Biocompatible α-Methylenation of Metabolic Butyraldehyde in Living Bacteria.

Small molecule organocatalysts are abundant in all living organisms. However, their use as organocatalysts in cells has been underexplored. Herein, we report that organocatalytic aldol chemistry can be interfaced with living Escherichia coli to enable the α-methylenation of cellular aldehydes using biogenic amines such as L-Pro or phosphate. The biocompatible reaction is mild and can be interfaced with butyraldehyde generated from D-glucose via engineered metabolism to enable the production of 2-methylenebutanal (2-MB) and 2-methylbutanal (2-MBA) by anaerobic fermentation, and 2-methylbutanol (2-MBO) by whole-cell catalysis. Overall, this study demonstrates the combination of non-enzymatic organocatalytic and metabolic reactions in vivo for the sustainable synthesis of valuable non-natural chemicals that cannot be accessed using enzymatic chemistry alone.

Palladium Nanoparticles from Desulfovibrio alaskensis G20 Catalyze Biocompatible Sonogashira and Biohydrogenation Cascades.

Transition-metal nanoparticles produced by living bacteria are emerging as novel catalysts for sustainable synthesis. However, the scope of their catalytic activity and their ability to be integrated within metabolic pathways for the bioproduction of non-natural small molecules has been underexplored. Herein we report that Pd nanoparticles synthesized by the sulfate-reducing bacterium Desulfovibrio alaskensis G20 (DaPdNPs) catalyze the Sonogashira coupling of phenyl acetylenes and aryl iodides, and the subsequent one-pot hydrogenation to bibenzyl derivatives using hydrogen gas generated from d-glucose by engineered Escherichia coli DD-2. The formal hydroarylation reaction is biocompatible, occurs in aqueous media at ambient temperature, and affords products in 70-99% overall yield. This is the first reported microbial nanoparticle to catalyze the Sonogashira reaction and the first demonstration that these biogenic catalysts can be interfaced with the products of engineered metabolism for small molecule synthesis.

Tyramine Derivatives Catalyze the Aldol Dimerization of Butyraldehyde in the Presence of Escherichia coli.

Biogenic amine organocatalysts have transformed the field of synthetic organic chemistry. Yet despite their use in synthesis and to label biomolecules in vitro, amine organocatalysis in vivo has received comparatively little attention - despite the potential of such reactions to be interfaced with living cells and to modify cellular metabolites. Herein we report that biogenic amines derived from L-tyrosine catalyze the self-aldol condensation of butanal to 2-ethylhexenal - a key intermediate in the production of the bulk chemical 2-ethylhexanol - in the presence of living Escherichia coli and outperform many amine organocatalysts currently used in synthetic organic chemistry. Furthermore, we demonstrate that cell lysate from E. coli and the prolific amine overproducer Corynebacterium glutamicum ATCC 13032 catalyze this reaction in vitro, demonstrating the potential for microbial metabolism to be used as a source of organocatalysts for biocompatible reactions in cells.

Micellar catalysis of the Suzuki Miyaura reaction using biogenic Pd nanoparticles from Desulfovibrio alaskensis.

Microorganisms produce metal nanoparticles (MNPs) upon exposure to toxic metal ions. However, the catalytic activity of biosynthesised MNPs remains underexplored, despite the potential of these biological processes to be used for the sustainable recovery of critical metals, including palladium. Herein we report that biogenic palladium nanoparticles generated by the sulfate-reducing bacterium Desulfovibrio alaskensis G20 catalyse the ligand-free Suzuki Miyaura reaction of abiotic substrates. The reaction is highly efficient (>99% yield, 0.5 mol% Pd), occurs under mild conditions (37 °C, aqueous media) and can be accelerated within biocompatible micelles at the cell membrane to yield products containing challenging biaryl bonds. This work highlights how native metabolic processes in anaerobic bacteria can be combined with green chemical technologies to produce highly efficient catalytic reactions for use in sustainable organic synthesis.

Interfacing non-enzymatic catalysis with living microorganisms.

Interfacing non-enzymatic catalysis with cellular metabolism is emerging as a powerful approach to produce a range of high value small molecules and polymers. In this review, we highlight recent examples from this promising young field. Specifically, we discuss demonstrations of living cells mediating redox processes for biopolymer production, interfacing solar-light driven chemistry with microbial metabolism, and intra- and extracellular non-enzymatic catalysis to generate high value molecules. This review highlights the vast potential of this nascent field to bridge the two disciplines of synthetic chemistry and synthetic biology for a sustainable chemical industry.

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies.

Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7-fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.