John E. Seip

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.

Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

ABSTRACT In the oleaginous yeast Yarrowia lipolytica, de novo lipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase in Y. lipolytica. Strains with a Y. lipolytica snf1 (Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into a Y. lipolytica strain engineered to produce omega-3 eicosapentaenoic acid (EPA), Ylsnf1 deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of the Y. lipolytica SNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of the Ylsnf1 mutant identified significantly differentially expressed genes during de novo lipid synthesis and accumulation in Y. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

Pyruvic acid production using methylotrophic yeast transformants as catalyst

Abstract Permeabilized transformants of the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris which express both the glycolate oxidase (( S )-2-hydroxyacid oxidase, EC 1.1.3.15) from spinach and an endogenous catalase (EC 1.11.1.6) have been used as catalysts for the oxidation of l -lactic acid to pyruvic acid. Oxidations of the sodium or ammonium salts of l -lactate at concentrations of up to 1.06 M were run in unbuffered aqueous solution without pH control and with oxygen sparging. The permeabilized transformant catalysts were recovered and recycled in up to 12 consecutive oxidations of the sodium or ammonium salts of 0.50 M l -lactate, where the initial selectivity to pyruvic acid was typically > 98% at 98% conversion of l -lactate. The pyruvic acid salt was readily recovered from unbuffered reaction mixtures in high yield and purity by separation of the catalyst from the reaction mixture, followed by removal of water by evaporation or freeze-drying.

Glyoxylic acid production using immobilized glycolate oxidase and catalase

A variety of methods for the immobilization of glycolate oxidase have been examined for the preparation of a catalyst for the oxidation of glycolic acid to glyoxylic acid. The co-immobilization of glycolate oxidase and catalase on oxirane acrylic beads produced a catalyst which was stable to the reaction conditions used for the oxidation, where glycolic acid and oxygen are reacted in aqueous solution in the presence of the immobilized enzyme catalyst and ethylenediamine. Under optimum reaction conditions, 99% yields of glyoxylic acid were obtained at greater than 99% conversion of glycolic acid, and the recovery and reuse of the co-immobilized enzyme catalyst was demonstrated.

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.

Enzymatic synthesis of cytidine 5'-diphosphate using pyrimidine nucleoside monophosphate kinase

Abstract Nucleoside monophosphate kinase (NMPK, EC 2.7.4.4, from bovine liver or yeast) has been used to prepare cytidine 5′-diphosphate (CDP). The enzyme catalyses the reversible (Keq ≅ 1) reaction of a pyrimidine nucleoside 5′-monophosphate and ATP to produce a nucleoside 5′-diphosphate and ADP. Equilibrium mixtures of CMP, CDP, ADP, and ATP were obtained from the reaction of CMP, ATP, and magnesium chloride with NMPK. The soluble enzyme could be recovered and reused but an enzyme activity half-life of only ca. 2 days was observed. Stabilization of enzyme activity by immobilization via covalent bonding to a carrier surface, and by gel entrapment, was examined. Immobilization yields were optimized by varying protein loading, pH, temperature, ionic strength, type of buffer, and concentration of enzyme substrates. The highest yields of immobilized NMPK activity were obtained by gel entrapment in a poly(acrylamide-co-N-acryloxysuccinimide) gel crosslinked with triethylenetetramine (PAN-500); NMPK immobilized using this method exhibited increased stability of enzyme activity compared to the unimmobilized enzyme.