Presentation Information
[P01-096]Engineering β-carotene hydroxylase and its electron supply improve zeaxanthin production in Yarrowia lipolytica
○Hojun Lee1, Hanbit Song1, Sangwoo Seo2,3,1,4,5 (1. Institute of Chemical Processes, Seoul National University (Korea), 2. School of Chemical and Biological Engineering, Seoul National University (Korea), 3. Interdisciplinary Program in Bioengineering, Seoul National University (Korea), 4. Bio-MAX Institute, Seoul National University (Korea), 5. Institute of Engineering Research, Seoul National University (Korea))
Keywords:
β-carotene hydroxylase,Zeaxanthin,Enzyme activity engineering,Redox partner
[Purpose] Zeaxanthin, a carotenoid associated with ocular protection, is increasingly researched using microbial production platforms. However, bottlenecks that prevent full conversion of β-carotene into zeaxanthin have not been thoroughly identified and addressed. In this study, we aimed to enhance the ability to produce zeaxanthin in a recombinant Y. lipolytica strain through emphasis on the enzyme, β-carotene hydroxylase, and its electron sources.
[Method] We compared multiple β-carotene hydroxylases in a β-carotene-producing Y. lipolytica strain. Sequence co-evolutionary analysis to control the efficiency of enzyme reactions (SCANEER) was used to screen a mutant of CrtZ with improved zeaxanthin titer. The effects of identified mutations on enzyme structure were analyzed by molecular dynamics simulation. Multiple copy integrations of the CrtZ mutant and β-carotene pool improvement were tested for bottleneck identification. Finally, a heterologous ferredoxin FD3 and the endogenous cytochrome b5 were compared for their ability to transfer electrons to CrtZ.
[Results] CrtZ from Pantoea ananatis was selected for further engineering based on the initial screening result. The SCANEER-predicted S62A mutant of CrtZ improved zeaxanthin titer by 1.2-fold. Subsequent increases of CrtZ gene copy number did not significantly improve zeaxanthin titer, implying a bottleneck elsewhere.
[Conclusion] This study identified a CrtZ mutant with enhanced zeaxanthin titer and demonstrated that modulating endogenous redox partner expression can be a viable strategy for improving CrtZ activity, offering a practical alternative to heterologous ferredoxin expression.
[Method] We compared multiple β-carotene hydroxylases in a β-carotene-producing Y. lipolytica strain. Sequence co-evolutionary analysis to control the efficiency of enzyme reactions (SCANEER) was used to screen a mutant of CrtZ with improved zeaxanthin titer. The effects of identified mutations on enzyme structure were analyzed by molecular dynamics simulation. Multiple copy integrations of the CrtZ mutant and β-carotene pool improvement were tested for bottleneck identification. Finally, a heterologous ferredoxin FD3 and the endogenous cytochrome b5 were compared for their ability to transfer electrons to CrtZ.
[Results] CrtZ from Pantoea ananatis was selected for further engineering based on the initial screening result. The SCANEER-predicted S62A mutant of CrtZ improved zeaxanthin titer by 1.2-fold. Subsequent increases of CrtZ gene copy number did not significantly improve zeaxanthin titer, implying a bottleneck elsewhere.
[Conclusion] This study identified a CrtZ mutant with enhanced zeaxanthin titer and demonstrated that modulating endogenous redox partner expression can be a viable strategy for improving CrtZ activity, offering a practical alternative to heterologous ferredoxin expression.
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