A cell-free synthetic biochemistry platform for raspberry ketone production

Tosi, Tomasso; Bell, David; Hleba, Yonek B; Polizzi, Karen M.; Freemont, Paul S.

doi:10.1101/202341

Cited by 10 publications

(9 citation statements)

References 60 publications

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“…The Nicotiana tabacum L. DBR ( Nt DBR, UniProt ID: Q9SLN8) has been shown to be active toward a variety of α,β-unsaturated alkenes ( Mansell et al, 2013 ), such as (−)-cinnamaldehyde and 1-nitrocyclohexene. In addition, Rubus idaeus L. raspberry DBR ( Ri DBR, UniProt ID: G1FCG0) catalyzes the reduction of 4-hydroxybenzalacetone and 3-methoxy-4-hydroxybenzalacetone to raspberry ketone and zingerone, respectively ( Simon et al, 2017 ). A DBR from Malus domestica L. ( Md DBR, UniProt ID: A0A5N5GUE7) has been isolated and characterized recently ( Caliandro et al, 2021 ) and suggested to be involved in the biosynthesis of polyphenolic compounds (e.g., dihydrochalcones) that are beneficial in human diet.…”

Section: Introductionmentioning

confidence: 99%

Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis

et al. 2021

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Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs like Mentha haplocalyx Briq., Nepeta tenuifolia Briq., Perilla frutescens (L.) Britt and Pogostemin cablin (Blanco) Benth. Pulegone reductase is the key enzyme in the biosynthesis of menthol and is required for the stereoselective reduction of the Δ2,8 double bond of pulegone to produce the major intermediate menthone, thus determining the stereochemistry of menthol. However, the structural basis and mechanism underlying the stereoselectivity of pulegone reductase remain poorly understood. In this study, we characterized a novel (−)-pulegone reductase from Nepeta tenuifolia (NtPR), which can catalyze (−)-pulegone to (+)-menthone and (−)-isomenthone through our RNA-seq, bioinformatic analysis in combination with in vitro enzyme activity assay, and determined the structure of (+)-pulegone reductase from M. piperita (MpPR) by using X-ray crystallography, molecular modeling and docking, site-directed mutagenesis, molecular dynamics simulations, and biochemical analysis. We identified and validated the critical residues in the crystal structure of MpPR involved in the binding of the substrate pulegone. We also further identified that residues Leu56, Val282, and Val284 determine the stereoselectivity of the substrate pulegone, and mainly contributes to the product stereoselectivity. This work not only provides a starting point for the understanding of stereoselectivity of pulegone reductases, but also offers a basis for the engineering of menthone/menthol biosynthetic enzymes to achieve high-titer, industrial-scale production of enantiomerically pure products.

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Section: Introductionmentioning

confidence: 99%

Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis

et al. 2021

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“…Recently, there has been emphasis on multi-enzyme complexes in cascades in the development of pathway engineering and it is clear that the rapid rise of synthetic biology owes much to the controllable cell-free environment, the ability to direct substrates toward the desired product and the facility with which superior combinations of often heterologous enzymes can be assembled. These issues have been reviewed in detail recently and the reader is referred to Jewett et al (2008); Carlson et al (2012), Gan and Jewett (2014), Hodgman and Jewett (2013), Jewett (2016, 2018), Kelwick et al (2016), Moore et al (2017b), and Silverman et al (2020). Further descriptions of pathway developments (for example, Dudley et al, 2019Dudley et al, , 2020 with limonene and isoprenoid synthesis).…”

Section: Prototypingmentioning

confidence: 99%

“…The PURE system relies on purified cellular components (108 in total, Shimizu et al, 2001) and is available commercially (New England Biolabs) but its high cost has prevented scale up. Its main use is in small scale prototyping (Moore et al, 2017b). A less expensive route is to employ crude cell lysates where the results are scalable into high volume fermentations (Zawada et al, 2011;Cai et al, 2015) coupled with inexpensive energy sources and an awareness of the problem of diversion of at least some of the target product into other reactions.…”

