Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

Bayer, Thomas; Milker, Sofia; Wiesinger, Thomas; Rudroff, Florian; Mihovilovic, Marko D.

doi:10.1002/adsc.201500202

Cited by 52 publications

(47 citation statements)

References 310 publications

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“…The attention in the biocatalysis community increased significantly towards cascade reactions in the past decade (Bayer et al 2015;Kohler and Turner 2015;Muschiol et al 2015). Synthesis of highly complex molecules based on multienzyme processes, inspired by Nature's paradigm, become more and more important.…”

Section: Bvmos Applied In Cascade Type Reactionsmentioning

confidence: 99%

Baeyer-Villiger oxidations: biotechnological approach

Bučko

Gemeiner

Schenkmayerová

et al. 2016

Appl Microbiol Biotechnol

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Baeyer-Villiger monooxygenases (BVMOs) are a very well-known and intensively studied class of flavin-dependent enzymes. Their substrate promiscuity, high chemo-, regio-, and enantioselectivity are prerequisites for the use in synthetic chemistry and should pave the way for successful industrial processes. Nonetheless, only a very limited number of industrial relevant transformations are known, mainly due to the lack of BVMOs stability and cofactor dependency. In this review, we focus on novel BVMO-mediated transformations, BVMOs in cascade type reactions, potential industrial applications, and how limitations have been tackled by the community. Special attention will be put on whole-cell immobilization strategies. We emphasize to bridge recent developments in fundamental research to industrial applications.

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Section: Bvmos Applied In Cascade Type Reactionsmentioning

confidence: 99%

Baeyer-Villiger oxidations: biotechnological approach

Bučko

Gemeiner

Schenkmayerová

et al. 2016

Appl Microbiol Biotechnol

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“…Although such in‐vitro cascade allows the highest flexibility to balance enzyme ratios for enhancing reaction performance (Muschiol et al, ), the necessity of several unit operations for the production of isolated enzymes and cofactors leads to high CapEx and OpEx costs. In this context, in‐vivo cascade offers economic perspectives by combining production and regeneration of enzymes and cofactors necessary to conduct cascade reactions in one single unit operation (Bayer, Milker, Wiesinger, Rudroff, & Mihovilovic, ; de Carvalho, ; Schrewe, Julsing, Bühler, & Schmid, ; Schrittwieser et al, ). However, for the efficient turnover of the in‐vivo cascade, the catalytic efficiency of the individual enzymes needs to be compatible to streamline substrate conversion toward the main product without accumulation of intermediate compounds.…”

Section: Introductionmentioning

confidence: 99%

Biocatalytic conversion of cycloalkanes to lactones using an in‐vivo cascade in Pseudomonas taiwanensis VLB120

Karande

Salamanca

Schmid

et al. 2017

Biotech & Bioengineering

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Chemical synthesis of lactones from cycloalkanes is a multi-step process challenged by limitations in reaction efficiency (conversion and yield), atom economy (by-products) and environmental performance. A heterologous pathway comprising novel enzymes with compatible kinetics was designed in Pseudomonas taiwanensis VLB120 enabling in-vivo cascade for synthesizing lactones from cycloalkanes. The respective pathway included cytochrome P450 monooxygenase (CHX), cyclohexanol dehydrogenase (CDH), and cyclohexanone monooxygenase (CHXON) from Acidovorax sp. CHX100. Resting (non-growing) cells of the recombinant host P. taiwanensis VLB120 converted cyclohexane, cyclohexanol, and cyclohexanone to ϵ-caprolactone at 22, 80-100, and 170 U g , respectively. Cyclohexane (5 mM) was completely converted with a selectivity of 65% for ϵ-caprolactone formation in 2 hr without accumulation of intermediate products. Promiscuity of the whole-cell biocatalyst gave access to analogous lactones from cyclooctane and cyclodecane. A total product concentration of 2.3 g L and a total turnover number of 36,720 was achieved over 5 hr with a biocatalyst concentration of 6.8 g L .

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“…Control experiments with E. coli BL21(DE3) in the presence of 1 a showed no endogenous oxidation activity under standard reaction conditions . Apart from the beneficial substrate uptake velocity of membrane‐associated AlkJ, this flavin adenine dinucleotide (FAD)‐dependent ADH promotes thermodynamically disfavored alcohol oxidation and takes advantage of irreversible O 2 reduction in the electron transport chain . With AlkJ, we identified a suitable ADH for the subsequent aldol reaction catalyzed by Fsa1‐A129S.…”

Section: Resultsmentioning

confidence: 99%

“…The introduction of synthetic cascades into living organisms, such as Escherichia coli , lead to so‐called designer cells, which offer advantages over in vitro systems. Tedious enzyme preparation, isolation, and purification can be omitted; no additional cofactor recycling system is required; and tools from synthetic biology and metabolic engineering can be used to optimize carbon flux through the cascade …”

Section: Introductionmentioning

confidence: 99%

Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds

et al. 2017

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A synthetic cascade for the transformation of primary alcohols into polyhydroxylated compounds in Escherichia coli, through the in situ preparation of cytotoxic aldehyde intermediates and subsequent aldolase-mediated C-C bond formation, has been investigated. An enzymatic toolbox consisting of alcohol dehydrogenase AlkJ from Pseudomonas putida and the dihydroxyacetone-/hydroxyacetone-accepting aldolase variant Fsa1-A129S was applied. Pathway optimization was performed at the genetic and process levels. Three different arrangements of the alkJ and fsa1-A129S genes in operon, monocistronic, and pseudo-operon configuration were tested. The last of these proved to be most beneficial with regard to bacterial growth and protein expression levels. The optimized whole-cell catalyst, combined with a refined solid-phase extraction downstream purification protocol, provides diastereomerically pure carbohydrate derivatives that can be isolated in up to 91 % yield over two reaction steps.

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Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

Abstract: An immense number of chemical reactions are carried out simultaneously in living cells. Natures optimization approache ncompasses the assemblyo fr eactions in cascades and to embed them in finely tuned metabolicn etworks

Cited by 52 publications

References 310 publications

Baeyer-Villiger oxidations: biotechnological approach

Baeyer-Villiger oxidations: biotechnological approach

Biocatalytic conversion of cycloalkanes to lactones using an in‐vivo cascade in Pseudomonas taiwanensis VLB120

Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds

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