Heterologous Synthesis of Polyunsaturated Fatty Acids for Aquaculture (2024)

Non Technical Summary
Non-technical summaryThe United States is a minor aquaculture producer, ranking 18th in global aquaculture production in 2022, but it is the leading importer of fish and fish products. Even though U.S. aquaculture is growing at a compound annual rate of 4%, we still depend heavily upon imports. By value, 90% of the seafood we eat originates abroad, and more than half of imported seafood is produced by foreign aquaculture. The USDA Aquaculture priorities are to identify and address the needs or constraints that limit U.S. aquaculture. These include "Improved production systems and management strategies for efficiency and reduced environmental impacts." One of the most critical needs for domestic aquaculture is to obtain an inexpensive source of essential fatty acids. One of the most important essential nutrients that humans require are omega-3 (w3), long-chain, polyunsaturated fatty acids (LC-PUFAs). These are fatty acids that our bodies do not produce in sufficient quantities, and they are critical in the development and maintenance of brain cells and neurons. Typically, we obtain these essential nutrients through the consumption of marine fish. These days, however, most of the fish eaten in the US is grown in aquaculture and imported. The most expensive component of aquaculture feeds is fish meal, which is the source of w3 fatty acids. Often, instead of fish meal, aquaculture farms substitute soybean oil, which does not contain long-chain polyunsaturated w3 fatty acids. As a result, the quality of foreign or even domestic fish grown in aquaculture lacks this highly important nutritional component. Xylome's proposed research and development aims directly at this nutritional and economic limitation by creating a novel pathway to provide high omega-3 long-chain polyunsaturated fatty acids for use in aquaculture feed. Currently, the only way to obtain w3 for aquaculture is by harvesting wild fish stocks, which are becoming rapidly depleted and more expensive to harvest. For aquaculture to be sustainable, a source of w3 that does not rely on wild fish stocks must be found. We propose to use byproducts from midwestern agriculture, drawing on stillage byproducts from ethanol production and soy protein to create a fish-free high protein, high w3 fatty acid diet for intensively cultivated salmon and other species. Xylome has developed a novel highly lipogenic yeast strain capable of producing 85% of its dry weight in lipid. We intend to use contemporary methods of synthetic biology and precision fermentation to alter the metabolism of our highly lipogenic yeast so that it produces omega-3 fatty acids.

Goals / Objectives
Goals:High ω3 LC-PUFA fatty acids are essential for neural development in children and adults and are critical for cardiovascular health, but the supplies of high ω3 fatty acids are insufficient to meet their demand. Marine fish oils are the main sources for LC-ω3 PUFAs in aquaculture. The competition between fish used for aquaculture feed vs. direct food consumption can create sustainability problems. Aquaculture feed represents 40-75% of aquaculture production cost and is one of the key factors in the aquaculture industry. Marine fish oils are the main sources for LC-ω3 PUFAs in aquaculture feeds. Aquaculture can convert human inedible byproducts into edible biomass, but the use of high-quality food-competing ingredients such as fishmeal, or soya bean products should be minimized in aquaculture feed.The most important nutritional constituents, eicosapentaenoic acid (EPA, 20:5 ω3) and docosahexaenoic acid (DHA, 22:6 ω3), are produced natively by marine protists belonging mainly to the Thraustochytrids, diatoms, algae, and marine bacteria. Thraustochytrids can be cultivated heterotrophically, but they grow slowly, and their oil contents are low. This makes the cost of their oils very high. For example, a commercial brand of DHA obtained from cultivation of Thraustochytrid (which does not contain EPA) costs about $25 for 12 grams. By comparison 90 grams of DHA along with 45 grams of EPA obtained from fish oil costs about $20. While these are retail costs, they reflect the underlying costs of supplying DHA and EPA to fish cultivated in aquaculture.The major goals of this project are as follows: (1) Make nutritionally vital long-chain polyunsaturated fatty acids (LC-PUFAs) high in ω3 fatty acids. (2) Engineer a highly lipogenic yeast, L. starkeyi, to express a heterologous polyketide synthase complex (PKS) that could produce LC-ω3 PUFAs efficiently. (3) Identify the factors or rate-limiting steps for LC-PUFA synthesis in the heterologous background. (4) Direct the flow of metabolites from the native lipogenic pathways into the heterologous LC-PUFA pathway.Technical ObjectivesThe technical objective in Phase II will be to (1) Create a new base strain for genetic engineering. This will consist of a recently constructed parental strain that has a disrupted ligase (LIG4) to enable targeted deletions and modification. (2) Identification of promoters that are active during lipogenesis and less active during growth, and vice-versa. This will enable us to separate the express ion of lipogenic genes from the growth phase and into the lipogenic (nitrogen starved) phase. In Phase I, we had used strong promoters that were expressed primarily in the growth phase. (3) Identification of integration sites that allows high expression of genes. With the lig4 we can target integration sites as well as promoters for modification. (4) Creation of multiple DPA/DHA producing loci. This will enable higher overall levels of omega-3 enzyme production. (5) Test novel targets to increase the flux of carbon into long-chain omega-3 fatty acids. We have identified additional proteins that could increase omega-3 production. (6) Express a Thraustochytrid Diacylglycerol Acyl Transferase (DAT). We suspect that the L. starkeyi DAT might not have high affinity for the poly unsaturated omega-3 moieties. (7) Knockdown expression of FAS I or FAS 2 genes to reduce competition with omega-3 FAS. (8) Construct and characterize final pre-commercial and commercial strains

