Metabolic Engineering for Ricinoleic Acid Production in Y. Lipolytica

Biofuels and oleochemicals produced by microorganisms have similar chemical structure and properties to petroleum-based products. However, producing oleochemicals in yields permitting their economic exploitation requires the engineering of the microorganism’s metabolism. Such engineering cannot be based on just one specific feedstock or host organism. Synthetic-biology approaches should be used to optimize both the host and pathways to maximize specific oleochemical and/or fuel production. Even though there are still challenges so as microbial biodiesel production can compete with conventional fuels, the production of an added value-oleochemical could be efficient enough to attain commercialization standards.

Here, we present the attempts on the metabolic engineering of the fatty acid pathway of the oleaginous yeast Yarrowia lipolytica for the production of the economically important ricinoleic acid (RA). Whereas numerous oleochemical applications for RA and its derivatives exist, their production is limited and subject to various safety legislations, mainly due to the extremely poisonous protein ricin, found in abundance in castor seeds. Y. lipolytica’s innate de novo lipogenesis may be modest (around 40% of cell dry weight in wild type strain) but nonetheless the rewiring of its lipid metabolism conducted by several groups, including ours, enhanced lipid accumulation up to 90% of the engineered strains’ cell dry weight. Adding this to its genetic tractability and the availability of genetic tools, make Y. lipolytica a good candidate as a platform organism for the production of oleochemicals.

Although Y. lipolytica is unable to naturally produce RA, the main fatty acids it synthesizes are oleic acid (the direct precursor of RA) and linoleic acid, the latter being the Δ12 desaturation product of the former. The study of RA incorporation in wild-type, triacylglycerol acyltransferase deleted (Dga1p, Dga2p and Lro1p) and fatty acid degradation invalidated (pox1-6Δ) Y. lipolytica strains led us to understand the enzymes specificities and the mechanisms governing the metabolic fate of RA. We therefore proceeded to the heterologous expression of the codon optimized Ricinus communis Δ12 hydroxylase (RcFAH12), under the control of the TEF constitutive promoter, after deletion of the sole native Δ12 desaturase (YlFAD2), in a strain combining all of the above modifications. However, RA constituted only 7% of the total lipids produced by this engineered strain. By contrast, expression of the codon optimized Claviceps purpurea (CpFAH12) in the same genetic background resulted in a strain able to accumulate RA to 29% of total lipids, and expression of an additional copy of CpFAH12 drove RA accumulation up to 35% of total lipids. The co-expression of the C. purpurea or R. communis type II diacylglycerol acyltransferase (RcDGAT2 or CpDGAT2) had negative effects on RA accumulation in this yeast, with RA levels dropping to below 14% of total lipids. The construction of the RA synthetic pathway in the heterologous host necessitated the fine-tuning of the CpFAH12 hydroxylase expression to the native Y. lipolytica PDAT acyltransferase (Lro1p) expression level to maximize metabolic flux. The strain co-expressing 3 copies of the CpFAH12 with 2 copies of YlLRO1, always under the control of the TEF constitutive promoter, not only restored the lipid accumulation capacity of the host strain, but also drove RA accumulation to over 50% of total lipid content. This preliminary work leads to the production of 12 g/L of RA in fermentor.

Furthermore, using functional-genomics approaches we identified the rate-limiting steps in RA production in an attempt to balance the flux between the oleic acid biosynthetic pathway and the oleic to ricinoleic hydroxylation pathway that is part of the Lands phospholipid cycle. Overexpression of the native diacylglycerol:cholineophosphotransferases (Ept1p, Cpt1p) accelerated the oleic acid flux from diacylglycerols (DAG) towards the phosphatidyl-choline (PC) where hydroxylation takes place. Similar results were obtained by overexpression of the native acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT) that facilitated the transport of fatty acids between lysophosphatidylcholine (LPC) and PC. Overexpression of the native A₂ phospholipase permitted the discharge of free RA from PC and resulted in the secretion of RA coupled with increased RA titres. Each of these modifications increased ricinoleic production of the engineered strain by at least 20%. The combination of all the aforementioned modifications should provide a robust ricinoleic producing cell factory with possible industrial applications. To our knowledge, this work reports the most efficient production of RA in an organism other than R. communis described to date. In order to achieve these rates of production, the lipid metabolism of Y. lipolytica necessitated substantial rerouting. The limiting steps of RA synthesis and accumulation are identified and will be discussed in an effort to provide fundamental understanding on the regulation and the interconnection of the lipogenic metabolic processes.