Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis

Hartley, Carol J.; French, Nigel G.; Scoble, Judith A.; Williams, Charlotte C.; Churches, Quentin I.; Frazer, Andrew R.; Taylor, Matthew C.; Coia, Gregory; Simpson, Gregory W.; Turner, Nicholas J.; Scott, Colin

doi:10.1371/journal.pone.0184183

Cited by 13 publications

(19 citation statements)

References 38 publications

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“…Prior comparison of enzymes that could be used for the first two steps of the model synthesis 18 had suggested that the most suitable enzymes for glycerol phosphorylation were a Thermococcus kodakarensis glycerol kinase (GlpK Tk ) and a Mycobacterium smegmatis acetate kinase (AceK Ms ). For the NAD + -dependent production of DHAP from glycerol-3-phosphate (G3P), E. coli glycerol-3-phosphate dehydrogenase (G3PD Ec ) and the water-forming NADH oxidase from Clostridium aminovalericum (NOX Ca ) were selected.…”

Section: Resultsmentioning

confidence: 99%

“…The pAF1 vector that encodes the DHAP-dependent fructose-1,6-biphosphate aldolase from Staphylococcus carnosus was a gift from Dr A. Frazer (University of Manchester, Manchester, UK). For all other constructs, the gene of interest was sourced as described previously 25 , codon-optimized for expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific, Germany), then cloned into pETCC2 26 (a modified version of pET14b, Novagen) using Nde I and Bam HI or Eco RI sites, to create an in-frame N -terminal hexa-histidine tag. Genes encoding fusions between the catalytic and cofactor-recycling domains (GlpK Tk -AceK M s and G3PD Ec -NOX Ca ) were synthesized.…”

Section: On-line Methodsmentioning

confidence: 99%

“…FruA Sc was selected as the preferred aldolase for the nanofactory based on its enantioselectivity (3 S , 4 R -ADHOP), oligomeric structure, stability, catalytic rate (Supplementary Table 2) and previous success using this enzyme in multi-enzyme cascades to produce similar chiral sugars 18 .…”

Section: On-line Methodsmentioning

confidence: 99%

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Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

Hartley

Williams

Scoble

et al. 2019

Preprint

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Biocatalysis is used for many chemical syntheses due to its high catalytic rates, specificities and 20 operation under ambient conditions 1, 2 . Continuous-flow chemistry offers advantages to 21 biocatalysis, avoiding process issues caused by substrate/product inhibition, equilibrium controlled 22 limitations on yield and allosteric control 3 . Modular continuous-flow biochemistry would also allow 23 the flexible assembly of different complex multistep reactions 3-6 . Here we tackle some technical 24 challenges that currently prohibit the wide-spread use of continuous flow biocatalysis; cofactor 25 immobilization and site-specific immobilization. We provide the first example of enzymes 26 engineered to retain and recycle their cofactors, and the use of these enzymes in continuous 27 production of chiral pharmaceutical intermediates. 28Enzyme immobilization for continuous-flow applications has been studied for some time; indeed, a 29 number of industrial processes are currently based on such technologies 4, 6-10 . However, such 30 processes have generally been limited to cofactor-independent enzymes, such as esterases. Cofactors, 31 such as nicotinamide adenine dinucleotide (NAD + ) and adenosine triphosphate (ATP), are used 32 stoichiometrically unless recycled, typically by a second enzyme: without recycling, cofactors become 33 prohibitively expensive for most industrial syntheses. As cofactors require diffusion for recycling, they 34 are ill-suited for use in continuous-flow reactors and the lack of a practical engineering solution for 35 the issue has stymied the use of cofactor-dependent enzymes in continuous-flow applications 4 ; 36 although, growing interest in immobilized biocatalysts for cell-free metabolic engineering has led to 37 the development of a variety of enzyme-cofactor-carrier combinations 5 . 38Herein, we propose a novel and generalizable chemo-genetic enzyme engineering approach that 39 enables the fabrication of modular, multistep, biocatalytic, continuous-flow reactors using cofactor-40 dependent enzymes, thereby extending the utility of biocatalysis for continuous-flow production 41 systems and cell-free metabolic engineering 11 . 42 Results 43Nanomachine design 44 Our design for a biocatalyst that can retain and recycle its cofactor (a 'nanomachine') was inspired by 45 enzymes that retain their substrates via covalent attachment during a reaction cascade involving 46 multiple active sites, e.g. phosphopantetheine-dependent synthases 12-14 and lipoic acid-dependent 47 dehydrogenases 15 . In such enzymes, the substrate is delivered from one active site to the next by a 48 3 flexible 'swinging arm' that is covalently attached to the protein. We have adopted a similar strategy, 49 whereby a flexible swinging arm covalently attaches a cofactor to a synthetic, multidomain protein 50 and delivers that cofactor to the different active sites of the fusion protein, allowing its simultaneous 51 use and recycling, while preventing its diffusion (Figure 1). 52The general design of our nanomachines...

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Section: Resultsmentioning

confidence: 99%

Section: On-line Methodsmentioning

confidence: 99%

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Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

Hartley

Williams

Scoble

et al. 2019

Preprint

Self Cite

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show abstract

“…Prior comparison of enzymes that could be used for the first two steps of the model synthesis 18 had suggested that the most suitable enzymes for glycerol phosphorylation were a Thermococcus kodakarensis glycerol kinase (GlpKTk) and a Mycobacterium smegmatis acetate kinase (AceKMs). For the NAD + -dependent production of DHAP from glycerol-3-phosphate (G3P), E. coli glycerol-3phosphate dehydrogenase (G3PDEc) and the water-forming NADH oxidase from Clostridium aminovalericum (NOXCa) were selected.…”

Section: Assembling the Nanomachinesmentioning

confidence: 99%

Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis

et al. 2019

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“…Starting from glycerol and pyrophosphate, a 57% yield of 5-deoxy-5-ethyl-D-xylulose was obtained from l-glycerol-3-phosphate. In another study, the conversion of glycerol to dihydroxyacetone phosphate (DHAP) was reported with a one-pot four-enzyme cascade composed of glycerol kinase (Thermococcus kodakarensis), acetate kinase (Mycobacterium smegmatis), glycerophosphate oxidase (Mycoplasma gallisepticum) and catalase (Micrococcus lysodeikticus) (Hartley et al, 2017). The cascade achieved 88% conversion of glycerol to DHAP in 1 h at 22 • C. The pathway was coupled with a DHAP-dependent fructose-1,6biphosphate aldolase enzyme from Staphylococcus carnosus to produce the rare chiral sugars D-fructose-1,6-biphosphate (3S, 4R) and 3,4-dihydroxyhexulose phosphate (3S, 4R).…”

Section: Substrates For Biomass Transformationmentioning

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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Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis

Cited by 13 publications

References 38 publications

Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis

Cell-Free Biocatalysis for the Production of Platform Chemicals

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