Polyunsaturated fatty acid production by Yarrowia lipolytica employing designed myxobacterial PUFA synthases

Abstract

Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica . Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains.

Current Knowledge about the Effect of Nutritional Status, Supplemented Nutrition Diet, and Gut Microbiota on Hepatic Ischemia-Reperfusion and Regeneration in Liver Surgery

“…PUFAs can be further categorized according to the location of the first double bond relative to the terminal methyl group: Omega-3 and omega-6 and are characterized by the presence of a double bond three and six atoms away from the methyl terminus, respectively [77]. Long-chain PUFAs (LC-PUFAs), particularly omega-3 LC-PUFAs EPA and DHA, are associated with beneficial health effects [78].…”

A common approach for absolute quantification of short chain CoA thioesters in industrially relevant gram-positive and gram-negative prokaryotic and eukaryotic microbes

“…First, the initially chosen small column geometry (100 × 2.1 mm) and particle size (1,5 µm) turned out incompatible with certain samples. Over rather few injections (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), the column pressure increased from initially 250 to 1000 bar, which required extensive cleaning with water to regenerate the separation column. However, despite such efforts, we faced a rapid loss of separation efficiency.…”

“…Strains used in this study were obtained from previous work. This included Streptomyces albus J1074 and its pamamycin producing derivative J1074/R2 [11], the two l-lysine producing strains Corynebacterium glutamicum LYS-9 and LYS-9 ∆sucCD [15], Pseudomonas putida KT2440 and its mutant KT2440 ∆gcd [13], and the docosahexaenoic acid (DHA) producing recombinant yeast Yarrowia lipolytica Po1h::Af4 [12]. All strains were maintained as glycerol stocks at -80 °C.…”

“…From a commercial perspective, CoA thioesters display building blocks of a wide range of industrially interesting products. Prominent examples are polyketides [11], polyunsaturated fatty acids (PUFAs) [12], polyhydroxyalkanoates (PHAs) [13], biofuels [14], amino acids [15], and dicarboxylic acids [16], among others [17]. This relevance might explain the increasing interest to trace CoA thioesters.…”

“…For this purpose, we adapted a previous protocol, used to assess a wide spectrum of CoA thioesters in the methylotrophic bacterium Methylobacterium extorquens [21]. After improvement and careful validation, we demonstrated the approach for industrially relevant microorganisms, which utilize CoA thioesters to form value-added products: the gram-positive bacteria Corynebacterium glutamicum [15], and Streptomyces albus [11], the gram-negative bacterium Pseudomonas putida [13], and the eukaryotic yeast Yarrowia lipolytica [12].…”