Section: Prototypingmentioning

confidence: 99%

Cell-Free Biocatalysis for the Production of Platform Chemicals

2020

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Genetically engineered host bacteria have an extensive history for the production of specific proteins including the synthesis of single enzymes for the modification of compounds produced for industrial purposes by biological or chemical processes. Such processes have been developed largely through the process of discovery. The ability to assemble multiple enzymes into synthetic pathways is a new development aided by the synthetic biology approach of constructing and assembling suitable enzymes into pathways that may or may not occur in Nature to provide a highimpact platform for bio-manufacturing of chemicals, biofuels and pharmaceuticals. Industry has depended on chemical catalysts because of the known constraints experienced frequently with biological catalysts but when high stereochemistry, mild synthesis conditions and environmentally friendly processes are significant, application of enzymes is preferred, especially in the case of drug synthesis where enantioselectivity is important. However, many whole cell production processes are beset by toxicity problems, metabolite competition, the production of side products, sub-optimal enzyme ratios and varying temperature optima. As a result, a cell-free biocatalysis allows the manipulation of substrate ratios, provision of regenerated cofactors and adjustment of high energy flux ratios that are difficult or impossible to control in whole cell synthesis. We discuss here the construction of cell free biocatalytic pathways as added free enzymes or multi-enzyme modules that may contain heterologous catalysts. We examine the status of applications leading to commercialisation, emphasizing the importance of economical cofactor regeneration. Nevertheless, problems remain, particularly protein post-translational modifications including glycosylation, phosphorylation, ubiquitination, acetylation, proteolysis, etc. The National Renewable Energy Laboratory and Pacific North West Laboratory have published lists of top value-added chemicals from biomass that include glucaric acid. There have been few reports on the combination of synthetic biology and cell-free protein synthesis to set up pathways for these valuable intermediate compounds. This observation is despite the existence of at least one large scale but specialized cell-free production of antibody conjugates. This review will provide a description of one successful attempt at the cell-free production of glucaric acid and will evaluate progress for other key intermediate and platform chemicals.

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“…These systems can also be an appropriate platform for production because of lower noise and toxicity and absence of resource competition between pathway and cell growth. To date, cell-free systems have been applied to implement pathways for violacein [49], 4-BDO [50], polyhydroxyalkanoates bioplastics [51], mevalonate [52], n-butanol [53] and raspberry ketone [54], using either transcriptiontranslation (TX-TL) systems, overexpressed enzymes in the crude extract or purified enzymes. Advantages and possibilities of cell-free systems for metabolic engineering has been reviewed elsewhere [55], and a methods chapter for pathway prototyping in cell-free systems has recently been published [56].…”

Section: Custom-made Biosensors' New Application Domain: Cell-free Mementioning

confidence: 99%

Custom-made transcriptional biosensors for metabolic engineering

Koch

Pandi

Borkowski

et al. 2019

Current Opinion in Biotechnology

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Transcriptional biosensors allow screening, selection or dynamic regulation of metabolic pathways, and are therefore an enabling technology for faster prototyping of metabolic engineering and sustainable chemistry. Recent advances have been made, allowing for routine use of heterologous transcription factors, and new strategies such as chimeric protein design allow engineers to tap into the reservoir of metabolite-binding proteins. However, extending the sensing scope of biosensors is only the first step, and computational models can help in fine-tuning properties of biosensors for custom-made behavior. Moreover, metabolic engineering is bound to benefit from advances in cell-free expression systems, either for faster prototyping of biosensors or for whole-pathway optimization, making it both a means and an end in biosensor design. Highlights • Successful examples of transcriptional biosensor implementations are presented • Various engineering strategies extend the space of detectable chemicals • Novel strategies exist to transform metabolite-responding proteins into biosensors • Mathematical models of varying complexity can help tune biosensor properties • Biosensors using or designed for cell-free systems are presented

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A cell-free synthetic biochemistry platform for raspberry ketone production

Cited by 10 publications

References 60 publications

Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis

Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis

Cell-Free Biocatalysis for the Production of Platform Chemicals

Custom-made transcriptional biosensors for metabolic engineering

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