Characterization of different biocatalyst formats for BVMO‐catalyzed cyclohexanone oxidation

Bretschneider, Lisa; Heuschkel, Ingeborg; Ahmed, Afaq; Bühler, Katja; Karande, Rohan; Bühler, Bruno

doi:10.1002/bit.27791

Cited by 7 publications

(6 citation statements)

References 88 publications

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“…. This is around 4 to 5 times lower than published for the heterotrophic host Pseudomonas taiwanensis VLB120 (Schäfer et al, 2020), which showed significantly higher BVMO expression levels (Bretschneider et al, 2021a) and features a highly active energy and redox metabolism based on organic acids and sugars (Volmer et al, 2014). It is however noteworthy that the BVMO exhibited a much higher in vivo activity in Syn6803 than in P. taiwanensis, that is, a 4.6 to 11 times higher k cat (6.9-17.6 s −1 vs. 1.5 s −1 (Bretschneider et al, 2021a)).…”

Section: Photosynthesis-driven Bvmo Catalysis Is Hampered By Reactant Inhibition Rather Than Host Metabolismcontrasting

confidence: 55%

“…This is around 4 to 5 times lower than published for the heterotrophic host Pseudomonas taiwanensis VLB120 (Schäfer et al, 2020), which showed significantly higher BVMO expression levels (Bretschneider et al, 2021a) and features a highly active energy and redox metabolism based on organic acids and sugars (Volmer et al, 2014). It is however noteworthy that the BVMO exhibited a much higher in vivo activity in Syn6803 than in P. taiwanensis, that is, a 4.6 to 11 times higher k cat (6.9-17.6 s −1 vs. 1.5 s −1 (Bretschneider et al, 2021a)). It should be noted that BVMO catalysis in Syn6803 appeared not to be limited by the metabolic capacity to supply reduction equivalents as the photoproduction of NADPH in Syn6803 is in the range of 9-18 U mg Chl a (Kauny and Setif, 2014), which translates to 106-212 U g CDW −1 .…”

Section: Photosynthesis-driven Bvmo Catalysis Is Hampered By Reactant Inhibition Rather Than Host Metabolismcontrasting

confidence: 55%

“…BVMO containing Syn6803 was found to feature a 3.7 times lower apparent uptake constant K S for C-one (80 ± 23 µM) than Pseudomonas taiwanensis VLB 120 containing the same enzyme (316 ± 21 µM) (Bretschneider et al, 2021a). This indicates a rather efficient substrate mass transfer into the cell.…”

Section: Photosynthesis-driven Bvmo Catalysis Is Hampered By Reactant Inhibition Rather Than Host Metabolismmentioning

confidence: 89%

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Maximizing Photosynthesis-Driven Baeyer–Villiger Oxidation Efficiency in Recombinant Synechocystis sp. PCC6803

et al. 2022

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Photosynthesis-driven whole-cell biocatalysis has great potential to contribute to a sustainable bio-economy since phototrophic cells use light as the only energy source. It has yet to be shown that phototrophic microorganisms, such as cyanobacteria, can combine the supply of high heterologous enzyme levels with allocation of sufficient reduction equivalents to enable efficient light-driven redox biocatalysis. Here, we demonstrated that the heterologous expression of an NADPH-dependent Baeyer–Villiger monooxygenase (BVMO) gene from Acidovorax sp. CHX100 turns Synechocystis sp. PCC6803 into an efficient oxyfunctionalization biocatalyst, deriving electrons and O2 from photosynthetic water oxidation. Several expression systems were systematically tested, and a PnrsB-(Ni2+)–controlled expression based on a replicative plasmid yielded the highest intracellular enzyme concentration and activities of up to 60.9 ± 1.0 U gCDW−1. Detailed analysis of reaction parameters, side reactions, and biocatalyst durability revealed—on the one hand—a high in vivo BVMO activity in the range of 6 ± 2 U mgBVMO−1 and—on the other hand—an impairment of biocatalyst performance by product toxicity and by-product inhibition. Scale-up of the reaction to 2-L fed-batch photo-bioreactors resulted in the stabilization of the bioconversion over several hours with a maximal specific activity of 30.0 ± 0.3 U g CDW−1, a maximal volumetric productivity of 0.21 ± 0.1 gL−1 h−1, and the formation of 1.3 ± 0.1 gL−1 of ε-caprolactone. Process simulations based on determined kinetic data revealed that photosynthesis-driven cyclohexanone oxidation on a 2-L scale under high-light conditions was kinetically controlled and not subject to a limitation by photosynthesis.

