Quinn Qun Zhu

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.

Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter

Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipid‐derived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1IN promoter (FBA1 sequence of −826 to +169) containing an intron (+64 to +165) showed five‐fold higher expression than the FBA1 promoter (−831 to −1). The transcriptional enhancement by the 5′‐region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC+ phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro‐region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC+ strains, our engineered stains secreted most of invertase into the medium. Copyright

Bioengineering of Oleaginous Yeast Yarrowia lipolytica for Lycopene Production

Oleaginous yeast Yarrowia lipolytica is capable of accumulating large amount of lipids. There is a growing interest to engineer this organism to produce lipid-derived compounds for a variety of applications. In addition, biosynthesis of value-added products such as carotenoid and its derivatives have been explored. In this chapter, we describe methods to integrate genes involved in lycopene biosynthesis in Yarrowia. Each bacterial gene involved in lycopene biosynthesis, crtE, crtB, and crtI, will be assembled with yeast promoters and terminators and subsequently transformed into Yarrowia through random integration. The engineered strain can produce lycopene under lipid accumulation conditions.

Omega-3 production by fermentation of Yarrowia lipolytica: From fed-batch to continuous

The omega‐3 fatty acid, cis‐5,8,11,14,17‐eicosapentaenoic acid (C20:5; EPA) has wide‐ranging benefits in improving heart health, immune function, and mental health. A sustainable source of EPA production through fermentation of metabolically engineered Yarrowia lipolytica has been developed. In this paper, key fed‐batch fermentation conditions were identified to achieve 25% EPA in the yeast biomass, which is so far the highest EPA titer reported in the literature. Dynamic models of the EPA fermentation process were established for analyzing, optimizing, and scaling up the fermentation process. In addition, model simulations were used to develop a two‐stage continuous process and compare to single‐stage continuous and fed‐ batch processes. The two stage continuous process, which is equipped with a smaller growth fermentor (Stage 1) and a larger production fermentor (Stage 2), was found to be a superior process to achieve high titer, rate, and yield of EPA. A two‐stage continuous fermentation experiment with Y. lipolytica strain Z7334 was designed using the model simulation and then tested in a 2u2009L and 5u2009L fermentation system for 1,008u2009h. Compared with the standard 2u2009L fed‐batch process, the two‐stage continuous fermentation process improved the overall EPA productivity by 80% and EPA concentration in the fermenter by 40% while achieving comparable EPA titer in biomass and similar conversion yield from glucose. During the long‐term experiment it was also found that the Y. lipolytica strain evolved to reduce byproduct and increase lipid production. This is one of the few continuous fermentation examples that demonstrated improved productivity and concentration of a final product with similar conversion yield compared with a fed‐batch process. This paper suggests the two‐stage continuous fermentation could be an effective process to achieve improved production of omega‐3 and other fermentation products where non‐growth or partially growth associated kinetics characterize the process. Biotechnol. Bioeng. 2017;114: 798–812.