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Fig. 3 | Microbial Cell Factories

Fig. 3

From: CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

Fig. 3

CRISPR–Cas9-mediated multiloci integration of 6-MSA and 3-methylcatechol biosynthetic genes. a The biosynthetic pathway for 6-MSA and 3-methylcatechol. 6-MSA can be synthesized from acetyl-CoA and malonyl-CoA by a polyketide synthase AtX and a phosphopantetheinyl transferase NpgA. 3-methylcatechol can be synthesized from 6-MSA by a salicylate 1-monooxygenase AtA. b Overview of pathway assembly for production of 6-MSA and 3-methylcatechol in Δku70 strain. The atX and npgA expression cassettes were simultaneously integrated at PTEF1UP-g1 and PAOX1UP-g2, respectively. The atX, npgA, and atA expression cassettes were simultaneously integrated at PTEF1UP-g1, PAOX1UP-g2, and PFLD1UP-g1, respectively, for production of 3-methylcatechol. c The CFUs and genotypes of the transformants obtained by multiloci integration. According to the genotypes identified by PCR (Additional file 1: Fig. S8), 27 correct strains (K-XN) among 41 transformants in DLI and 4 correct strains (K-XNA) among 17 transformants in TLI. d HPLC analysis of organic extracts from culture broth. The strain K-XN 2# integrating the atX and npgA expression cassettes and the strain K-XNA 3# integrating the atX, npgA, and atA expression cassettes were cultured in YPD medium for 72 h. Samples extracted from culture broth were analyzed for UV absorbance at 254 nm. The HPLC analysis of other correct strains are shown in Additional file 1: Fig. S9

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