Exploration of Transaminase Diversity for the Oxidative Conversion of Natural Amino Acids into 2-Ketoacids and High-Value Chemicals

Article abstract of DOI:10.1021/acscatal.0c01895

The use of 2-ketoacids is very common in feeds, food additives, and pharmaceuticals, and 2-ketoacids are valuable precursors for a plethora of chemically diverse compounds. Biocatalytic synthesis of 2-ketoacids starting from l-amino acids would be highly desirable because the substrates are readily available from biomass feedstock. Here, we report bioinformatic exploration of a series of aminotransferases (ATs) to achieve the desired conversion. Thermodynamic control was achieved by coupling an l-glutamate oxidation reaction in the cascade for the recycling of the amine acceptor. These enzymes were able to convert a majority of proteinogenic amino acids into the corresponding 2-ketoacids with high conversion (up to 99percent) and atom-efficiency. Furthermore, this enzyme cascade was extendable, and one-pot two-step processes were established for the synthesis of d-amino acids and N-methylated amino acids, achieving great overall conversion (up to 99percent) and high ee values (>99percent). These developed enzymatic methodologies offer convenient routes for utilizing amino acids as synthetic reagents.

Full text of DOI:10.1021/acscatal.0c01895

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Letter
Exploration of transaminase diversity for the oxidative conversion
of natural amino acids into 2-ketoacids and high-value chemicals
Tao Li, Xuexian Cui, Yinglu Cui, Jinyuan Sun, Yanchun Chen, Tong Zhu, Chuijian Li, Ruifeng Li, and Bian Wu
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.0c01895 • Publication Date (Web): 30 Jun 2020
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Exploration of Transaminase Diversity for the Oxidative
Conversion of Natural Amino Acids into 2-Ketoacids and
High-Value Chemicals
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Tao Li1,2, Xuexian Cui1,2, Yinglu Cui1, Jinyuan Sun1, Yanchun Chen1,2, Tong Zhu1,2,
Chuijian Li1, Ruifeng Li1,2, Bian Wu1*
1 CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key
Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, PR China
2 University of Chinese Academy of Sciences, Beijing, 100101, PR China.
*Corresponding Author
Bian Wu*
Email Address: wub@im.ac.cn
Telephone: +8610-64806035
Fax: +8610-64806035
ORCID: 0000-0002-6524-2049
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ABSTRACT:
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The use of 2-ketoacids is very common in feeds, food additives and
pharmaceuticals, and 2-ketoacids are valuable precursors for a plethora of chemically
diverse compounds. Biocatalytic synthesis of 2-ketoacids starting from L-amino acids
would be highly desirable, since the substrates are readily available from biomass feedstock.
Here, we report bioinformatic exploration of a series of aminotransferases (ATs) to achieve
the desired conversion. Thermodynamic control was achieved by coupling a L-glutamate
oxidation reaction in the cascade for the recycling of the amine acceptor. These enzymes
were able to covert a majority of proteinogenic amino acids into corresponding 2-ketoacids
with high conversion (up to 99%) and atom-efficiency. Furthermore, this enzyme cascade
was extendable, and one-pot two step processes were established for the synthesis of D-
amino acids and N-methylated amino acids, achieving great overall conversion (up to 99%)
and high e.e. values (> 99%). These developed enzymatic methodologies offer convenient
routes for utilizing amino acids as synthetic reagents.
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KEYWORDS: amino acids, 2-ketoacids, enzymatic cascade, transaminase, bioinformatic
enzyme discovery
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Given the finite nature of petroleum, the biosynthetic production of chemical
compounds from sustainable biomass is receiving remarkable attention.1 In the last decade,
innovations in synthetic biology have enabled the industrial production of various fuels
and platform chemicals from renewable resources.2 Among those bio-based routes, L-
amino acids fermentation had exceptional success and become a huge industry.3 The
worldwide production of amino acids is at 8.5 million metric tons in 2017. Traditionally,
L-amino acids have been applied in their native forms.3 As the manufacturing cost of L-
amino acids continues to decline,4 the demands for using L-amino acids as raw material to
produce value-added products are increasing.5 For instance, L-alanine has been
successfully utilized to produce the commodity chelating agent methylglycinediacetic acid
(Trilon® M Liquid) by BASF.
