N-terminal truncation of prenyltransferase enhances the biosynthesis of prenylnaringenin / 生物工程学报
Chinese Journal of Biotechnology
; (12): 1565-1575, 2022.
Article
em Zh
| WPRIM
| ID: wpr-927801
Biblioteca responsável:
WPRO
ABSTRACT
8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values. It also serves as an important precursor for many prenylated flavonoids. Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors. In this work, a SfN8DT-1 from Sophora flavescens was used to improve the efficiency of (2S)-naringenin prenylation. The predicted structure of SfN8DT-1 showed that its main body is comprised of 9 α-helices and 8 loops, along with a long side chain formed by nearly 120 amino acids. SfN8DT-1 mutants with different side-chain truncated were tested in Saccharomyces cerevisiae. A mutant expressing the truncated enzyme at K62 site, designated as SfND8T-1-t62, produced the highest 8-PN titer. Molecular docking of SfN8DT-1-t62 with (2S)-naringenin and dimethylallyl diphosphate (DMAPP) showed that K185 was a potentially crucial residue. Alanine scanning within a range of 0.5 nm around these two substrates showed that the mutant K185A may decrease its affinity to substrates, which also indicated K185 was a potentially critical residue. Besides, the mutant K185W enhanced the affinity to ligands implied by the simulated saturation mutation, while the saturated mutation of K185 showed a great decrease in 8-PN production, indicating K185 is vital for the activity of SfN8DT-1. Subsequently, overexpressing the key genes of Mevalonate (MVA) pathway further improved the titer of 8-PN to 31.31 mg/L, which indicated that DMAPP supply is also a limiting factor for 8-PN synthesis. Finally, 44.92 mg/L of 8-PN was produced in a 5 L bioreactor after 120 h, which is the highest 8-PN titer reported to date.
Palavras-chave
Texto completo:
1
Base de dados:
WPRIM
Assunto principal:
Saccharomyces cerevisiae
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Flavonoides
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Sophora
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Dimetilaliltranstransferase
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Prenilação
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Simulação de Acoplamento Molecular
Tipo de estudo:
Prognostic_studies
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2022
Tipo de documento:
Article