A DNA fragment carrying the third mutated ACP domain of from orfA Schizochytrium sp. was amplified by overlap PCR with primers (SP48/SP49/SP50/SP51) and pET-orfA as a template. The fragment was digested with and and inserted into the and FspI EagI, AfeI EagI sites of pHSG298-ACP2. The replacement of the and fragments was done as EagI BamHI described above. Plasmid construction of pET-SopfaA-S1 A DNA fragment carrying sequence S1 (accession number NC_004347, 118th to hlyB 224th amino acids of HlyB, Supplementary Table 4), which is an ABC transporter from S. was amplified by PCR with primers (SP52/SP53) and the genome as oneidensis, S. oneidensis a template. The fragment was digested with and and inserted into the and PvuII EagI, AfeI sites of pHSG298-ACP2. The replacement of the and fragments was done EagI EagI BamHI as described above. Plasmid construction of pET-SofaA-S2 A DNA fragment carrying sequence S2 (accession number NC_004347, to hlyB 489th amino acids of HlyB, Supplementary Table 4) was amplified by PCR with primers (SP54/SP55) and the genome as a template. After this, the protocol for S. oneidensis was employed. References Hauvermale, A. Fatty acid production in sp.: involvement of a et al. Schizochytrium polyunsaturated fatty acid synthase and a type I fatty acid synthase. 41, 739–747 Lipids (2006) Amann, E., Ochs, B. & Abel, K. J. Tightly regulated promoter vectors useful for the tac expression of unfused and fused proteins in 69, 301–315 (1988) Escherichia coli. Gene Ujihara, T., Nagano, M., Wada, H. & Mitsuhashi, S. Identification of a type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis. FEBS Lett. 588, 4032–4036 (2014)
A DNA fragment carrying the third mutated ACP domain of orfA from Schizochytrium sp. was amplified by overlap PCR with primers (SP48/SP49/SP50/SP51) and pET-orfA as a template. The fragment was digested with FspI and EagI, and inserted into the AfeI and EagI sites of pHSG298-ACP2. The replacement of the EagI and BamHI fragments was done as described above. Plasmid construction of pET-SopfaA-S1 A DNA fragment carrying hlyB sequence S1 (accession number NC_004347, 118th to 224th amino acids of HlyB, Supplementary Table 4), which is an ABC transporter from S. oneidensis, was amplified by PCR with primers (SP52/SP53) and the S. oneidensis genome as a template. The fragment was digested with PvuII and EagI, and inserted into the AfeI and EagI sites of pHSG298-ACP2. The replacement of the EagI and BamHI fragments was done as described above. Plasmid construction of pET-SofaA-S2 A DNA fragment carrying hlyB sequence S2 (accession number NC_004347, to 489th amino acids of HlyB, Supplementary Table 4) was amplified by PCR with primers (SP54/SP55) and the S. oneidensis genome as a template. After this, the protocol for was employed. References Hauvermale, A. et al. Fatty acid production in Schizochytrium sp.: involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase. Lipids 41, 739–747 (2006) Amann, E., Ochs, B. & Abel, K. J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69, 301–315 (1988) Ujihara, T., Nagano, M., Wada, H. & Mitsuhashi, S. Identification of a type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis. FEBS Lett. 588, 4032–4036 (2014)
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