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The penultimate intermediates of the L-amino acids biosynthetic pathway are 2-
ketoacids, which play an important role in human and animal metabolism, and are widely
used as medicines (2-ketoisocaproate, chronic glomerulonephritis6 α-keto-γ-
;
methylthiobutyric acid, anti-cancer drug7), food-feed additives (pyruvate, nutraceutical
with annual worldwide production about 400 tons8 2-ketoisovalerate, feed of animal9) and
;
agricultural products (indole-3-pyruvic acid and phenylpyruvate,10 precursor of indole-3-
acetic acid with the market size of 10.7 million dollars in 2016). The 2-ketoacids are also
attractive from a synthetic standpoint as follow-up transformations can grant access to a
multitude of synthons,11,12 e.g., aldehydes (via decarboxylation),13 hydroxyl acids (via
reduction),14 and D-amino acids (via transamination).15 The biosynthetic pathway for 2-
ketoacids is similar to pathways for the corresponding L-amino acids.16 However,
fermentative production of 2-ketoacids is limited to laboratory research due to several
restrictions, such as low cell growth rate associated with accumulation of 2-ketoacids, the
lack of efficient transporters, and the instability of 2-ketoacids inside the cell.17,18 To date,
most 2-ketoacids have been synthesized by chemical processes, including the hydrolysis
of acyl cyanide, hydrolysis of azalactones, and oxidation of 2-keto aldehydes.11,19
Generally, these methods require expensive catalysts or special starting compounds, which
results in high production costs and associated environmental hazards.
Enzymatic transformation of natural L-amino acids is an alternative approach for
2-ketoacids production that has prominent advantages for sustainability and economical
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fashion (Table S1).20 With this approach, four classes of enzymes can be used, namely,
amino acids dehydrogenase (ADH),21 L-amino acids oxidase (LAAO),22 L-amino acids
deaminase (LAAD)23 and aminotransferase (AT).24 The use of ADH is not cost-effective
because an expensive cofactor is required, and the reaction tends to produce amino acids
from 2-ketoacids rather than the reverse process.25 LAAO catalyzes the oxidative
deamination of L-amino acids into corresponding 2-ketoacids; the re-oxidation of reduced
cofactor FADH2 by molecular oxygen then generates hydrogen peroxide.22 However, the
broad-spectrum LAAOs are difficult to express in recombinant hosts, and the microbial
counterparts have a strict substrate spectrum.26 LAAD catalyzes similar reaction as LAAO,
but the electrons of the reduced cofactor are transferred to a cytochrome. LAAD is
considered a promising alternative to LAAO for biotechnological applications.27
Nevertheless, it is a membrane-bound protein and requires a physiological membrane as
the electron acceptor.27 In addition, only a few LAADs are found in the Proteus species,23
and such a limited gene pool severely hinders the discovery of new enzymes with an
alternative substrate specificity or enhanced robustness. Finally, ATs are ubiquitous
enzymes found in all organisms and are broadly used as promising biocatalysts in the
organic synthesis of chiral amino compounds,24 but the application of AT for the
preparation of 2-ketoacids has rarely been studied. The absence of literature reporting AT
activities for deamination reactions suggests that their potential has been simply
overlooked, particularly in cases in which the amine substrates are less expensive
compounds compared to the corresponding ketones or aldehydes.
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In this study, we aimed to design and construct an ATs-based enzymatic route for
the conversion of L-amino acids into 2-ketoacids and related high-value chemicals. The
major obstacle that hinders the development of this process is the neutral equilibrium
position of most AT reactions (the equilibrium constant for the conversion of L-amino
acids into 2-ketoacids and vice versa with ATs is close to 1). To achieve good conversion,
it was demonstrated that removal of the coproduct by enzymatic conversions or
spontaneous reactions could facilitate an equilibrium shift.28 However, these strategies are
still not ideal because of the need for another ketoacid in excess as the amino acceptor.
