Whole cell biotransformation for reductive amination reactions

Abstract

Whole cell biotransformation systems with enzyme cascading increasingly find application in biocatalysis to complement or replace established chemical synthetic routes for production of, e.g., fine chemicals. Recently, we established an Escherichia coli whole cell biotransformation system for reductive amination by coupling a transaminase and an amino acid dehydrogenase with glucose catabolism for cofactor recycling. Transformation of 2-keto-3-methylvalerate to l-isoleucine by E. coli cells was improved by genetic engineering of glucose metabolism for improved cofactor regeneration. Here, we compare this system with different strategies for cofactor regeneration such as cascading with alcohol dehydrogenases, with alternative production hosts such as Pseudomonas species or Corynebacterium glutamicum, and with improving whole cell biotransformation systems by metabolic engineering of NADPH regeneration.

Reductive Whole Cell Biotransformation: An Upcoming Tool of Biocatalysis

Biocatalysis has come more and more into focus as a green and sustainable technology for the functionalization of chemical compounds complementing and sometimes replacing established chemical synthetic routes. For the synthesis of the product of interest, chemical catalysts may be replaced by biological catalysts, which often make use of the exquisite stereoselectivity of enzymes. Biocatalysts comprise either cell-free systems with isolated enzymes or whole cells. Isolated enzymes are preferably used for hydrolytic or isomerization reactions since they are independent of costly cofactors.¹ For reactions requiring cofactor regeneration, e.g., redox reactions, whole cell systems are preferable since, e.g., redox equivalents may be regenerated by cellular metabolism. Whole cell systems also offer the advantage of (1) providing the native environment, e.g., with respect to pH to keep the enzymes stable and (2) avoiding costly isolation and purification of reaction biocatalysts, intermediates, and cofactor addition. Whole cell systems using genetically amenable organisms such as E. coli, C. glutamicum, or the yeast Saccharomyces cerevisiae may be optimized for cofactor provision by genetic engineering, which will be discussed in detail later.

Whole cell biotransformations employing ATP as an energy source have been implemented less often because recycling reactions might be too complex for economical purpose. For conversion of Xanthosine monophosphate (XMP) obtained from fermentation with Corynebacterium ammoniagenes to Guanosine monophosphate (GMP), E. coli cells overproducing ATP-dependent GMP synthetase can be used and ATP can be regenerated from glucose via glycolysis by C. ammoniagenes cells.² Polyphosphate kinase, which in C. glutamicum and other bacteria also phosphorylates nucleoside diphosphates using inexpensive polyphosphate as a phospho-donor yielding NTPs³ was applied, e.g., for ATP recycling biotransformation of amino acids to dipeptides using l-amino acid ligase Lal.⁴

Redox reactions have been implemented as whole cell biotransformations employing a variety of systems for redox cofactor recycling. Also, cell-free systems have been developed by cascading redox reactions for redox cofactor recycling, e.g., for reduction of ketones to alcohols, respectively, or racemization via ketone intermediates^5,6 or employing electrochemical, chemical, or photochemical reactions.⁷ Here, most prominently four cofactor recycling types are highlighted due to their redox self-sufficient character provided by enzymes or the cellular metabolism.

First, a single enzyme may be used as substrate-coupled recycling system (Fig. 1A) when, e.g., reduction of a substrate such as cinnamaldehyde to the respective product cinnamyl alcohol by yeast alcohol dehydrogenase is coupled to oxidation of a second substrate such as propanol or ethanol to the respective aldehydes by the same enzyme.⁸

Figure 1. Enzyme cascading for cofactor recycling in reductions and reductive aminations. Cascading substrate conversion and cofactor recycling by a single enzyme (A) or by two sequential reactions (B). Coupling of a reduction reaction with glucose driven cofactor regeneration by the cellular metabolism directly (C) or via an electron transfer mediator such as ferredoxin (D). Coupling a transaminase reaction and an amino acid dehydrogenase to either an alcohol dehydrogenase (E) or cellular glucose catabolism (F).

