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.
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 β-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 β-carotene, upregulated endogenous β-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.
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