This prompted us to consider a different approach in which the coproduct of the AT
reaction is not sacrificed but converted back to the cosubstrate. To this end, L-glutamate
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oxidase (LGOX) was selected as the cosubstrate recycling enzyme because it happens to
catalyze the conversion of L-glutamate (coproduct of AT reaction) to 2-ketoglutarate
(cosubstrate of AT reaction).29 Coupling AT with oxidase can be applied for
deracemization of amines30, developing biosensors31, 32 and colorimetric AT assays.33-35
Since the produced hydrogen peroxide would severely affects overall conversion, catalase
was also included in the cascade to remove peroxide (Scheme 1). In theory, the overall net
reaction of the designed cascade is deamination of L-amino acids at the expense of oxygen,
and only catalytic amounts of L-glutamate are necessary to kick-start the reaction.
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Scheme 1. The enzymatic cascade for the synthesis of 2-ketoacids from L-amino acids.
The proof-of-concept experiment was performed with the synthesis of 2-
ketoisovalerate with branched-chain amino acid aminotransferase from Escherichia coli
(EcBCAT), LGOX from Streptomyces species29 and catalase from Novozyme. In the
presence of all three enzymes, 100 mM L-valine was converted to 2-ketoisovalerate
effectively within 5.5 h, with a product conversion up to 98% (Figure S1). This result
demonstrated that coupling LGOX and catalase with ATs successfully shifted the
unfavourable equilibrium of ATs towards the desired direction, rendering the atom-
efficient production of 2-keto acids.
Encouraged by the initial success, we decided to develop a complementary enzyme
toolbox from the rich source of ATs to cover all natural L-amino acids substrate. ATs
belong to the large and diverse group of pyridoxal 5’-phosphate (PLP)-dependent enzymes,
with more than 370,000 amino acid sequences of ATs stored in the Uniprot database and
more than 800 structures available in the PDB Bank. Despite plenty of AT gene sequences
available, the spectrum of natural L-amino acids has not been fully annotated. Thus, we
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performed extensive exploration of the sequence space and structural information from AT
databases. Multiple sequence alignment analyses, which were conducted with the Protein
Family Database (Pfam), and structure/sequence-function relationships analyses36 suggest
that ATs are distinguished with six classes. Representatives of ATs related to amino acids
from class I and II are AlaATs, AspATs and ARATs, whereas BCATs belong to class IV.
Since functional evolution of proteins often occurs by branching out from an
existing family to a neighbourhood,37,38 the evolutionary relationships between sequences
in class I and II were examined, which may provide a clue for the identification of novel
ATs with different substrate specificities. Primary sequence analysis indicated that AspATs
and ARATs share a high sequence similarity, whereas AlaATs are divergent. This
discrepancy characterized the collected AT sequences in class I and II into two subgroups
(AspAT/ARAT subgroup and AlaAT subgroup). To scatter AspATs/ARATs and AlaATs
in a sequence space, 2-dimensional coordinates were assigned to each sequence by scoring
them with hidden Markov models (HMM) profiles (Figure 1A). The sequences with biased
scores towards AspAT/ARAT or AlaAT seemed to be typical AspATs/ARATs or AlaATs.
On the other hand, the sequences with scores similar to both subfamily profiles were
predicted as evolutionary intermediates, which may lead to a promiscuous substrate
specificity. Based on the distribution of sequences, 15 ATs belonging to class I and II were
selected as potential catalysts (Table S3).
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For the BCATs, the sequences and structures were nonhomologous to the previous
subgroups.39,40 As more crystal structures of BCATs were solved, the structure-based
method was performed for mining novel BCATs. Bornscheuer et al. identified the
sequence motif of PLP-dependent fold type IV subfamily based on the comprehensive
analysis of the structure/sequence-function information. The key amino acids of BCATs
for substrate binding were predicted to make a distinction with (R)-ATs.41 Specifically, the
107-XGX-109 motif located in the inter-subunit loop was involved in the hydrophobic
surface of the large active site pocket and was suggested to determine the substrate
specificity of BCATs (Figure 1B).41 Therefore, the sequences of irredundant BCATs were
scattered by searching the diversity of positions 107 and 109. The sequences that matched
the motif (Y(I/L/V)RP(X)9GX) can be divided into 74 clusters (Figure 1B). Subsequently,
these clusters were further classified into 9 groups by the hydropathy index of the amino
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acids. The hot spot of group 1 indicated that most of the 107 and 109 residues were
hydrophobic amino acids (I, L, V, M), which are typical BCATs. For the other 8 groups, a
phylogenetic tree was constructed, and the representative sequence was selected for further
analysis (Figure S2-S8, Table S4). Collectively, 12 BCATs were chosen for further
identification.
