Nervonic acid is a very long-chain fatty acid (VLCFA) with a wide range of potential applications in the pharmaceutical, food, and cosmetics industries. However, the natural sources of nervonic acid are limited and expensive. Metabolic engineering offers a promising approach to the sustainable production of nervonic acid. Nervonic acid (NA), also known as cis-15-tetracosenoic acid (C24:1 Δ15), is an omega-9 very-long-chain monounsaturated fatty acid (VLCFA) that is found abundantly in the myelin sheath of the human brain. NA plays a crucial role in maintaining the structure and function of the nervous system. Studies have shown that NA deficiency can lead to neurological disorders such as adrenoleukodystrophy (ALD) and multiple sclerosis (MS).
In a recent study, researchers employed the oleaginous yeast Yarrowia lipolytica as a host to de novo synthesizing nervonic acid. They used a systematic metabolic engineering approach to engineer the fatty acid elongation (FAE) pathway and modulate endogenous pathways to increase the production of nervonic acid.The researchers first introduced a heterologous β-ketoacyl-CoA synthase gene (CgKCS) into Y. lipolytica. This gene enabled the yeast to elongate fatty acids up to 24 carbons in length, which is necessary to produce nervonic acid.
Next, the researchers modulated endogenous pathways by expressing a C16:0-acyl-CoA preferred fatty acid elongase (gELOVL6) and a C18:0-acyl-CoA preferred fatty acid desaturase (MaOLE2). These enzymes increased the production of nervonic acid by directing the elongation and desaturation of fatty acids towards the synthesis of nervonic acid. Finally, the researchers optimized the expression of the key enzymes involved in nervonic acid synthesis. They also identified and overexpressed a gene (YlINO2) that regulates the structure of the endoplasmic reticulum (ER), which is the site of fatty acid synthesis. This overexpression further increased the production of nervonic acid.
As a result of these systematic metabolic engineering efforts, the researchers achieved a nervonic acid titre of 17.3 g/L, the highest reported titre to date for de novo nervonic acid production. This study demonstrates the potential of oleaginous yeasts as sustainable hosts to produce nervonic acid and other VLCFAs. Currently, the primary sources of NA are the seeds of a few plants, such as Sapium sebiferum and Vernonia galamensis. However, these plant-based sources are limited and often expensive. Therefore, there is a growing need for alternative methods to produce NA.
Metabolic Engineering of Yarrowia lipolytica for NA Production
Yarrowia lipolytica is an oleaginous yeast, which can accumulate high levels of lipids, including fatty acids. This makes it an attractive host to produce NA through metabolic engineering.Researchers have employed various metabolic engineering strategies to enhance NA production in Yarrowia lipolytica. These strategies involve manipulating the yeast’s metabolic pathways to increase the flux of carbon towards NA synthesis.
Metabolic Engineering: A Powerful Tool for Bioproduction
Metabolic engineering involves manipulating the metabolic pathways of an organism to enhance the production of a desired compound. In this study, the researchers focused on engineering the fatty acid elongation (FAE) pathway in Yarrowia lipolytica, a yeast known for its ability to accumulate lipids.
They introduced a heterologous β-ketoacyl-CoA synthase gene (CgKCS) from the plant Cuphea carthagenesis into the yeast’s genome. This gene encodes an enzyme that catalyzes the elongation of fatty acids, enabling the production of NA chains.
Key Strategies for NA Overproduction
Several key strategies have been employed to achieve high-level production of NA in Yarrowia lipolytica:
- Engineering the Fatty Acid Elongation (FAE) Pathway:
The FAE pathway is responsible for elongating fatty acyl chains. By introducing heterologous β-ketoacyl-CoA synthase (KCS) genes from other organisms, such as CgKCS from the marine diatom Thalassiosira pseudonana, NA production can be enabled in Yarrowia lipolytica.
- Modulating Endogenous Pathways:
Expression of genes encoding fatty acid elongase (ELOVL) and fatty acid desaturase (OLE) can further enhance NA production. For instance, co-expression of gELOVL6, a C16:0-acyl-CoA preferred ELOVL, and MaOLE2, a C18:0-acyl-CoA preferred OLE, has been shown to increase NA content in total fatty acids (TFA).
- Iterative Expression of Key Genes:
Repeatedly expressing genes encoding KCS, ELOVL, and OLE at different genomic loci can further boost NA production. This iterative approach has led to significant increases in NA titers.
- Enhancing Lipid Biosynthesis:
Expression of genes encoding glycerol-3-phosphate acyltransferases (GPAT) and diacylglycerol acyltransferases (DGAT) from Malania oleifera in the endoplasmic reticulum (ER) membrane can further enhance the biosynthesis of both NA and lipids.
- Regulating ER Structure:
Overexpression of a newly identified ER structure regulator gene YlINO2 in Yarrowia lipolytica has been shown to increase lipid production by 39.3%, potentially contributing to NA production.
- Disrupting SNF1 Gene:
Disruption of the AMP-activated S/T protein kinase gene SNF1 can increase the ratio of NA (C24:1) to lignoceric acid (C24:0) by 61.6%.
Proof-of-Concept Purification and Separation
Pilot-scale fermentation in a 50-L reactor using the strain YLNA9 exhibited a lipid titer of 96.7 g/L and an NA titer of 17.3 g/L (17.9% of total fatty acids), the highest reported titer to date for de novo NA production. A proof-of-concept purification and separation of NA were performed, and the purity of NA reached 98.7%. This demonstrates the feasibility of producing high-purity NA from engineered Yarrowia lipolytica
Potential applications of nervonic acid
Nervonic acid has a wide range of potential applications, including:
- Pharmaceuticals: Nervonic acid is a potential treatment for neurological diseases such as Alzheimer’s disease and Parkinson’s disease. It is also being investigated as a treatment for demyelinating diseases such as multiple sclerosis.
- Food: Nervonic acid is a valuable source of omega-9 fatty acids, which are beneficial for cardiovascular health. It can be used as a food additive or an ingredient in functional foods.
- Cosmetics: Nervonic acid is used in various cosmetic products, such as moisturizers and sunscreens. It is also being investigated as a potential anti-aging agent.
The development of a sustainable and cost-effective method for producing nervonic acid is essential for realizing the potential of this valuable compound. Metabolic engineering of oleaginous yeasts offers a promising approach to achieving this goal.
Conclusion
Systematic metabolic engineering of Yarrowia lipolytica has emerged as a promising approach to producing NA. By manipulating the yeast’s metabolic pathways, researchers have achieved significant increases in NA titre, paving the way for cost-effective and sustainable production of this valuable fatty acid for potential therapeutic applications
References:
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- High-level production of nervonic acid in the oleaginous yeastYarrowia lipolyticaby systematic metabolic engineering – Abstract – Europe PMC
- High-level production of nervonic acid in the oleaginous yeastYarrowia lipolyticaby systematic metabolic engineering | Sciety Labs (Experimental)
- High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering | bioRxiv
- High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering (nature.com)
