Removing the Obstacle to (−)‐Menthol Biosynthesis by Building a Microbial Cell Factory of (+)‐<i>cis</i>‐Isopulegone from (−)‐Limonene

Shou, Chao; Zheng, Yu‐Cong; Zhan, Jing-Ru; Li, Chun-Xiu; Xu, Jian‐He

doi:10.1002/cssc.202101741

Cited by 8 publications

(2 citation statements)

References 37 publications

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“…Previous research showed that Mentha plants use the methylerythritol phosphate pathway as the main source of C5 raw material for essential oil production and other monoterpene biosynthesis [ 23 , 24 ]. Geranyl diphosphate produces menthol and pulegone via two pathways catalyzed by a series of enzymes [ 25 ] ( Fig. 4d ).…”

Section: Resultsmentioning

confidence: 99%

A haplotype-resolved gap-free genome assembly provides novel insight into monoterpenoid diversification in Mentha suaveolens ‘Variegata’

Yang,

Wang,

Zhou

et al. 2024

Horticulture Research

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Mentha is a commonly used spice worldwide, which possesses medicinal properties and fragrance. These characteristics are conferred, at least partially, by essential oils such as menthol. In this study, a gap-free assembly with a genome size of 414.3 Mb and 31,251 coding genes was obtained for Mentha suaveolens ‘Variegata’. Based on its high heterozygosity (1.5%), two complete haplotypic assemblies were resolved, with genome sizes of 401.9 Mb and 405.7 Mb, respectively. The telomeres and centromeres of each haplotype were almost fully annotated. In addition, we detected a total of 41,135 structural variations. Enrichment analysis demonstrated that genes involved in terpenoid biosynthesis were affected by these structural variations. Analysis of volatile metabolites showed that Mentha suaveolens mainly produces piperitenone oxide rather than menthol. We identified three genes in the Mentha suaveolens genome which encode isopiperitenone reductase (ISPR), a key rate-limiting enzyme in menthol biosynthesis. However, the transcription levels of ISPR were low. Given that other terpenoid biosynthesis genes were expressed, Mentha suaveolens ISPRs may account for the accumulation of piperitenone oxide in this species. The findings of this study may provide a valuable resource for improving the detection rate and accuracy of genetic variants, thereby enhancing our understanding of their impact on gene function and expression. Moreover, our haplotype-resolved gap-free genome assembly offers novel insights into molecular marker-assisted breeding of Mentha.

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Section: Resultsmentioning

confidence: 99%

A haplotype-resolved gap-free genome assembly provides novel insight into monoterpenoid diversification in Mentha suaveolens ‘Variegata’

Yang,

Wang,

Zhou

et al. 2024

Horticulture Research

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“…Interestingly they used a robotics-driven semirational design strategy to identify a PpKSI variant with four active site mutations that conferred a 4.3 fold increase in activity over the WT. The final piece of the microbial menthol production puzzle was recently solved when Shou et al (2021) filled in the gap between limonene and cis -isopulegone by replacing the microbially inefficient L3H and IPDH with a bacterial P450 monooxygenase from P. putida and a bacterial IPDH from Pseudomonas aeruginosa , respectively ( Shou et al, 2021 ). Their system was able to produce 163.3 mg of cis -isopulegone from 0.54 g of limonene at a purity of 90% when analysed through GC-MS. Other work prior to limonene production has been done by Rolf et al (2020) who transformed E. coli with a plasmid containing the genes encoding the enzymes in the MVA pathway and MsLimS.…”

Section: Engineering Mint For the Futurementioning

confidence: 99%

Genetic Manipulation of Biosynthetic Pathways in Mint

et al. 2022

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In recent years, the study of aromatic plants has seen an increase, with great interest from industrial, academic, and pharmaceutical industries. Among plants attracting increased attention are the Mentha spp. (mint), members of the Lamiaceae family. Mint essential oils comprise a diverse class of molecules known as terpenoids/isoprenoids, organic chemicals that are among the most diverse class of naturally plant derived compounds. The terpenoid profile of several Mentha spp. is dominated by menthol, a cyclic monoterpene with some remarkable biological properties that make it useful in the pharmaceutical, medical, cosmetic, and cleaning product industries. As the global market for Mentha essential oils increases, the desire to improve oil composition and yield follows. The monoterpenoid biosynthesis pathway is well characterised so metabolic engineering attempts have been made to facilitate this improvement. This review focuses on the Mentha spp. and attempts at altering the carbon flux through the biosynthetic pathways to increase the yield and enhance the composition of the essential oil. This includes manipulation of endogenous and heterologous biosynthetic enzymes through overexpression and RNAi suppression. Genes involved in the MEP pathway, the menthol and carvone biosynthetic pathways and transcription factors known to affect secondary metabolism will be discussed along with non-metabolic engineering approaches including environmental factors and the use of plant growth regulators.

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Discovery and Engineering of a Bacterial (+)‐Pulegone Reductase for Efficient (−)‐Menthol Biosynthesis

Wu,

Li,

Liu

et al. 2024

ChemSusChem

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The biosynthesis of valuable plant‐derived monoterpene (–)‐menthol from readily available feedstocks (e.g., (–)‐limonene) is of great significance because of the high market demand for this product. However, biotransforming (+)‐pulegone into (–)‐menthone, the (–)‐menthol precursor, through (+)‐pulegone reductase (PGR) catalysis is inefficient because of the poor protein expression or catalytic efficiency (kcat/Km) of plant origin PGRs. In this study, a novel bacterial PGR from Pseudomonas resinovorans (PrPGR) was identified, and the most successful variant, PrPGRM2‐1 (A50V/G53W), was obtained, showing respective 20‐fold and 204‐fold improvements in specific activity and catalytic efficiency. PrPGRM2‐1 was employed to bioreduce (+)‐pulegone, resulting in 4.4‐fold and 35‐fold enhancements in (–)‐menthone titers compared with the bioreductions catalyzed by wild‐type (WT) PrPGR and MpPGR, respectively. Furthermore, a whole‐cell biocatalyst containing PrPGRM2‐1, MpMMR, and BstFDH was constructed and achieved the highest (–)‐menthol titer reported to date without externally supplemented NADPH/NADP+. Overall, this study details an efficient PGR with high catalytic efficiency that possesses great potential for (–)‐menthol biosynthesis.

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Removing the Obstacle to (−)‐Menthol Biosynthesis by Building a Microbial Cell Factory of (+)‐cis‐Isopulegone from (−)‐Limonene

Cited by 8 publications

References 37 publications

A haplotype-resolved gap-free genome assembly provides novel insight into monoterpenoid diversification in Mentha suaveolens ‘Variegata’

A haplotype-resolved gap-free genome assembly provides novel insight into monoterpenoid diversification in Mentha suaveolens ‘Variegata’

Genetic Manipulation of Biosynthetic Pathways in Mint

Discovery and Engineering of a Bacterial (+)‐Pulegone Reductase for Efficient (−)‐Menthol Biosynthesis

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