**1. Introduction**

Knowledge-based bioeconomy implies the conversion of cheap renewable resources into biotechnological products with added value.

Docosahexaenoic acid (DHA) is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), with numerous health benefits such as reducing the risk of cardiovascular diseases, cancer, and rheumatoid arthritis; alleviating depression symptoms and post-natal depression; and contributing to immune-modulatory effects [1]. DHA also has an important role in the healthy development of the fetal brain and retina, and thus is commonly used in infant-related food products. The global EPA/DHA market was estimated at USD 2.49 billion in 2019, with a projected annual growth rate of 7% until 2027 [2]. Currently, cold-water marine fish oil is a source of 96% of DHA, but it is not able to meet the increasing demand of DHA for human consumption [3] due to the depletion of wild fish stocks and pollution of the marine environment (with lipophilic environmental

**Citation:** Berzins, K.; Muiznieks, R.; Baumanis, M.R.; Strazdina, I.; Shvirksts, K.; Prikule, S.; Galvanauskas, V.; Pleissner, D.; Pentjuss, A.; Grube, M.; et al. Kinetic and Stoichiometric Modeling-Based Analysis of Docosahexaenoic Acid (DHA) Production Potential by *Crypthecodinium cohnii* from Glycerol, Glucose and Ethanol. *Mar. Drugs* **2022**, *20*, 115. https://doi.org/ 10.3390/md20020115

Academic Editors: Maria do Rosário Domingues and Philippe Soudant

Received: 5 January 2022 Accepted: 28 January 2022 Published: 1 February 2022

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pollutants, dioxins, heavy metals, etc.). Moreover, fish and other animals lack certain fatty acid desaturases that are required for the de novo synthesis of LC-PUFAs. Plants, although a commercially important source of oils and fats, do not synthesize LC-PUFAs.

Efforts to explore alternative sources of DHA have been made in the last decade, including the generation of transgenic oilseed plants [4] and large-scale production of DHA-producing microalgae and protists [5]. As microbes synthesize all of their cell lipid fatty acids de novo, the profile of these lipids is relatively simple, more predictable, and can be very rich in specific fatty acids, including LC-PUFAs. Among the protists, *Crypthecodinium cohnii*, a marine heterotrophic dinoflagellate, is successfully used for the industrial production of DHA because it can accumulate DHA at high concentrations within the cells [6]. In contrast to photosynthetic microalgae, heterotrophs, such as *C. cohnii*, do not require light; hence, a high biomass density can be reached in conventional bioreactors.

The established carbon substrates for the growth of *C. cohnii* are glucose, ethanol, and acetate. Ethanol and acetate are found to be superior to glucose for the production of DHA, likely because of their short conversion pathway to acetyl-CoA, the key precursor of fatty acid synthesis [7]. No or marginal growth on sucrose, glycerol, fructose, maltose, rhamnose, arabinose, lactose, and galacturonic acid has been reported previously [6,8,9]. However, several recent papers [10–12] demonstrated *C. cohnii* growth and abundant DHA synthesis in glycerol. Glycerol is an interesting renewable substrate since it is a by-product of biodiesel production and other industries, and is generated globally in large quantities. The contradictory information in the literature about the consumption of glycerol by *C. cohnii*, and DHA production from glycerol, calls for a closer look at this substrate. Notably, glycerol consumption requires just two additional reactions (glycerol kinase and glycerol-3-phosphate dehydrogenase) until it enters the metabolic "highway" of glycolysis.

The systems biology approach is used to gain a mechanistic understanding of the functioning of metabolic pathways and the theoretical limitations of different biotechnologically used but insufficiently explored organisms by combining laboratory experiments and mathematical modeling [13–16]. The implementation of the systems biology approach in education and production can lead to improvements in industrial biotechnology facilitated by interdisciplinary synergy [15,17]. The applications of different modeling approaches shed light on different aspects of the process of interest [18], enabling the implementation of different types of case-specific constraints [19].

In the present work, the authors focused on the experimental work and mathematical modeling of *C. cohnii*-central metabolic fluxes with three substrates: (i) glucose, as the most widely used carbon substrate for laboratory cultivation of this dinoflagellate [6]; (ii) ethanol, reported to be the best substrate for accumulation of DHA [20]; and (iii) glycerol, as an important renewable substrate, yet with somewhat contradictory evidence on its consumption and DHA production in *C. cohnii* [9,11,12]. The enzymatic capacity of metabolic pathways towards Acetyl-CoA (DHA precursor) is analyzed by a kinetic model. The availability of metabolic resources at the central metabolism scale is assessed by a stoichiometric model.
