Howard Glenn Damude

Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica

The availability of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is currently limited because they are produced mainly by marine fisheries that cannot keep pace with the demands of the growing market for these products. A sustainable non-animal source of EPA and DHA is needed. Metabolic engineering of the oleaginous yeast Yarrowia lipolytica resulted in a strain that produced EPA at 15% of dry cell weight. The engineered yeast lipid comprises EPA at 56.6% and saturated fatty acids at less than 5% by weight, which are the highest and the lowest percentages, respectively, among known EPA sources. Inactivation of the peroxisome biogenesis gene PEX10 was crucial in obtaining high EPA yields and may increase the yields of other commercially desirable lipid-related products. This technology platform enables the production of lipids with tailored fatty acid compositions and provides a sustainable source of EPA.

Enhancing Plant Seed Oils for Human Nutrition

The age of true designer plant oils has arrived. Using the tools of biotechnology, it is now possible to modify the fatty acid content of oilseed plants to change the relative abundance of individual fatty acids in seed oil for health purposes or to produce nutritional fatty acids not normally found

Engineering Oilseed Plants for a Sustainable, Land-Based Source of Long Chain Polyunsaturated Fatty Acids

Numerous clinical studies have demonstrated the cardiovascular and mental health benefits of including very long chain omega-3 polyunsaturated fatty acids, namely eicospentaenoic acid (EPA) and docosohexaenoic acid (DHA) in the human diet. Certain fish oils can be a rich source of omega-3 long chain polyunsaturated fatty acids although processed marine oils are generally undesirable as food ingredients because of the associated objectionable flavors and contaminants that are difficult and cost-prohibitive to remove. Oilseed plants rich in omega-3 fatty acids, such as flax and walnut oils, contain only the 18-carbon omega-3 polyunsaturated fatty acid alpha-linolenic acid, which is poorly converted by the human body to EPA and DHA. It is now possible to engineer common omega-6 rich oilseeds such as soybean and canola to produce EPA and DHA and this has been the focus of a number of academic and industrial research groups. Recent advances and future prospects in the production of EPA and DHA in oilseed crops are discussed here.

Stacking Multiple Transgenes at a Selected Genomic Site via Repeated Recombinase-Mediated DNA Cassette Exchanges

Recombinase-mediated DNA cassette exchange (RMCE) has been successfully used to insert transgenes at previously characterized genomic sites in plants. Following the same strategy, groups of transgenes can be stacked to the same site through multiple rounds of RMCE. A gene-silencing cassette, designed to simultaneously silence soybean (Glycine max) genes fatty acid ω-6 desaturase 2 (FAD2) and acyl-acyl carrier protein thioesterase 2 (FATB) to improve oleic acid content, was first inserted by RMCE at a precharacterized genomic site in soybean. Selected transgenic events were subsequently retransformed with the second DNA construct containing a Yarrowia lipolytica diacylglycerol acyltransferase gene (DGAT1) to increase oil content by the enhancement of triacylglycerol biosynthesis and three other genes, a Corynebacterium glutamicum dihydrodipicolinate synthetase gene (DHPS), a barley (Hordeum vulgare) high-lysine protein gene (BHL8), and a truncated soybean cysteine synthase gene (CGS), to improve the contents of the essential amino acids lysine and methionine. Molecular characterization confirmed that the second RMCE successfully stacked the four overexpression cassettes to the previously integrated FAD2-FATB gene-silencing cassette. Phenotypic analyses indicated that all the transgenes expressed expected phenotypes.

Metabolic engineering of an oleaginous yeast for the production of omega-3 fatty acids.

Publisher Summary Numerous clinical studies have demonstrated that the omega-3 fatty acids in fish oil significantly reduce the risk of cardiovascular disease in adults. This chapter discusses the approach to introduce the genes encoding an omega-3 fatty acid biosynthesis pathway into an oleaginous yeast that synthesizes and stores triglycerides as an energy reserve when starved for nitrogen in the presence of an excess carbon source, such as glucose. It explains the development of a clean and sustainable source of omega-3 fatty acids by fermentation, which uses a metabolically engineered strain of the oleaginous yeast Y. lipolytica. While certain strains of Y. lipolytica can accumulate oil up to 40% of the dry cell weight, the only Polyunsaturated Fatty Acid (PUFA) normally synthesized by the organism is Linoleic Acid (LA). Coordinate expression of desaturase genes and elongase genes comprising a “delta6 pathway” was sufficient to demonstrate the synthesis of Eicosapentaenoic Acid (EPA). However, only an integrated strategy, based on the use of strong promoters, an increase in gene copy numbers, the push and pull of carbon into the engineered pathway, and the use of oleaginous condition, resulted in the generation of a high EPA production strain.

Modifying Vegetable Oils for Food and Non-food Purposes

Oils and fats are an important source of energy for the human diet and alsocontributesignificantlytothesensorycharacteristicsoffood.Manyoilsarealsoused for non-food applications, although industrial use currently accounts foronly a small proportion of the world vegetable oil production, less than 5% oftotal production, mostly for biodiesel. About 80% of edible oils are derivedfrom plant sources and temperate annual oil seeds (soy, rapeseed, sunflowerand peanut) account for about 60% of this total. Soybean oil is by far thedominantoilinthiscategory,accountingforoverhalfoftheworldvegetableoilproduction.Improving the functional and nutritional qualities of vegetable oils hasgarnered much attention over the last 15 years or so. This chapter will describesome of the attempts to genetically improve plant seed oils, with specialemphasis on soybean oil, for food and non-food uses.

Metabolic Engineering of Seed Oil Biosynthetic Pathways for Human Health

Multiple studies have shown that inclusion of omega-3 long chain polyunsaturated fatty acids in the diet can have multiple health benefits including positive effects on cardiovascular and mental health. Although marine oils can be a rich source of omega-3 long chain polyunsaturated fatty acids, processed fish oil is undesirable as a food ingredient because of the associated objectionable flavors and contaminants that are difficult and cost-prohibitive to remove. Oilseed plants engineered to produce omega-3 LC PUFAs offer a safe, sustainable and cost-effective alternative to fish oils as a source of LC PUFA for food ingredients. Given the potential benefit, much recent effort from both academic and industrial research labs has been directed towards producing a land-based, oilseed-derived source of LC-PUFAs and the approaches, recent advances and future prospects will be discussed here