Chapter 25
A Novel Method of Amino Acids in a Cell Factory Motoko Hayashi and Hiroaki Yamamoto Himeji Research Center, Daicel Chemical Industries, Ltd., 1239 Shinzaike, Aboshi-ku, Himeji, Hyogo 671-1283, Japan
A novel method for the synthesis of amino acids is described: deracemization from racemate to a single enantiomer with only one recombinant E. coli. The plasmid harbored 4 genes required for the reaction, which code D–amino acid oxidase, L– amino acid dehydrogenase, formate dehydrogenase, and catalase on a single operon. With this recombinant E. coli, the entire deracemization process was carried out in a single cell, i.e., "cell factory."
Introduction Generally, many steps are needed to synthesize a desired compound. A "one-pot reaction" is a strategy that seeks to achieve sequential reactions in a single reactor. There is no need for purification at each step. Instead, the reaction must be checked, the next compounds are added, and the reaction conditions are adjusted. Enzymatic reactions are attractive methods in organic synthesis because they exhibit high chemo-, regio-, and enantioselectivity. Such high selectivity reduces the need for purification. Another attraction is that such reactions can be carried out under the ambient conditions, i.e., under similar conditions. Thus, the reactions with some enzymes are suitable for use as one-pot reactions. Amino acids are popular starting materials for asymmetric synthesis, (the so-called chiral pool method) because they are natural compounds with chiral centers (1). Since amino acids have common structures, we can plan a process from amino acids regardless of whether they are natural or unnatural. When we © 2009 American Chemical Society
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408 need an unnatural aromatic amino acid, we can easily synthesize it, e.g. by amination of the corresponding α-keto acid. On the other hand, we cannot use this synthetic route for industrial production due to the cost of the corresponding α-keto acid when we need an unnatural amino acid with an alkyl chain (Figure 1). As a result, we select deracemization from a racemic amino acid, where the undesired enantiomer is converted into the desired one. Turner reported one-pot amino acid synthesis by deracemization (2). This process consisted of the
Figure 1. Synthesis of (a) α-keto acid and (b) racemic amino acid, (a) R = Aromatic group: a-Keto acid should be obtained without side reactions. R = Aliphatic group: Aldol condensation should occur since aliphatic aldehyde has a-hydrogen(s). (b) Racemic amino acid is obtained by Strecker reaction.
enantioselective oxidation of D-amino acid to imine by D-amino acid oxidase and the reduction of imine to racemic amino acid by a hydride reducing agent or metal catalyst (Scheme 1). We used an unnatural amino acid, L-norvaline (L-2-aminopentanoic acid, LNva), as a target material. We designed a reaction so that all of the steps of deracemization for the production of L-Nva could be performed using only enzymes (3, 4). We used whole recombinant cells as a biocatalyst, since living cells, which contain enzymes, can multiply by cultivation. In addition, since the
409 NH, ^ R C0 H
oxidation by D-amino acid oxidase
2
Hp
NH reduction [H]
R
C0 H 2
H0
Ο
Λ.C0 H
R
2
2
NH ^ R C0 H
oxidation by L-amino acid oxidase
2
2
Hp
NH
reduction [H]
A.C0 H
R
2
Hp
Ο
.A.C0 H
R
2
Scheme J. Reproducedfrom reference 2. By permission of The Royal Society of Chemistry.
genes coding the necessary enzymes were expressed on a single operon, all of the necessary catalysts could be obtained by a single cultivation. Since all of the steps of deracemization proceeded in a single cell rather than in one pot, we call this approach "a cell factory."
Results and Discussion Screening of Enzymes Several routes are possible for deracemization (Scheme 2). Our enzyme library had one D-amino acid dehydrogenase, two D-amino acid oxidases, six amino acid dehydrogenases, and two amino acid transaminases. Enzyme screening was performed with these enzymes. Escherichia coli was used as a host microorganism for the expression of these genes.
