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Aurantiochytrium limacinum  has received attention because of its abundance of polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). DHA is synthesized through the polyketide synthase (PKS) pathway in  A .  limacinum . The related enzymes of the PKS pathway are mainly expressed by three gene clusters, called  pks1 ,  pks2  and  pks3 . In this study, the full-length  pks3  gene was obtained by polymerase chain reaction amplification and Genome Walking technology. Based on a domain analysis of the deduced amino acid sequence of the  pks3  gene, 3-ketoacyl-ACP reductase (KR) and dehydratase (DH) enzyme domains were identified. Herein,  A .  limacinum  OUC168 was engineered by gene knock-in of KR and DH using the 18S rDNA sequence as the homologous recombination site. Total fatty acid contents and the degree of unsaturation of total fatty acids increased after the  kr  or  dh  gene was knocked in. The cloning and functional study of the  pks3  gene of  A .  limacinum  establishes a foundation for revealing the DHA synthetic pathway. Gene knock-in of the enzyme domain associated with PKS synthesis has the potential to provide effective recombinant strains with higher DHA content for industrial applications.

Cloning of the pks3 gene of Aurantiochytrium limacinum and functional study of the 3-ketoacyl-ACP reductase and dehydratase enzyme domains