Recent developments in enzymatic and microbial biosynthesis of flavor and fragrance molecules.

Flavors and fragrances are an important class of specialty chemicals for which interest in biomanufacturing has risen during recent years. These naturally occurring compounds are often amenable to biosynthesis using purified enzyme catalysts or metabolically engineered microbial cells in fermentation processes. In this review, we provide a brief overview of the categories of molecules that have received the greatest interest, both academically and industrially, by examining scholarly publications as well as patent literature. Overall, we seek to highlight innovations in the key reaction steps and microbial hosts used in flavor and fragrance manufacturing.

Reductive amination cascades in cell-free and resting whole cell formats for valorization of lignin deconstruction products.

The selective introduction of amine groups within deconstruction products of lignin could provide an avenue for valorizing waste biomass while achieving a green synthesis of industrially relevant building blocks from sustainable sources. Here, we built and characterized enzyme cascades that create aldehydes and subsequently primary amines from diverse lignin-derived carboxylic acids using a carboxylic acid reductase (CAR) and an ω-transaminase (TA). Unlike previous studies that have paired CAR and TA enzymes, here we examine multiple homologs of each of these enzymes and a broader set of candidate substrates. In addition, we compare the performance of these systems in cell-free and resting whole-cell biocatalysis formats using the conversion of vanillate to vanillyl amine as model chemistry. We also demonstrate that resting whole cells can be recycled for multiple batch reactions. We used the knowledge gained from this study to produce several amines from carboxylic acid precursors using one-pot biocatalytic reactions, several of which we report for the first time. These results expand our knowledge of these industrially relevant enzyme families to new substrates and contexts for environmentally friendly and potentially low-cost synthesis of diverse aryl aldehydes and amines.

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells.

Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C-C bond formation. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as expected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for reduced aromatic aldehyde reduction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supplemented to resting cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits reduced oxidation and reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions - the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard ß-hydroxy-α-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.

Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications.

The formation of inclusion bodies (IBs) during expression of recombinant therapeutic proteins using E. coli is a significant hurdle in producing high-quality, safe, and efficacious medicines. The improved understanding of the structure-function relationship of the IBs has resulted in the development of novel biotechnologies that have streamlined the isolation, solubilization, refolding, and purification of the active functional proteins from the bacterial IBs. Together, this overall effort promises to radically improve the scope of experimental biology of therapeutic protein production and expand new prospects in IBs usage. Notably, the IBs are increasingly used for applications in more pristine areas such as drug delivery and material sciences. In this review, we intend to provide a comprehensive picture of the bio-processing of bacterial IBs, including assessing critical gaps that still need to be addressed and potential solutions to overcome them. We expect this review to be a useful resource for those working in the area of protein refolding and therapeutic protein production.