D-Amino acid dehydrogenase /D-Amino acid oxidase C0 H
C0 H
2
2
NAD(P)H Formate dehydrogenase NAD(P)*
Amino acid dehydrogenase NH
2
COjn Route 2 NH,
D-Amino acid dehydrogenase /D-Amino acid oxidase
NH
2
C0 H
COjn
2
C0 H 2
Amino acid transaminase NH
2
C0 H 2
Scheme 2
The enzyme for the first step [conversion of D-Nva to 2-oxopentanoic acid (2-ketovaleric acid, KVA)] might be a D-amino acid dehydrogenase or a Damino acid oxidase. Since the high expression of these enzymes affects the metabolism of D-amino acid and inhibits the synthesis of cytomembrane, they might be toxic for the host. In fact, since transformants that expressed D-amino acid dehydrogenase with high efficiency did not exhibit constant enzymatic activities, D-amino acid oxidase from Candida boidinii (CbDAO) (5) was selected. Fortunately, CbDAO was not inhibited by L-Nva. The second step (conversion of KVA, the product of the first step, to L-Nva) was thought to be transamination with amino acid transaminase and reductive amination with L-amino acid dehydrogenase. Transamination is an equilibrium reaction and L-glutamate is usually used as an amino-donor. If L-aspartate is used as an amino-donor, the resulting oxaloacetate may be decomposed into
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pyruvate, and thus the equilibrium is probably shifted to the right (Scheme 3). In fact, the reaction with transformants that highly expressed amino acid transaminase and L-aspartate as an amino-donor did not show the expected equilibrium shift. In the best result, 17 equiv. of L-glutamate was needed to reach >99% conversion with a branched-chain transaminase from Escherichia coli, which is unfavorable for an industrial process. We also screened amino acid dehydrogenase, including alanine dehydrogenases (AlaDH), leucine dehydrogenases (LeuDH), and phenylalanine dehydrogenase (PheDH). AlaDHs exhibited low activities for KVA. Although LeuDHs had adequate enzymatic activity, a reaction with over 4% substrate did not run to completion due to substrate inhibition or inactivation of the enzyme by a high concentration of substrate. PheDH from Thermoactinomyces intermedius (TiPheDH) (6) had sufficient activity and inhibition or inactivation was not observed. It also showed perfect enantioselectivity, in that the D-form
Scheme 3
was not detected in the reaction. A mutant of formate dehydrogenase from Mycobacterium vaccae (McFDH) which was modified to resist organic solvents was used to regenerate coenzyme NADH (7), since this reaction was essential for reductive amination with L-amino acid dehydrogenase. In summary, three enzymes (CbDAO to oxidize D-Nva, TiPheDH to produce L-Nva, and McFDH to regenerate coenzyme) were selected.
Coexpression of Three Genes Comprising a Single Operon We constructed plasmids that expressed the genes of the above three enzymes. Figure 2 shows maps of these plasmids. pSE420U is a versatile vector that enables the expression of these three genes like an operon (8). The first plasmid contained a CbDAO-gene, an McFDH-gene, and a TiPheDH-gene, in
Figure 2. Maps of the plasmids pSFTPCOJ, pSFTPC02, and pSFTPC03 harbored 3 genes, CbDAO, TiPheDH, and McFDH. CbDAO, D-amino acid oxidase from Candida boidinii; McFDH, formate dehydrogenasefromMycobacterium vaccae; TiPheDH, phenylalanine dehydrogenasefromThermoactinomyces intermedius; P, trc promoter; T(rrnB), rrnB terminator; amp, ampicilli- resistance gene; ori, origin of replication; rop, rop protein gene; laqF, lactose repressor.
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that order, and was named pSFTPCOl. The second contained an McFDH-gene, a CbDAO-gene, and a TiPheDH-gene and was named pSFTPC02. The third contained an McFDH-gene, a TiPheDH-gene, and a CbDAO-gene and was named pSFTPC03. Escherichia coli HB101 was transformed with each plasmid. The McFDH gene was put at the first or second cistron, since it had less molecular activity than the others. The obtained transformants were cultured and the activities of enzymes were determined. Cells reached full-broth under the general conditions, and the activities of the enzymes were expressed with higher efficiency (Table I).
Deracemization with the Three Enzymes We attempted to deracemize DL-Nva with the above recombinant E. coli that coexpressed the three genes. Since the oxidation with D-amino acid oxidase required oxygen, the speed of stirring was an important factor in the reaction: the harder a reaction mixture was stirred, the faster D-Nva was decreased. DO was 0% in the oxidation of D-Nva, and an increase in DO indicated the disappearance of D-Nva. However, the oxidation product, KVA, did not disappear with further prolongation of the reaction. An analysis of the reaction mixture showed that LNVA was produced in 60% yield and KVA was produced in 3% yield. These results suggested that the steric inversion of D-Nva into the L-form occurred only partly, and by-product(s) was formed. When D-Nva was used as a substrate, the total yield of D-, L-Nva, and KVA was only 40%. When Nva and the other materials were stirred with E. coli harboring an empty vector, pSE420U, almost all Nva remained in the mixture. These results may be accounted for by the decomposition of Nva and/or an intermediate by hydrogen peroxide. Hydrogen peroxide is generated along with oxidation by oxidase, and it must be decomposed to increase the yield. The best pH for the reaction was 8, since the reaction was slow below pH 7 and the product yield became low above pH 9. This supports the notion that hydrogen peroxide decomposed Nva and/or an intermediate since it is stable under a basic conditions. In addition, butyrate, which is the oxidation product of KVA with hydrogen peroxide, was detected in the reaction mixture.
