Enzyme evolution for industrial biocatalytic cascades

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Highlights

  • In vitro, multi-enzyme cascades of industrial relevance are reviewed.

  • Enzyme evolution allows combining multiple enzymatic steps in a single reaction step within a single vessel.

  • Biocatalytic retrosynthesis aids cascade development.

  • Sustainable chemical synthesis enabled by enzyme engineering.

Multi-step, biocatalytic cascades are poised to lead to further adoption of enzymes by the chemical industry. Over the past twenty years, the promise of in vitro enzyme evolution for the sustainable biocatalytic synthesis of complex chemicals at large scale has materialized. Recently, the field of biocatalysis is seeing further expansion, with biocatalytic processes becoming more complex and involving multiple consecutive enzymatic conversions. These biocatalytic cascades are assembled in single reaction vessels to accomplish difficult chemistry under mild reaction conditions, with minimal waste generation and attractive economics. Advances in enzyme engineering have enabled the increasingly efficient optimization of enzymes in the context of such cascades, where each enzyme operates in the presence of others, under continuously changing conditions as substrate, reaction intermediates, and product concentrations fluctuate over the course of the reaction. Enzyme evolution has provided biocatalysts with greatly improved traits, including activity, selectivity, and stability. This review focuses on recently developed, industrially relevant enzyme cascades.

Introduction

The potential of the unique specificity and selectivity of enzymes for the synthesis of complex molecules was already recognized in the late 19th century. However, for enzymes to be used for commercial purposes, the arrival of recombinant DNA technologies and the PCR was crucial. Since then, advances in enzyme engineering, through iterative rounds of library generation combined with high throughput screening, have enabled economic biocatalytic manufacturing of complex chemicals and were recognized by the 2018 Nobel Prize in Chemistry being awarded to Frances Arnold for her pioneering work [1••]. Advanced protein engineering technologies have led to the development of biocatalysts for use in large scale chemical manufacture [2].

In nature, enzymes work together in metabolic pathways, such as the pentose-phosphate shunt, β-oxidation, and the nucleotide salvage pathway, to efficiently convert available carbon sources to a multitude of intermediates to generate energy and building blocks for sustaining life. Recapitulating this concept in industry would enable the conversion of inexpensive raw materials into complex chemicals, in a single vessel. In a perspective on industrial biocatalysis in 2001, Schmid et al. [3] posited that continuous regeneration of co-factors would impact industrial catalytic reductions and indeed, today, many enzymatic processes that use co-factor recycling run at commercial scale.

At that time, the use of whole cells as biocatalysts for multi-enzyme processes was anticipated to see increased implementation, especially given their advantages of co-factor recyclability, simple starting material requirements, and ability to accommodate multi-step enzymatic pathways [4]. Fermentative processes using recombinant microorganisms for the production of lactic acid, isobutanol, poly-hydroxyalkanoates, and biodiesel constituents have been scaled up, yet such processes are often compromised by relatively low yields and a need for complex downstream processes that add significant cost [5]. The use of cell-free extracts of such strains can overcome the challenges of cellular toxicity and increase yield and product titer [6], and additional process design space can be derived from using combinations of isolated enzymes to optimize each step for highest efficiency.

Advances in enzyme engineering [1••], an abundance of genomic information that provides a plethora of starting genes, and maturation of retrosynthesis concepts in biocatalysis [7, 8, 9] have enabled assembly of multiple enzymes into biocatalytic cascades, where the product of one enzymatic reaction, is the substrate for the next enzyme. Non-natural pathways are now readily designed and realized for the efficient syntheses of complex chemicals. A recent review summarizes considerations for choosing a preferred route for production of a molecule of interest [5]. In this contribution, we provide an overview of the progression of biocatalytic cascade process development that has culminated in the industrialization of in vitro, multi-enzyme cascades.

Section snippets

One-step cascade reactions involving co-factor recycling

The simplest examples of industrial enzyme cascades involve combinations of a biocatalyst with a co-factor recycling enzyme and a co-substrate.

NAD(P)H is a redox cofactor widely used by many enzymes. One of the most frequently studied enzymes to rely on NAD(P)H regeneration are ketoreductases (KREDs), which use NAD(P)H as a co-factor to reduce ketones and aldehydes to their corresponding, typically chiral, alcohols. The resulting NAD(P) co-product is the substrate for a second enzyme, such as

Multi-step cascade reactions involving multiple enzymes

Building on these one-step cascades, several groups have assembled multi-step cascades to further simplify chemical processes by eliminating the need for isolation of reaction intermediates. This often requires solving challenges traditionally associated with combining multiple enzymes, for instance where conditions optimal for one enzyme may not be favorable for others. Engineering multiple enzymes individually to work alongside other enzymes in a cascade enabled their efficient, concerted

Complex cascade reactions involving many enzymatic steps

Fermentations generally start from economically attractive starting materials, however, achieving commercial viability may be challenging as high product yields may be compromised by the complexity of microbial physiology, product toxicity to the host, and cumbersome downstream processing steps. To circumvent such constraints, more complex cascades are being pursued in which metabolic pathways are taken out of the cellular context and run as biocatalytic processes employing sometimes as many as

Conclusions

The drive for sustainable chemical synthesis has contributed to biocatalysis being widely used in the pharmaceutical industry and its fast expansion into the fine chemical sector. Biocatalysis is now viewed broadly as an attractive technology for commercial-scale synthetic chemistry. To further the adoption of biocatalysis, enabling the routine use of enzyme cascades is crucial [49••]. While most examples of industrial-scale biocatalysis have thus far employed only a handful of enzymes in a

Conflict of interest statement

JN, JL, and GH are employees and shareholders of Codexis.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

CRediT authorship contribution statement

Jovana Nazor: Writing - original draft, Writing - review & editing. Joyce Liu: Writing - original draft, Writing - review & editing. Gjalt Huisman: Writing - original draft, Writing - review & editing.

Acknowledgements

The authors would like to thank David Entwistle and Stefan Lutz for their helpful comments and suggestions on the manuscript and Nandhitha Subramanian for her help with the figures.

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