Second, two redox reactions generating or converting the same intermediate may be coupled with cofactor recycling (Fig. 1B). Based on this principle a general strategy for racemization of secondary alcohols under mild reaction conditions was developed by coupling a pair of stereocomplementary alcohol dehydrogenases, e.g., for racemization of ®- and (S)-2-octanol.⁹ Redox cofactor recycling may involve coupling of different types of redox reactions, e.g., an alcohol dehydrogenase and an amino acid dehydrogenase. For conversion of d-mandelic acid to l-phenylglycine the oxidation of d-mandelic acid to phenylglyoxylate by a d-specific alcohol dehydrogenase was coupled to the reductive amination of phenylglyoxylate to l-phenylglycine by a l-specific amino acid dehydrogenase.⁵ As extension, addition of mandelate racemase enabled deracemization of d,l-mandelic acid to l-phenylglycine.⁵ For coupling a number of further oxidation reactions are typically used, e.g., oxidation of formate, phosphite, glucose, glucose 6-phosphate, alanine, and glutamate by the respective dehydrogenases^10,11

Redox factor regeneration may involve coupling of a reduction reaction to cellular metabolism in a whole cell biotransformation since reducing equivalents are generated in glycolysis (Fig. 1C), e.g., coupling reduction of methyl acetoacetate to (R)-methyl 3-hydroxybutyrate with cellular glucose catabolism.¹²

Third, for the use of different cofactors (NADH, FMN, reduced ferredoxin, etc.) in the enzyme cascade, mediating enzymes are required for electron transfer (Fig. 1D). Monooxygenases that use molecular oxygen to insert one oxygen atom into a substrate while the second oxygen atom is reduced to water with electrons derived from NADH or NADPH can efficiently be coupled to cellular metabolism for redox factor recycling. Highly efficient styrene epoxidation by recombinant E. coli cells carrying the styrene monooxygenase (SMO) genes styAB from Pseudomonas sp. strain VLB120 driven by cellular glucose catabolism may serve as an example.^13,14 Some cytochrome P450 monooxygenases require ferredoxin and ferredoxin reductase for electron transfer from NAD(P)H. Thus, e.g., hydroxylation reactions based on cytochrome P450 monooxygenase from Sphingomonas sp. recombinant E. coli overproducing ferredoxin and ferredoxin reductase besides the cytochrome P450 monooxygenase enzyme were necessary.¹⁵

Fourth, two redox reactions generating or converting two different intermediates—that, in turn, need to be interconverted by a transfer reaction—may be coupled (Fig. 1E). Redox self-sufficient amination of primary alcohols—e.g., coupled oxidation of an alcohol such as hexanol—to the respective aldehyde with reductive amination of another aldehyde, such as pyruvate, to l-alanine requires an aminotransferase for interconversion of hexanal and l-alanine to pyruvate and the final product hexylamine.¹¹ Similarly, in a whole cell system we have coupled an aminotransferase reaction with reductive amination of pyruvate to l-alanine and glucose catabolism (Fig. 1F) by metabolically active E. coli cells to synthesize l-isoleucine from 2-keto-3-methyvalerate.¹⁶

Hosts in Whole Cell Biotransformation

Among the broad spectrum of enzymes from microorganisms hydrolases like lipases, oxidoreductases like oxygenases or dehydrogenases and lyases like hydratases are the most commonly used enzymes in industrial biotechnology (63%, 25%, and 5%, respectively).¹⁷ Industrial biocatalytic cell-free systems can be found in the production of the low-calorie sweetener aspartame catalyzed by a Bacillus subtilis thermolysin (DSM, Netherlands), in the synthesis of semisynthetic β-lactame antibiotics catalyzed by acylases (DSM, Netherlands) and in the reductive amination of trimethylpyruvate to l-tert-leucine by a leucine dehydrogenase coupled with a formate dehydrogenase for cofactor recycling (Evonik Industries AG, Germany). For instance, the synthesis of L-Dopa by a tyrosine-phenol lyase in immobilized whole cell Erwinia herbicola (Ajinomoto, Japan) or the nicotinamide production by a nitrile hydratase in immobilized Rhodococcus rhodochrous (Lonza, Switzerland) are industrially relevant biotransformations in whole cells.

