Modern Biocatalysis

Advances Towards Synthetic Biological Systems

Gavin Williams, Mélanie Hall

• Publisher: Royal Society of Chemistry
• Year: 2018
• Print ISBN:
  9781782627265
• Edition: 1
• eBook ISBN:
  9781788014533
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

The synergy between synthetic biology and biocatalysis is emerging as an important trend for future sustainable processes. This book reviews all modern and novel techniques successfully implemented in biocatalysis, in an effort to provide better performing enzymatic systems and novel biosynthetic routes to (non-)natural products. This includes the use of molecular techniques in protein design and engineering, construction of artificial metabolic pathways, and application of computational methods for enzyme discovery and design. Stress is placed on current 'hot' topics in biocatalysis, where recent advances in research are defining new grounds in enzyme-catalyzed processes. With contributions from leading academics around the world, this book makes a ground-breaking contribution to this progressive field and is essential reading for graduates and researchers investigating (bio)catalysis, enzyme engineering, chemical biology, and synthetic biology.

Section IV: Biocatalysis for Modern Synthesis

Chapter 12

Self-contained Biocatalysts

Jon D. Stewart^*

University of Florida, Department of Chemistry, P.O. Box 117200, Gainesville, FL 32611, USA

^* Email: [email protected]

12.1 Introduction

While hydrolases, mutases, epimerases and isomerases generally operate without exogenous cofactors, many synthetically important conversions involve redox reactions that demand either a source or destination for electrons. This is particularly the case for nicotinamide-dependent reactions, which demand a continuing supply of NAD(P)⁺ or NAD(P)H to support substrate oxidations or reductions, respectively. Due to their high costs, nicotinamides are almost never used stoichiometrically; instead, provision is made for their in situ regeneration from a sacrificial substrate or by direct coupling to an electrode. For many practical reasons, the former remains much more common, and will be the focus of this review. Before tackling the problem of self-contained redox biocatalysis, the three main strategies for cofactor regeneration are outlined below. These are illustrated schematically, using ketone reduction as an example of a biocatalytic conversion, in Figure 12.1.

Figure 12.1 Schematic illustrations of cofactor regeneration strategies.

12.1.1 The “One-enzyme” Solution

The simplest approach to nicotinamide regeneration requires only a second enzyme-catalyzed reaction that occurs in the complementary redox direction. For those alcohol dehydrogenases (also known as ketoreductases or KREDs) that accept isopropanol/acetone as well as the substrate of synthetic interest, a single enzyme can accomplish both the desired transformation as well as cofactor regeneration (illustrated for a carbonyl reduction in Scheme 12.1). This is a biochemical equivalent to the Meerwein–Pondorf–Verley reduction/Oppenauer oxidation. In addition to requiring only a single biocatalyst, this approach also allows the highly polar nicotinamide cofactor to remain bound to the enzyme active site throughout the reaction, opening the possibility of carrying out these reactions in non-aqueous solvents. Moreover, because acetone is usually the most volatile reaction component, its removal by vacuum or pervaporation can be used to drive the equilibrium position toward the desired direction.

Scheme 12.1 One- and two-enzyme strategies for NAD(P)H regeneration.

One disadvantage of the “one-enzyme” solution to nicotinamide regeneration is that it is only applicable to the subset of NAD(P)⁺/NAD(P)H-dependent reactions that are readily reversible. While carbonyl reductions/alcohol oxidations fall within this scope, Baeyer–Villiger oxidations, cytochrome P450-mediated oxidations, alkene reductions,^†etc., cannot be readily accommodated by this strategy. The other major drawback to the “one-enzyme” approach is that the equilibrium for the desired reaction depends on the thermodynamics and reactant concentration of the sacrificial substrate (usually i-PrOH/acetone). A large excess of the second substrate not only detracts from the economics of the process but can also result in enzyme deactivation.

12.1.2 The “Two-enzyme Solution”

Using a second enzyme—one tailored for nicotinamide cofactor regeneration—in addition to the one catalyzing the desired synthetic reaction eliminates the drawbacks of the “one-enzyme” approach (Scheme 12.1). Because the two enzymes catalyze different reactions, there is no requirement that their substrate specificities overlap. This also allows for choosing sacrificial substrates based on their costs, reduction potentials (to drive the overall reaction equilibrium in the desired direction without resorting to large molar excesses) and lack of detrimental effects on enzyme activity and stability. In addition, because a single cofactor regeneration enzyme/substrate pair can be coupled to many different synthetic transformations, process optimization (see Chapter 17) can be simplified since reaction conditions for only one of the two reactions must be developed. The two major drawbacks of the “two-enzyme” strategy are the additional costs of the second (cofactor regeneration) enzyme and the requirement that it has good activity and stability under the reaction conditions demanded by the synthetically important enzyme. In practice, these requirements have been easily met by a variety of cofactor regeneration enzymes.

12.1.3 Whole Microbial Cells

Whole cells (usually microbial) are a logical extension of the “two-enzyme” strategy for cofactor regeneration (for recent reviews, see ref. ^4–7). In favorable cases, the synthetically important enzyme is produced directly by the microbial cells; otherwise, cloning and molecular biology strategies are used for its heterologous expression in a suitable microbial host. In practice, even when native cells produce the enzyme of interest, they are re-engineered to increase its relative concentration, which often increases the volumetric productivity of the process. When whole microbial cells are used as the biocatalyst, a sacrificial substrate is nearly always included whose metabolism provides a continuous supply of the cofactor required by the synthetically important enzyme.⁸ In most cases, the sacrificial substrate is a carbon source, e.g., glucose, sucrose, glycerol, etc., that also supports growth of the organism. In this scheme, the “cofactor regeneration enzyme” actually encompasses all of the enzyme-catalyzed, catabolic processes involved in carbon source utilization such as glycolysis and the citric acid cycle.² There are two key advantages to using whole cells as the “cofactor regeneration enzyme.” First, the enzyme that catalyzes the synthetically useful transformation, the cofactor regeneration system and the required cofactor are all supplied by the cells, which minimizes their costs. Second, because entire metabolic processes are available for carbon source metabolism, the sacrificial substrate can be used completely. For example, glucose oxidation by whole cells yields 10 NAD(P)H when consumed by glycolysis and the citric acid cycle as compared to 1 NAD(P)H when oxidized by glucose dehydrogenase. These advantages, however, must be balanced against some important drawbacks. First, because entire metabolic networks must be preserved during the biotransformation reaction, it is almost always essential that whole cells remain intact. High substrate concentrations desirable for synthetic purposes often cause cell membrane leakage and/or destruction. In addition, the presence of many enzymes in the reaction can lead to side-products and/or undesired stereoisomers. It is for this reason that the synthetically important enzyme is often overproduced so that its catalytic activity dominates, even when competing with all others in the cell. In some cases, this may make cofactor regeneration rate-limiting. Membrane transport of the substrate and/or product can also be partly or completely rate-limiting. Finally, the larger quantity of extraneous biomass inherent in using whole cells can complicate downstream processing and product isolation (see Chapter 17). For all of these reasons, the use of unmodified whole cells for redox biocatalysis has declined in popularity as other cofactor regeneration schemes have become better developed.

One additional difficulty with using whole cells is that their growth phase can have major impacts on the efficiency of the biocatalytic process. Specifically, it is commonly observed that activel...

Table of contents

• Cover
• Title
• Copyright
• Contents
• Section I: Accessing New Enzymes
• Section II: Understanding and Engineering Enzymes
• Section III: Enzymes from Secondary Metabolism
• Section IV: Biocatalysis for Modern Synthesis
• Section V: Applied Biocatalysis
• Subject Index