Project Methods
MethodsIn Phase I of this project, we synthesized four genes for the synthesis of DHA (FasA, FasB, FasC and FasE) and transformed the constructs into a native Lipomyces starkeyi host. We have also created a lig4 mutant of the wild-type strain that will enable us to conduct targeted integrations and deletions. We expect to use advanced vector construction techniques (a Golden Gate Cloning system called El Dorado) to develop integration cassettes. This enables the rapid construction of expression vectors promoters, terminators, open reading frames, selection markers, and insertional pieces to quickly build expression, integration, and deletion cassettes for the genetic engineering of Lipomyces starkeyi.We expect to screen our transformants in 250 ml Erlenmeyer flasks and use lipid extraction followed by gas chromatography to identify and quantify EPA and DHA products. We will take advantage of the native regulation of lipogenesis in L. starkeyi to cultivate cells under a nitrogen-rich phase and shift them into lipogenesis during the nitrogen starvation phase. We will promoters that are active during lipogenesis and less active during growth, and vice-versa. a transcriptomic experiment comparing mRNA pools isolated from a fermentor-grown Lipomyces starkeyi culture in the growth phase to a culture in the lipogenic phase. We will extract mRNA from cells and send the material to a sequencing facility for mRNA sequencing and transcriptomics analysis. We will select potential promoters based on the fold increase or decrease in the lipogenic phase versus the growth phase, and the overall expression level at the lipogenic or growth phase. The top ten promoters for each type will be domesticated into our Golden Gate Cloning system and synthesized. We will place a reporter gene under the promoter's control to test the promoters and integrate the construct into a specific region in the genome using our LIG4 deletion strain.Using the transcriptomics data generated in the previous method, we will identify areas of the genome that are highly transcribed during lipogenesis. We will identify potential integration sites between highly transcribed genes or genes that are non-essential and not required for lipogenesis and target these regions for integration of our expression cassettes.In addition to the basic DHA synthase, it may be necessary to express a Thraustochytrid diacylglycerol acyl transferase or other genes to attain product formation. It may also be necessary to disable or delete genes in the native Lipomyces lipid synthesis pathway to increase the flux of metabolites into the heterologous omega-3 biosynthetic pathway. This could be accomplished for example by replacing the endogenous diacylglycerol acyl transferase with the Thraustochytrid gene and by knocking down the FAS1 or FAS2 biosynthetic genes.It may also be necessary to try alternative EPA/DHA biosynthetic genes in order to enable heterologous expression.