For whole cell biotransformation processes the choice of the production host is important. An ideal host is genetically amenable, grows fast in simple media, allows high enzyme production levels, is recalcitrant to a wide range of substrates and products, allows operation in two-phase systems, and is compatible to subsequent downstream processing regimens. For coupling of the biotransformation reactions to the host’s cellular metabolism, the ideal host allows easy import of substrates and export of products and possesses no or reduced endogenous catalytic activity toward the substrates, intermediates, and products of the biotransformation.

Due to the ease of genetic manipulation and high levels of enzyme overproduction, E. coli is the first choice as host for whole cell biotransformations.

The eukaryotic bakers’ yeast S. cerevisae was screened for reductases and thus became the most common whole cell biocatalyst for reduction of β-keto-esters to chiral alcohols.¹⁸ Individual reductases of this microorganism are highly stereoselective and mostly NADPH-specific but as the yeast produces several reductases with the same substrate specificity but different stereoselectivity, the production of enantiomeric pure products is important, e.g., pharmaceuticals became a challenging task for yeasts’ scientists.¹⁸

Pseudomonas is an attractive biotransformation host due to its recalcitrance to many solvents used in biocatalytic two-phase systems like octanol and toluene, but also to toxic substrates and products, which is partly due to solvent efflux pumps such as SrpABC from Pseudomonas putida.¹⁹

Transport through the cell wall barrier is critical, e.g., for the often large and lipophilic building blocks in pharmaceutical industry such as hydroxylated long chain fatty acids.²⁰ Heterologous production of, e.g., the fatty acid uptake system of Pseudomonas oleovorans in E. coli, improved hydroxylation of pentadecanoic acid.²⁰ Alternatively, cell permeabilization, e.g., by the bacteriostatic antimycobacterial drug ethambutol, improved cyclohexanone whole cell biotransformation by C. glutamicum.²¹ This Gram-positive bacterium is attractive as whole cell catalyst as its mycolic acid haboring cell wall protects against toxic compounds. It has a history of five decades of safe use in the food and feed industries for the annual production of 2,930,000 tons of l-glutamate and 1,950,000 tons of l-lysine (Ajinomoto, Inc., available at: http://www.ajinomoto.com/en/ir/pdf/FY13Q1_data_E.pdf, cited 05 September 2013).

Metabolic Engineering of Biotransformation Hosts

The reductive whole-cell-based biotransformation processes for production of stereoselective chemical and pharmaceutical compounds, amino acids, chiral alcohols, and fine chemicals, have developed rapidly through process optimization and engineering of cellular metabolic pathways.^22,23 Metabolic engineering focused on core biosynthetic pathways, cofactor-regeneration systems, uptake and export systems, and further optimization of the cellular interaction. For whole cell biotransformation involving redox reactions, regeneration of reduction equivalents, mostly the nicotinamide adenine dinucleotide coenzymes NADH and NADPH, is critical.²⁴

With E. coli, different strategies for cofactor regeneration have been applied, e.g., cascading of reductive biotransformations with oxidation of formate to carbon dioxide by NAD-dependent formate dehydrogenase. Glucose dehydrogenase (GDH) from Bacillus megaterium enhanced NADPH-dependent bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE).²⁵

