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Biochemical analysis and genetic engineering of oleaginous fungi for the production of eicosapentaenoic acid and free fatty acid derivatives

Brian, King Himm Mo 京都大学 DOI:10.14989/doctor.k23253

2021.03.23

概要

The thesis describes biochemical analysis and genetic engineering of oleaginous fungi for the production of functional lipids and oleo-chemicals. ω3-polyunsaturated fatty acids (ω3- PUFAs) and free fatty acid (FFA) derivatives hold great industrial importance as pharmaceutical intermediates and fine oleochemicals, but they are currently obtained from unsustainable sources. For example, eicosapentaenoic acid (EPA) is obtained mainly from depleting marine fisheries, and ricinoleic acid is obtained mainly from castor bean which produces the potentially lethal toxic ricin. Their production from alternative sustainable sources by oleaginous microorganisms is therefore necessary. The results obtained from this thesis can be applied to establishing microbial production of the ω3-PUFA, EPA, and the FFA derivative, 13-hydroxy-cis-9-octadecenoic acid (13-OH LA).

　Chapter 1 describes characterization of ω3 fatty acid desaturases from oomycetes and their application toward EPA production in Mortierella alpina. ω3 desaturases with efficient desaturase activity are vital to produce ω3-PUFAs such as EPA from ω6-PUFAs, but such gene resources are limited. With an aim to isolate efficient ω3 desaturases, oomycetes were selected based on intracellular EPA/arachidonic acid (ARA) ratios. Two oomycetes with the highest EPA/ARA ratios in their respective orders, Pythium sulcatum and Plectospira myriandra, were selected, and the ω3 desaturase genes, psulω3, pmd17c, and pmd17g, were cloned.

　The enzymes were expressed in the oleaginous fungus M. alpina, which produces approx. 70 % of total fatty acids (TFAs) as ω6-PUFAs at 28 °C. Two resulting transformants produced EPA comprising 38 % (psulω3) and 40 % (pmd17g) of TFAs, which are the highest reported EPA/TFA values for M. alpina to date. Compared to the strains expressing pmd17c and pmd17g, the strain expressing psulω3 showed less accumulation of the C18 ω6 byproducts, linoleic acid (LA) and γ-linolenic acid, as they were presumably converted to the C18 ω3 fatty acids, α-linolenic acid (ALA) and stearidonic acid (SDA). In contrast, M. alpina expressing pmd17c and pmd17g accumulated EPA as a major product with minimal accumulation of C18 ω3 byproducts such as ALA and SDA.

　These results show that PSULω3 is a non-specific ω3 desaturase that shows high potential for EPA production through C18 and C20 fatty acids, while PMD17C and PMD17G are of great value for EPA biosynthesis in M. alpina via C20 ω6-PUFAs due to their ability to convert endogenous C20 ω6-PUFAs to C20 ω3-PUFAs with minimal byproducts and high conversion efficiency. These ω3 desaturases should thus facilitate the advent of more sustainable EPA sources.

　Chapter 2 describes development of a platform host microorganism Basidiobolus meristosporus for production of FFA derivative. FFA-derived oleochemicals fermented from biomass are potential alternative sources for various functional lipids and industrial oleochemicals, but no reported organisms possess the metabolic environment to accumulate high levels of FFA without extensive metabolic engineering.

　Section 1 of this chapter describes screening and characterization of oleaginous filamentous fungus B. meristosporus accumulating high levels of FFAs.

　After characterization and optimization of FFA production, a fed-batch fermentation at 300 mL flask scale was conducted. B. meristosporus rapidly grew to a maximum of 60.5 g/L dry cell weight (DCW) after 312 h cultivation at 28 °C. FFAs were produced to high levels from glucose, reaching an FFA titer of 10.0 g/L, a productivity of 61.7 mg/L/h, and a yield of 0.05 g FFA/g glucose (~17 % of the theoretical maximum) after 162 h. These FFA production metrics are the highest reported in any wild type organism to date, with the titer, productivity and yield representing ~500,000-fold, ~100-fold and ~330-fold increases, respectively, compared to the wild types of the current platforms Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica.

　Section 2 of this chapter describes a transformation system for B. meristosporus utilizing a plasmid vector containing a carboxin resistance gene as a selection marker and a β- glucuronidase (GUS) gene as an expression reporter.

　After transformation of the ballistospores by biolistic bombardment, the resulting transformants could be recovered on carboxin selective plates after 72 h and showed stable GUS activity. Transformation efficiency reached approximately 237 transformants/106 ballistospores.

　Lactobacillus acidophilus FA-HY1 catalyzes the hydration of LA to 13-OH LA, with biochemical characterization and homology models strongly suggesting FFAs to be the sole substrate. To demonstrate that endogenously accumulated FFAs in B. meristosporus could be utilized as substrates for FFA derivative production, de novo biosynthesis of the hydroxy fatty acid 13-OH LA from glucose was evaluated with B. meristosporus transformants. The expression vector pBIG_CarR_FA-HY1 was utilized, which contained FA-HY1 in place of GUS. This vector was introduced into B. meristosporus ballistospores. Analysis of the intracellular lipids of a resulting transformant yielded a novel peak with a similar retention time to the 13-OH LA standard. The mass spectrum of the peak produced by the FA-HY1 transformant showed the molecular ions characteristic of 13-OH LA.

　13-OH LA production was then evaluated by fed-batch cultivation of the FA-HY1 transformant at 300 mL flask scale. During fermentation, the wild type strain showed no 13- OH LA production and the LA FFA titer reached 1250 mg/L after 162 h. In comparison, the FA-HY1 transformant showed 13-OH LA production throughout fermentation, reaching a maximum of 311 mg/L 13-OH LA after 186 h.

　Thus, B. meristosporus and the transformation system for the stain facilitate sustainable production of industrially relevant FFA derivatives.

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