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Figure 1. The bioinformatic procedure for rationally mining novel ATs. A: Overall
analysis scheme for scattering sequences from class I and II by subfamily HMM profiles.
“in” and “jn” mean there is a profile analysis score of sequence n against the AspAT/ATAR
profile and AlaAT profile, respectively. B: Overall analysis flowchart for scattering BCATs
and the sequence diversity of screened BCATs in positions 107 and 109 (the numeration
of EcBCAT). C: The activities of screened ATs toward 18 L-amino acids.
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In the next step, the selected 27 ATs were heterologously expressed in E. coli and
then underwent His-tag purification. Most of the ATs could be expressed in sufficient
amounts (except AlaAT7, ATI2, and BCAT3) (Table S6). The purified ATs were examined
concerning activity and specificity towards 18 L-amino acids except for L-proline and L-
glutamate (Figure 1C, Table S7), using 2-ketoglutarate as the amine acceptor. Fortunately,
the ATs obtained from gene mining displayed complementary specificity and were able to
pair all L-amino acid substrates with 2-ketoglutarate together. It should be noted that the
enzymes screened in class I and II exhibited typical AspATs/ARATs or AlaATs features,
especially for AlaATs, the sequences with higher scores to the subfamily profile were
found to have higher activities toward L-alanine and relative strict substrate specificity.
However, some AlaATs also showed activity toward polar residues. Similar cases obtained
in the BCATs (BCAT7, BCAT8, and BCAT9) exhibited significantly expanded substrate
spectrum toward a set of aromatic and polar amino acids, inferring that the substitution of
107 residue with hydrophilic amino acids (proline, aspartate, arginine) in the characteristic
motif dramatically enlarge the substrate spectrum of BCATs. This finding was further
confirmed by the characterization of another BCAT in subgroup 8 (BCAT8-2), which has
similar substrate specificity with BCAT8 (Table S7). Given that the rapidly growing
genomic databases may contain vague sequence annotations and miss-annotations, e.g.
ATI1 that should belong to AlaAT, the main benefit of this bioinformatic exploration lies
in the efficient sampling of particular enzymatic activity and identification of a wealth of
functional novelty when abnormalities present in the database with miss-annotations.
Scoring sequences with subfamily HMM profiles could have an output for the typical
enzymes with higher activities and the substrate specificity of BCATs implied that
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scattering sequences by binding pocket motif diversity would be a more effective approach
to expand the substrate scope. The integrated system (subfamily profile or motif sequence)
can provide biocatalysts with broad catalytic diversity showing unique substrate specificity
profiles and be adapted to other enzyme families for exploration of their biocatalytic
diversity in a large sequence space.
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To illustrate the synthetic usefulness of these ATs, 18 proteinogenic L-amino acids
were tested for conversion to the corresponding 2-ketoacids by using the best performing
AT for the individual substrate. Setting up the biocatalytic reactions in a shaker with 200
rpm was sufficient for the oxygen supplement. The concentration of L-glutamate had an
effect on the final conversion, and thus, the concentration of L-glutamate for several amino
acids was further optimized. Under optimized conditions, most L-amino acids were
efficiently converted by the enzymatic cascade to the corresponding 2-ketoacids with high
conversion (>99%) and good product conversion (95~99%, Table 1). For glyoxylic acid,
oxaloacetic acid and β-indolylpyruvic acid that undergo mild decarboxylation at neutral
pH, moderate product conversion could be achieved through kinetically controlled
conversions. The only exceptions were the corresponding 2-ketoacids of L-cysteine and L-
lysine, because 3-Mercaptopyruvate was extremely unstable in aqueous solution and 6-
amino-2-oxohexanoic acid was in equilibrium with the cyclic form. Interestingly, the
developed enzyme cascade also allowed for the synthesis of mixed 2-ketoacids
simultaneously based on the promiscuity of ATs (Figure S14). Furthermore, we performed
reactions on a preparative scale. The enzyme cascade smoothly converted four grams L-
leucine and L-isoleucine, which produced 4.38 g 2-ketoisocaproate sodium salt (medical
supplement) with a 95% isolated yield and 4.18 g 2-keto-3-methylvalerate sodium salt
(animal feed) with a 91% isolated yield, respectively (see the Supporting Information). As
compared with the rival biocatalytic routes (Table S8), the identified AT panel provides a
more versatile platform, supporting all possible L-amino acids to 2-ketoacids conversions.