Coupling to cellular metabolism for cofactor regeneration can be optimized by metabolic engineering, e.g., for NADPH generation. Since two molecules of NADPH are generated in the oxidative pentose phosphate pathway (PPP), overproduction of glucose-6-phosphate dehydrogenase (G6PDH) in engineered E. coli improved ε-caprolactone production.²⁶ Alternatively, the competing pathway glycolysis may be abrogated by deletion of the gene of the first glycolytic enzyme phosphoglucose isomerase (pgi) such that each imported glucose molecule is catabolized via the oxidative PPP before entering glycolysis at the levels of fructose 6-phosphate and triosephosphates (Fig. 2A). The subsequent increase of NADPH regeneration improved production of two polyphenols, leucocyanidin and catechin, from dihydroquercetin.²⁷ Extending this concept, the oxidative PPP was partially cyclized by deletion of the genes for phosphofructokinases pfkA and pfkB, which entails that only glyceraldehyde-3-phosphate produced in the PPP enters glycolysis while fructose 6-phosphate re-enters the oxidative PPP (Fig. 2B). This partial cycling enhanced the reduction of the prochiral ketoester methyl acetoacetate (MAA) to the chiral hydroxy ester (R)-methyl-3-hydroxybutyrate (MHB).²⁸

Figure 2. Metabolic engineering of host cell metabolism for improved biotransformations. NADPH regeneration can be improved by forcing glucose catabolism to the oxidative pentose phosphate pathway (PPP) by deletion of pgi, the gene for phosphoglucoisomerase (A) or by deletion of the genes for phosphofructokinase genes pfkA and pfkB resulting in partial cyclization of the PPP (B). Improved biotransformation capacity resulted from various gene deletions primarily encoding tricarboxylic acid enzymes (C).

In order to investigate the metabolic redox capacity of recombinant E. coli for asymmetric styrene epoxidation deletion, mutants lacking genes relevant for the NADH regeneration were compared in silico and in vivo according to NADH regeneration rates.¹³ Based on this approach, several genes that are directly involved in NADH regeneration were selected to assess their potential to increase reductive amination of 2-keto-3-methylvalerate to l-isoleucine¹⁶ (Fig. 2C).

Amino Acid Production

Amino acids are commercially important as they are widely used as additives in food, feed, pharmaceuticals, cosmetics, polymer, and other industries with an annual market growth rate of 5–7% worldwide.²⁹ Natural proteinogenic amino acids are mostly produced from sugars by fermentation.⁴ For the production of non-natural amino acids and their derivatives, whole cell biotransformations can be employed. Non-natural amino acids find application as synthons for chemical synthesis of active pharmaceutical ingredients (APIs) and as orthogonal modules for genetic code expansion in synthetic biology.³⁰

Enzyme-catalyzed amino acid production may proceed via ammonium addition to alkenes by ammonia lyases.³¹ Commercial production of l-aspartate is based on aspartate ammonia lyase.³² This enzyme adds ammonium to the double bond of fumaric acid to yield l-aspartate and, therefore, does not depend on reduction equivalents such as NAD(P)H. The aspartate ammonia lyase process is also the basis for commercial l-alanine production since the l-aspartate obtained from fumarate can be decarboxylated to l-alanine by l-aspartate-β-decarboxylase.³²

Transamination or reductive amination of keto acids may be pursued by enzyme catalysis or by whole cell biotransformation. Amino acid dehydrogenases catalyze reductive amination using ammonium in an NAD(P)H-dependent manner. These enzymes are rather substrate specific, such as l-alanine dehydrogenase, l-glutamate dehydrogenase or l-phenylalanine dehydrogenase.³³ l-leucine dehydrogenase, e.g., has been used for production of l-tert-leucine and l-neopentylglycine.³⁴ Since amino acid dehydrogenases depend on NAD(P)H, cofactor recycling is required by coupling to formate dehydrogenase.³⁴

Transaminases, on the other hand, are often active with a broad spectrum of keto acids as substrates, but they require stoichiometric supply of an amino acid as amino group donor. Amino acid dehydrogenases may be coupled with transaminases to enable reductive amination of many keto acids. For example, we have described an E. coli whole cell biotransformation system for reductive amination of 2-keto-3-methylvalerate (KMV) to the amino acid l-isoleucine¹⁶ by coupling endogenous l-alanine dependent transaminase AvtA with NADH-dependent l-alanine dehydrogenase from B. subtilis. Recycling of the cofactor NADH was ensured by glucose catabolism of the host E. coli. Thus, the E. coli host which produces heterologous NADH-dependent l-alanine dehydrogenase and catabolizes glucose may serve as a chassis for a wide range of reductive amination reactions by coupling to the respective transaminase.

