This content will become publicly available on November 21, 2024
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.
more » « less- NSF-PAR ID:
- 10475275
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- ISSN:
- 0006-3592
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract In the presence of a thiourea–carboxylic acid catalyst, N-9-fluorenyltryptamines undergo highly enantioselective Pictet–Spengler reactions with a range of aldehydes. The reaction works particularly well with aromatic aldehydes, tolerating electronically diverse substituents in all ring positions. Electron-deficient tryptamines are viable substrates. Removal of the fluorenyl protecting group is readily accomplished without deterioration of product ee.more » « less
-
Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings, incurring large processing costs for the production of industrial-scale biofuels or biochemicals. Manipulating surface charge interactions to minimize nonproductive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability but with limited demonstrated success to date. Here, we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe, Thermobifida fusca. A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of −52 to 37 was computationally designed using Rosetta macromolecular modeling software. Activity for all mutants was rapidly characterized as soluble cell lysates, and promising mutants (containing mutations on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2- and 5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using a protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM-containing endocellulases exhibited a 5–10 °C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity toward complex lignocellulosic biomass-derived substrates.more » « less
-
Abstract Enzymes from secondary metabolic pathways possess broad potential for the selective synthesis of complex bioactive molecules. However, the practical application of these enzymes for organic synthesis is dependent on the development of efficient, economical, operationally simple, and well‐characterized systems for preparative scale reactions. We sought to bridge this knowledge gap for the selective biocatalytic synthesis of β‐hydroxy‐α‐amino acids, which are important synthetic building blocks. To achieve this goal, we demonstrated the ability of ObiH, an
l ‐threonine transaldolase, to achieve selective milligram‐scale synthesis of a diverse array of non‐standard amino acids (nsAAs) using a scalable whole cell platform. We show how the initial selectivity of the catalyst is high and how the diastereomeric ratio of products decreases at high conversion due to product re‐entry into the catalytic cycle. ObiH‐catalyzed reactions with a variety of aromatic, aliphatic and heterocyclic aldehydes selectively generated a panel of β‐hydroxy‐α‐amino acids possessing broad functional‐group diversity. Furthermore, we demonstrated that ObiH‐generated β‐hydroxy‐α‐amino acids could be modified through additional transformations to access important motifs, such as β‐chloro‐α‐amino acids and substituted α‐keto acids. -
Abstract While ketones are among the most versatile functional groups, their synthesis remains reliant upon reactive and low‐abundance starting materials. In contrast, amide formation is the most‐used bond‐construction method in medicinal chemistry because the chemistry is reliable and draws upon large and diverse substrate pools. A new method for the synthesis of ketones is presented here that draws from the same substrates used for amide bond synthesis: amines and carboxylic acids. A nickel terpyridine catalyst couples N‐alkyl pyridinium salts with in situ formed carboxylic acid fluorides or 2‐pyridyl esters under reducing conditions (Mn metal). The reaction has a broad scope, as demonstrated by the synthesis of 35 different ketones bearing a wide variety of functional groups with an average yield of 60±16 %. This approach is capable of coupling diverse substrates, including pharmaceutical intermediates, to rapidly form complex ketones.