases the rate of homologous recombination up to 100% in Neurospora strains (Ninomiya 2004).The kusA gene deletion vector, comprised of three different DNA fragments, namely the upstream region of KusA gene, the antibiotic selection marker and downstream region of KusA gene, was assembled by yeast-gap repair method. The gene deletion vector yeast genomic DNA mixtures were rescued by E. coli transformation. The isolated plasmid DNA was confirmed by PCR and digested with endonuclease restriction enzyme PmeI for M. alpina transformation. The protoplast isolation was optimized by adjustment of the enzymes concentration and protoplast buffers. The optimization of protoplast transformation is still undergoing. A successful deletion of KusA gene within biotechnologically important M. alpina could enable homologous recombination of other genes of interest in a higher frequency. This capacity may also improve the advancing the production of microbial oils for bioenergy and arachidonic acid human health applications.
Biology, general | Biotechnology
Pacific Northwest National Laboratory (PNNL)
This material is based upon work supported by the S.D. Bechtel, Jr. Foundation and by the National Science Foundation under Grant No. 0952013. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the S.D. Bechtel, Jr. Foundation or the National Science Foundation. This project has also been made possible with support of the National Marine Sanctuary Foundation. The STAR program is administered by the Cal Poly Center for Excellence in Science and Mathematics Education (CESaME) on behalf of the California State University (CSU).