This concept of reductive amination, however, is dependent on the provision of the keto acids, e.g., by chemical synthesis, as precursors of the desired amino acids. Both the non-natural keto acid or the non-natural amino acid may be toxic to the reductive amination host cell as observed for the keto acids ketoisocaproate and ketoisovalerate,¹⁶ therefore, use of alternative hosts such as C. glutamicum or Pseudomonas may be needed. Moreover, uptake of the non-natural keto acid into the host cell and export of the produced non-natural amino acid out of the cell may slow or even preclude effective reductive amination and may necessitate using alternative hosts or co-expression of transport genes. Currently, redox factor recycling only occurs by glucose catabolism. Besides coupling to favorable oxidation reactions such as formate dehydrogenase which drive the reaction to completion since the generated carbon dioxide gases out, the reaction may start with a hydroxy acid instead of the keto acid. Using enzyme preparations, a transaminase/l-alanine dehydrogenase pair has been coupled to an alcohol dehydrogenase, which recycles the reduction equivalent and provides the precursor for the transaminase.¹¹ This three-enzyme-cascade catalyzes redox-neutral amination of an alcohol to the amine. It should be possible to transfer this principle to a whole cell biotransformation setup for amination of hydroxyl acids to amino acids.

Outlook

Our demonstration of efficient transamination in an E. coli whole cell biotransformation system with the enzyme couple alanine dehydrogenase AlaDH and transaminase AvtA provides a basis for further applications. While shown exemplarily for amino acid production, it should be feasible to transfer this approach more broadly to production of amine functionalized chemicals. Redox cofactor regeneration via glucose catabolism by the biotransformation host E. coli was improved by genetically engineering its metabolism. As discussed here, alternative redox cofactor recycling systems—e.g., by coupling to oxidation reactions catalyzed by glutamate dehydrogenase and formate dehydrogenase—may be employed to enhance the described whole cell biotransformation system. Moreover, reductive amination by whole cell biotransformation may involve alternative hosts, such as the more solvent resistant pseudomonads or C. glutamicum, which has been used safely in food and feed industries for more than 50 years, since these alternative hosts are also amenable to genetic and metabolic engineering.

Glossary

Abbreviations:

G6P

glucose-6-phosphate

6PG

6-phosphogluconase

P5P

pentose-5-phosphate

F6P

fructose-6-phosphate

T3P

triose-3-phosphate

pgi

phosphoglucoisomerase

pfkA

phosphofructokinase 1

pfkB

phosphofructokinase 2

zwf

glucose-6-phosphate dehydrogenase

gnd

6-phosphogluconate dehydrogenase

dld

d-lactate dehydrogenase

ldhA

d-lactate dehydrogenase

adhE

acetaldehyde dehydrogenase

pntAB

subunit of pyridine nucleotide transhydrogenase

nuoG

subunit of NADH dehydrogenase I

sucC

subunit of succinyl-CoA synthetase

aceA

isocitrate lyase

fumA

fumarase A

mdh

malate dehydrogenase

mqo

malate:quinone oxidoreductase

MQ

menaquinone

MQH₂

menaquinol

10.4161/bioe.27151

Lorenz E, Klatte S, Wendisch VF. Reductive amination by recombinant Escherichia coli: Whole cell biotransformation of 2-keto-3-methylvalerate to l-isoleucine. J Biotechnol. 2013;168:289–94. doi: 10.1016/j.jbiotec.2013.06.014.