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

Abstract

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.

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

“…The gene encoding squalene synthase (SQS) was upregulated, while the gene encoding GGPP synthase (CrtE) was downregulated as cells grew (Figure and Table S4). We have also succsessfully increased β-carotene and astaxanthin by upregulating endogenous crtE and crtIBY . CrtIBY from strain M326 is likely a trifunctional enzyme, because it has predicted active domains for phytoene synthase, phytoene dehydrogenase, and lycopene cyclase.…”

“…We presumed that more acetyl-CoA was utilized for the formation of DHA-enriched lipids instead of carotenoids under the tested conditions. We have upregulated the ratio of astaxanthin to DHA-enriched TAG by diverting more acetyl-CoA into the MVA pathway . The gene encoding squalene synthase (SQS) was upregulated, while the gene encoding GGPP synthase (CrtE) was downregulated as cells grew (Figure and Table S4).…”

“…We have upregulated the ratio of astaxanthin to DHA-enriched TAG by diverting more acetyl-CoA into the MVA pathway. 21 The gene encoding squalene synthase (SQS) was upregulated, while the gene encoding GGPP synthase (CrtE) was downregulated as cells grew (Figure 7 and Table S4). We have also succsessfully increased β-carotene and astaxanthin by upregulating endogenous crtE and crtIBY.…”

“…We have also succsessfully increased β-carotene and astaxanthin by upregulating endogenous crtE and crtIBY. 21 CrtIBY from strain M326 is likely a trifunctional enzyme, because it has predicted active domains for phytoene synthase, phytoene dehydrogenase, and lycopene cyclase. A similar trifunctional CrtIBY has been identified from Aurantiochytrium sp.…”

“…Carotenoids were extracted and detected by using high-performance liquid chromatograph (HPLC 1200 series, Agilent Technologies, USA) analysis according to our previous report. 21 TLC was used to separate various carotenoids using solvent systems of diethyl ether and hexane (40:60, v/v). If needed, astaxanthin esters were recovered from silica plates and methylated for GC analysis.…”