US 12,649,936 B2 · P450 cytochrome enzyme for andrographolide synthesis and its application

Abstract

The present disclosure provides a P450 cytochrome enzyme for andrographolide synthesis and its application, belonging to the field of bioengineering. The present disclosure uses Saccharomyces cerevisiae CEN.PK2-1D as a host, and implements knockout of ROX1 and GAL80 genes on the genome, and integrative expression of GGPP synthase encoding gene and CPS diterpene synthase encoding gene at ROX1 site; and implements free expression of ApCPR and CYP71A8 and CYP71D10 both with truncated signal peptides, successfully constructing recombinant S. cerevisiae, and achieving de novo synthesis of 3,15,19-Trihydroxy-8(17),13-ent-labdadiene-16-oic acid. Compared with the blank, a response value of a product peak reaches 1.9*106, and this strategy provides necessary reference for analyzing biosynthetic pathway of andrographolide and using metabolic engineering to synthesize andrographolide and related derivatives thereof.

Claims6

Dependent claims

A recombinant Saccharomyces cerevisiae, comprising knockout of ROX1 and GAL80 genes on the genome;

integrative expression of a GGPP synthase encoding gene and CPS diterpene synthase encoding gene;
and free expression of CYP71A8 encoding gene CYP71A8t with N-terminal truncated by 32 amino acids, CYP71A10 encoding gene CYP71D10t with N-terminal truncated by 28 amino acids, and CPR encoding gene ApCPR;
wherein a nucleotide sequence of the CYP71A8 with N-terminal truncated by 32 amino acids is set forth in SEQ ID NO:3 and a nucleotide sequence of the CYP71D10 with N-terminal truncated by 28 amino acids being shown in SEQ ID NO:4;
a nucleotide sequence of the ApCPR is set forth in SEQ ID NO:7;
a nucleotide sequence of the GGPP synthase is set forth in SEQ ID NO:8;
and a nucleotide sequence of the CPS diterpene synthase is set forth in SEQ ID NO:9.

3 more elements

Dependencies are inferred from the claim text.

P450 cytochrome enzyme for andrographolide synthesis and its application

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P450 cytochrome enzyme for andrographolide synthesis and its application

Life of this patent

Abstract

Claims6