Improvement of Transformant Coexpressing Four Genes of Enzymes To increase the yield of L-Nva, two methods were considered. First, the activities of TiPheDH and McFDH could be increased, and the reaction from K V A to L-Nva could be accelerated. Plasmids were constructed with different orders of the genes. Although these plasmids gave different balances of enzymatic activities, the reaction yields of L-Nva were reduced. Thus, the
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Table I. Enzymatic activities of transformants Plasmid pSFTPCOl pSFTPC02 pSFTPC03 pSCCBDOl pSFCPCOl
McFDH
TiPheDH
CbDAO
EcKatE
0.46 0.53 0.63
5.56 3.27 4.07
3.15 0.66 0.75 2.01 2.40
_
-
-
0.63
5.30
-
218 399
N O T E : Units are Units per mg-protein.
decomposition of K V A and/or Nva with hydrogen peroxide was faster than the conversion to L-Nva with TiPheDH and McFDH. Otherwise, pSFTPCOl gave the best balance of enzymatic activities. The other method was the removal of hydrogen peroxide from the reaction mixture. Hydrogen peroxide is an oxidant, and a scavenger may be added to the reaction mixture to remove it. On the other hand, catalase can convert hydrogen peroxide to oxygen. Therefore, E. coli was transformed by the plasmid pSCCBDOl comprised of the genes for D-amino acid oxidase and catalase from E. coli (EcKatE) (9). The enzymatic activities could be measured without disturbing each other (Table I). The fourth gene, catalase, was inserted into the above plasmid pSFTPCOl, and the resulting plasmid was named pSFCPCOl. Its gene was placed in the fourth frame which is furthest from the promoter since it showed high activity (Figure 3). In general, the farther a gene is from the promoter, the lower its expression. Its transformant showed an ash green appearance because of heme in catalase. The activities of the enzymes are shown in Table I. Those of all the enzymes were expressed with high efficiency even in the transformant with pSFCPCOl.
Deracemization in a Cell Factory The deracemization process consists of two steps: oxidation of D-Nva to K V A and reductive amination of K V A to L-Nva. We attempted to carry out these reactions under the same conditions. Under aerobic conditions, L-Nva was obtained in 77% yield with 100% e.e. However, the yield of K V A remained at 21% and prolongation of the reaction did not convert it to L-Nva. These facts indicate that while catalase was effective, L-amino acid dehydrogenase and formate dehydrogenase were not.
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Figure 3. Map of the plasmid pSFCPCOl, which harbors 4 genes, CbDAO, TiPheDH, McFDH, and EcKatE. EcKat E, catalase ΕfromEscherichia coli.
In general, enzymatic reduction prefers anaerobic conditions, since NADH reduced with FDH might be reoxidized by a respiratory chain. A two-step reaction was then carried out. We sought to achieve the oxidation of D-Nva to K V A in the first step under aerobic conditions. Next, aeration was stopped and reductive amination of K V A to L-Nva occurred in the second step. In the first step, L-Nva was obtained in 60% yield, K V A was obtained in 32% yield, and DNva was not detected. The conversion of K V A to L-Nva was achieved in the additional reaction step, and L-Nva was obtained in a final yield of 93% with >99.9% e.e. (8). The time course of deracemization process was shown in Figure 4. The deracemization of Nva was carried out in a single cell that expressed four enzymes. Racemic Nva was incorporated into a cell and L-Nva was released, and this cell acted as an amino acid-producing factory.
Conclusions Deracemization from racemate to a single enantiomer is a simple process because an additional step, such as racemization in kinetic resolution, is not necessary. Deracemization with a single cell is a versatile process because it can be applied to other target compounds with suitable sets of enzymes.
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Time/h Figure 4. Time course of deracemization process. Closed circle, L-Nva; open circle, D-Nva; closed square, KVA. 1st step is indicated the reaction with aeration, and 2nd step is indicated without aeration.
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