The Biosynthesis of Astaxanthin Esters in Schizochytrium sp. is Mediated by a Bifunctional Diacylglycerol Acyltransferase.

Astaxanthin esters are a major form of astaxanthin found in nature. However, the exact mechanisms of the biosynthesis and storage of astaxanthin esters were previously unknown. We found that Schizochytrium sp. synthesized both astaxanthin and docosahexaenoic acid (DHA)-enriched lipids. The major type of astaxanthin produced was free astaxanthin along with astaxanthin-DHA monoester and other esterified forms. DHA accounted for 41.0% of the total fatty acids from astaxanthin monoesters. These compounds were deposited mainly in lipid droplets. The biosynthesis of the astaxanthin esters was mainly carried out by a novel diacylglycerol acyltransferase ScDGAT2-1, while ScDGAT2-2 was involved only in the production of triacylglycerol. We also identified astaxanthin ester synthases from the astaxanthin-producing algae Haematococcus pluvialis and Chromochloris zofingiensis, as well as a thraustochytrid Hondaea fermentalgiana with an unknown carotenoid profile. This investigation enlightens the application of thraustochytrids for the production of both DHA and astaxanthin and provides enzyme resources for the biosynthesis of astaxanthin esters in the engineered microbes.

Modulating DHA-Producing Schizochytrium sp. toward Astaxanthin Biosynthesis via a Seamless Genome Editing System.

Schizochytrium sp. is commercially used for the production of docosahexaenoic acid (DHA). Some strains of Schizochytrium sp. are also known to produce low amounts of carotenoids, including astaxanthin and ß-carotene. In order to enhance the production of astaxanthin in Schizochytrium sp., we established a seamless genome editing system with a dual selection marker for rapid screening of positive transformants. By using this system, we strengthened the endogenous mevalonate pathway, enhanced the supply of geranylgeranyl diphosphate and ß-carotene, upregulated endogenous ß-carotene hydroxylase, and introduced the algal astaxanthin pathway. The highest astaxanthin production in the engineered Schizochytrium sp. was achieved at 8.1 mg/L (307.1 µg/g dry cell weight) under shake-flask conditions, which was 2.6-fold higher than that in the start strain. Meanwhile, the percentage of DHA to total fatty acids was not obviously affected. We then eliminated the dual selection marker by using the Cre-loxP recombination system, and the engineered strain was ready for iterative editing. The developed system could be applied to seamlessly engineer DHA-producing Schizochytrium sp. toward astaxanthin and other value-added terpenoids, which broadens the application of this strain.

Production of High Levels of 3S,3'S-Astaxanthin in Yarrowia lipolytica via Iterative Metabolic Engineering.

Astaxanthin is a highly value-added keto-carotenoid compound. The astaxanthin 3S,3'S-isomer is more desirable for food additives, cosmetics, and pharmaceuticals due to health concerns about chemically synthesized counterparts with a mixture of three isomers. Biosynthesis of 3S,3'S-astaxanthin suffers from limited content and productivity. We engineered Yarrowia lipolytica to produce high levels of 3S,3'S-astaxanthin. We first assessed various ß-carotene ketolases (CrtW) and ß-carotene hydroxylases (CrtZ) from two algae and a plant. HpCrtW and HpCrtZ from Haematococcus pluvialis exhibited the strongest activity in converting ß-carotene into astaxanthin in Y. lipolytica. We then fine-tuned the HpCrtW and HpCrtZ transcriptional expression by increasing the rounds of gene integration into the genome and applied a modular enzyme assembly of HpCrtW and HpCrtZ simultaneously. Next, we rescued leucine biosynthesis in the engineered Y. lipolytica, leading to a five-fold increase in biomass. The astaxanthin production achieved from these strategies was 3.3 g/L or 41.3 mg/g dry cell weight under fed-batch conditions, which is the highest level reported in microbial chassis to date. This study provides the potential for industrial production of 3S,3'S-astaxanthin, and this strategy empowers us to build a sustainable biorefinery platform for generating other value-added carotenoids in the future.

Lipid Distribution Pattern and Transcriptomic Insights Revealed the Potential Mechanism of Docosahexaenoic Acid Traffics in Schizochytrium sp. A-2.

Schizochytrium sp. A-2 is a heterotrophic marine fungus used for the commercial production of docosahexaenoic acid (DHA). However, the pattern of the distribution of DHA and how DHA is channeled into phospholipid (PL) and triacylglycerol (TAG) are unknown. In this study, we systematically analyzed the distribution of DHA in TAG and PL during the growth of the cell. The migration of DHA from PL to TAG was presumed during the fermentation cycle. DHA and docosapentaenoic acid were accumulated in both TAG and phosphatidylcholine (PC), whereas eicosapentaenoic acid was mainly deposited in PC. RNA seq revealed that malic enzyme may provide lipogenic NADPH. In addition, long-chain acyl-CoA synthase and acyl-CoA:lysophosphatidylcholine acyltransferase may participate in the accumulation of DHA in PL. No phosphatidylcholine:diacylglycerol cholinephosphotransferase was identified from the genome sequence. In contrast, phospholipid:diacylglycerol acyltransferase-mediated acyl-CoA-independent TAG synthesis pathway and phospholipase C may contribute to the channeling of DHA from PC to TAG.