A β-carotene hydroxylase mutant, genetically engineered bacteria and its applications

By introducing the codon-optimized β-carotene hydroxylase mutant EuCrtZ-M92L-SC into a Saccharomyces cerevisiae strain and integrating it into the genome, the problem of poor enzyme stability was solved, and high-yield biosynthesis of zeaxanthin was achieved, with a yield of 445.2 mg/L.

CN122303164APending Publication Date: 2026-06-30TIANJIN UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
TIANJIN UNIV
Filing Date
2026-04-10
Publication Date
2026-06-30

AI Technical Summary

Technical Problem

During microbial fermentation, β-carotene hydroxylase (CrtZ) is unstable due to attack by reactive oxygen species (ROS), resulting in low corn xanthine yield and conversion rate, which is difficult to meet industrial needs.

Method used

By introducing the codon-optimized β-carotene hydroxylase mutant EuCrtZ-M92L-SC into a Saccharomyces cerevisiae strain and integrating it into the genome at a specific site using CRISPR/Cas9 gene editing technology, a recombinant engineered strain producing high levels of zeaxanthin was constructed, enhancing the enzyme's antioxidant capacity.

Benefits of technology

The enzyme stability and zeaxanthin yield were significantly improved. The mutant engineered strain achieved a yield of 445.2 mg/L in shake-flask fermentation, solving the problems of low enzyme stability and activity and realizing efficient biosynthesis of zeaxanthin.

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Abstract

This invention discloses a β-carotene hydroxylase mutant, a genetically engineered strain, and its applications. The methionine (Met) at position 92 of the codon-optimized EuCrtZ gene of *Saccharomyces cerevisiae* is mutated to leucine (Leu), yielding the β-carotene hydroxylase mutant gene EuCrtz-M92L-SC. Using *Saccharomyces cerevisiae* SyBE_Sc0014CY06 as the substrate strain, the β-carotene hydroxylase mutant gene EuCrtz-M92L-SC of claim 1 is integrated into the genome at the TKL2 and Int14 sites via CRISPR / Cas9 gene editing technology, while simultaneously integrating the HSC82 gene at the DAK2 site. This invention, through enzyme engineering and metabolic engineering, obtained a β-carotene hydroxylase mutant (M92L) with a key site mutation and a high-yield Saccharomyces cerevisiae engineered strain. This mutation significantly improved the enzyme's tolerance to intracellular oxidative stress and maintained the high stability of enzyme activity during fermentation. The engineered strain achieved a zeaxanthin yield of 445.2 mg / L through shake-flask fermentation.
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Patent Citations

  • CN104962488B