Development of an Ene Reductase-Based Biocatalytic Process for the Production of Flavor Compounds

Abstract

Ene reductases catalyze the biocatalytic reduction of activated alkenes, offering a powerful biobased alternative to metal-catalyzed and organocatalyzed double-bond reductions. With the aim to utilize the natural catalysts for the development of sustainable industrial processes for the flavor and fragrance (F&F) industry, we investigated the synthetic potential of a wild-type ene reductase library consisting of 20 enzymes to produce flavor compounds, including decanal. In our library screening, we identified several ene reductases that could efficiently reduce 2E-decenal as well as other investigated target substrates. Five of the characterized enzymes exhibited high reduction activities even at increased substrate concentrations (10 g/L). By analyzing additional enzyme characteristics (thermostability, solubility, and activity at 10 °C), enzyme Pbr-ER from Pseudomonas brassicacearum was chosen for further characterization and process optimization. Using optimized reaction conditions, the Pbr-ER-catalyzed reduction of 2Edecenal was performed at 100 mL scale at 40 g/L substrate concentration, achieving a high conversion yield (>93%) within 24 h.

A Collaborative Journey towards the Late‐Stage Functionalization of Added‐Value Chemicals Using Engineered Halogenases

“…The Competence Center for Biocatalysis at ZHAW (CCBIO) is dedicated to the industrialization of biocatalytic processes through the discovery and optimization of enzymes, with special focus on automated and algorithm‐assisted enzyme engineering and the utilization of oxidoreductases for C−H activation [19–22] . Given the favorable geographical location, and driven by a common interest in enzymatic halogenation and sustainable synthesis, Novartis Pharma AG , Syngenta AG , and CCBIO decided to engage in a collaboration to explore the possibility to engineer enzymes capable to halogenate molecular scaffolds of agrochemical and pharmaceutical interest.…”

“…[18] The Competence Center for Biocatalysis at ZHAW (CCBIO) is dedicated to the industrialization of biocatalytic processes through the discovery and optimization of enzymes, with special focus on automated and algorithm-assisted enzyme engineering and the utilization of oxidoreductases for CÀ H activation. [19][20][21][22] Given the favorable geographical location, and driven by a common interest in enzymatic halogenation and sustainable synthesis, Novartis Pharma AG, Syngenta AG, and CCBIO decided to engage in a collaboration to explore the possibility to engineer enzymes capable to halogenate molecular scaffolds of agrochemical and pharmaceutical interest. The collaboration between industry and academia was made possible by the Project-bound Contributions 2017-2020 (Swiss State Secretariat for Education, Research and Innovation) which funded pre-competitive, biocatalysis-themed research projects in the frame of the network project 'Innovation in Biocatalysis'.…”

Emerging Technologies for Biocatalysis in the Pharmaceutical Industry

“…coli cells (800 wt % with respect to substrate) . However, in recent years, ERED reactions with conditions more amenable to scale-up have been reported: in 2022, ERED reactions producing the flavor compound decanal were reported at 40–60 g/L substrate loading and 10 g/L wet cells, achieving >93% conversion in a 100 mL reaction …”

“…35 However, in recent years, ERED reactions with conditions more amenable to scale-up have been reported: in 2022, ERED reactions producing the flavor compound decanal were reported at 40−60 g/L substrate loading and 10 g/L wet cells, achieving >93% conversion in a 100 mL reaction. 36 Similarly to IRED enzymes, it is thought that the biocatalytic potential of EREDs is limited by poor stability under industrial conditions as well as low specific activity with desired substrates. 37 While these problems are addressable through enzyme engineering, the time it takes to engineer an enzyme for both improved activity and stability can make a project incompatible with the demanding timelines needed for pharmaceutical development.…”

“…33 However, despite the long history of the study of EREDs, reports of multikilogram scale ERED reactions are rare. 34 Instead, examples of preparativescale reactions reported in the literature are more often performed at gram scale 35,36 and often require low substrate loading and large amounts of enzyme to achieve high yields. In a report from Pfizer describing a preparative-scale ERED reaction to access a chiral intermediate to pregabalin (Scheme 3), the process afforded quantitative yield from 150 mL containing 7.5 g/L substrate and 60 g/L wet E. coli cells (800 wt % with respect to substrate).…”