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Section: Photosynthesis-driven Bvmo Catalysis Is Hampered By Reactant Inhibition Rather Than Host Metabolismcontrasting

confidence: 55%

Section: Photosynthesis-driven Bvmo Catalysis Is Hampered By Reactant Inhibition Rather Than Host Metabolismcontrasting

confidence: 55%

Section: Photosynthesis-driven Bvmo Catalysis Is Hampered By Reactant Inhibition Rather Than Host Metabolismmentioning

confidence: 89%

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Maximizing Photosynthesis-Driven Baeyer–Villiger Oxidation Efficiency in Recombinant Synechocystis sp. PCC6803

et al. 2022

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“…Crude cell extracts were obtained as previously described, [9b] but with extended centrifugation for 30 min after cell disruption. Enzyme activity was determined based on the decrease in NAD(P)H absorption at 340 nm with a Cary Bio 300 UV‐visible spectrophotometer (Varian, Palo Alto, USA) following a described procedure: [37] Assay mixtures of 2 ml total volume in a screw capped cuvette contained 200 μM NAD(P)H and 50–100 μl crude cell extract (0.1–0.3 mg protein) in 100 mM KPi buffer. Assays were initiated by adding 2.5 μl pure cyclohexane and were performed for at least 2 min.…”

Section: Methodsmentioning

confidence: 99%

Enlighting Electron Routes In Oxyfunctionalizing Synechocystis sp. PCC 6803

Tüllinghoff,

Toepel,

Bühler

2023

ChemBioChem

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Phototrophic microorganisms, like cyanobacteria, are gaining attention as host organisms for biocatalytic processes with light as energy source and water as electron source. Redox enzymes, especially oxygenases, can profit from in‐situ supply of co‐substrates, i. e., reduction equivalents and O2, by the photosynthetic light reaction. The electron transfer downstream of PS I to heterologous electron consuming enzymes in principle can involve NADPH, NADH, and/or ferredoxin, whereas most direct and efficient transfer is desirable. Here, we use the model organism Synechocystis sp. PCC 6803 to investigate, to what extent host and/or heterologous constituents are involved in electron transfer to a heterologous cytochrome P450 monooxygenase from Acidovorax sp. CHX100. Interestingly, in this highly active light‐fueled cycloalkane hydroxylating biocatalyst, host‐intrinsic enzymes were found capable of completely substituting the function of the Acidovorax ferredoxin reductase. To a certain extent (20 %), this also was true for the Acidovorax ferredoxin. These results indicate the presence of a versatile set of electron carriers in cyanobacteria, enabling efficient and direct coupling of electron consuming reactions to photosynthetic water oxidation. This will both simplify and promote the use of phototrophic microorganisms for sustainable production processes.

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“…Depending on the reaction(s) to be catalyzed, resting cells can be easier to handle and reusable but often suffer from poor stability (Julsing et al, 2012;Zhang et al, 2012). Recently, mixed-species concepts have been used to produce the monomer adipic acid, 6-aminohexanoic acid, or 1,6-hexanediol from cyclohexane (Wang et al, 2020;Zhang et al, 2020;Bretschneider et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Conversion of Cyclohexane to 6-Hydroxyhexanoic Acid Using Recombinant Pseudomonas taiwanensis in a Stirred-Tank Bioreactor

et al. 2021

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6-hydroxyhexanoic acid (6HA) represents a polymer building block for the biodegradable polymer polycaprolactone. Alternatively to energy- and emission-intensive multistep chemical synthesis, it can be synthesized directly from cyclohexane in one step by recombinant Pseudomonas taiwanensis harboring a 4-step enzymatic cascade without the accumulation of any intermediate. In the present work, we performed a physiological characterization of this strain in different growth media and evaluated the resulting whole-cell activities. RB and M9* media led to reduced gluconate accumulation from glucose compared to M9 medium and allowed specific activities up to 37.5 ± 0.4 U gCDW−1 for 6HA synthesis. However, 50% of the specific activity was lost within 1 h in metabolically active resting cells, specifying growing cells, or induced resting cells as favored options for long-term biotransformation. Furthermore, the whole-cell biocatalyst was evaluated in a stirred-tank bioreactor setup with a continuous cyclohexane supply via the gas phase. At cyclohexane feed rates of 0.276 and 1.626 mmol min−1 L−1, whole-cell biotransformation occurred at first-order and zero-order rates, respectively. A final 6HA concentration of 25 mM (3.3 g L−1) and a specific product yield of 0.4 g gCDW−1 were achieved with the higher feed rate. Product inhibition and substrate toxification were identified as critical factors limiting biocatalytic performance. Future research efforts on these factors and the precise adjustment of the cyclohexane feed combined with an in situ product removal strategy are discussed as promising strategies to enhance biocatalyst durability and product titer and thus to enable the development of a sustainable multistep whole-cell process.

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Characterization of different biocatalyst formats for BVMO‐catalyzed cyclohexanone oxidation

Cited by 7 publications

References 88 publications

Maximizing Photosynthesis-Driven Baeyer–Villiger Oxidation Efficiency in Recombinant Synechocystis sp. PCC6803

Maximizing Photosynthesis-Driven Baeyer–Villiger Oxidation Efficiency in Recombinant Synechocystis sp. PCC6803

Enlighting Electron Routes In Oxyfunctionalizing Synechocystis sp. PCC 6803

Conversion of Cyclohexane to 6-Hydroxyhexanoic Acid Using Recombinant Pseudomonas taiwanensis in a Stirred-Tank Bioreactor

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