Moreover, the reactions can proceed with only a catalytic amount of L-glutamate, which
simplifies the process of product purification and has the potential for conducting
sequential reactions in the same pot.
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Table 1. Oxidation of L-amino acids to 2-ketoacids using an AT-based cascade[a].
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Amino Enzyme
acids
Substrate L-glutamate Conversion
Product
/mM
100
100
100
100
100
/mM
/%
conversion[c]/%
Met
Ala
Ile
PsBCAT
AlaAT5
PsBCAT
PsBCAT
PsBCAT
1
>99 (12 h)
>99 (6.5 h)
>99 (11 h)
>99 (10 h)
>99 (12 h)
>99 (8 h)
>99 (9.5 h)
>99 (9 h)
>99 (9 h)
>99 (21 h)
>99 (21 h)
>99 (21 h)
98.6 (40 h)
>99 (9 h)
80.6 (1 h)
36.6 (6 h)
99.6
99.5
99.4
99.3
99.3
95.4
92.4
n.d.[b]
n.d.
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1
2
Leu
Val
Phe
Tyr
Asn
Gln
Arg
His
Thr
Ser
Asp
Trp
Gly
3
2
EcAspAT 100
10
10
1
ARAT2
ARAT2
BCAT9
BCAT9
EcARAT
BCAT9
AlaAT5
EcARAT
ARAT2
AlaAT5
100
100
100
100
100
100
100
100
30
1
1
n.d.
1
n.d.
5
n.d.
10
1
92.0
40.8
78.9
31.9
5
10
1
[a]
The branched-chain, aromatic and polar amino acids were represented in the rows
marked in blue, yellow and green, respectively. The corresponding 2-ketoacids of the
amino acids marked in gray were unstable in aqueous solution.
[b]
n.d.: The identity of the 2-ketoacid product was confirmed by LC-MS (see the
Supporting Information), but the product conversion was not determined due to the lack of
an authentic reference compound.
[c]
HPLC assayed conversion to desired product.
Consequently, we explored the possibilities of extending the enzymatic cascade for
the synthesis of 2-ketoacids derivatives. Since there is an increasing interest in chiral
unnatural amino acids in the pharmaceutical and agrochemical industries, cost-effective
methods for the synthesis of D-amino acids and N-methylated amino acids are highly
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desirable.42 We anticipated that these compounds can be readily synthesized by introducing
additional reductive amination enzymes into the cascade. Accordingly, the double-site
mutant of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum
(StDAPDH W121L/H227I) with improved activities43 and N-methylamino acid
dehydrogenase from Pseudomonas fluorescens (PfNMAADH) with a broad substrate
scope44 were selected. Bacillus megaterium glucose dehydrogenase (GDH) was included
for cofactor regeneration in the second step. Considering both dehydrogenases were able
to accept 2-ketoglutarate, which would disorganize the first enzymatic cascade, we
developed a one-pot two step sequential process to synthesize the desired products (Figure
2). Pleasingly, the designed cascade proceeded with high substrate conversion and the
desired products were obtained with an almost quantitative product conversion and
excellent e.e. values (>99%). In addition to the analytical-scale reactions, we carried out
gram-scale preparative reactions using L-alanine as the substrate. Both stereo-inversion
and N-alkylation were achieved with high conversions. Through a straightforward work-
up and purification, D-alanine (1.21 g, 91% yield, e.e.>99%) and N-methyl-L-alanine (1.40
g. 90% yield, e.e.>99%) were obtained (see the Supporting Information). These results
demonstrate that the ATs-based cascade can be compatible with sequential biocatalytic
cascades and the modularity of this cascade highlights an immense potential for
synthesizing related high-value added compounds from L-amino acids.
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Figure 2. One-pot, two-step enzymatic cascade for the synthesis of chiral unnatural
amino acids. The first steps in the cascade reactions were carried out at 30°C, 200 rpm in
0.2 M phosphate buffer (pH=7.5), with 30 mM L-amino acids, 1 mM 2-ketoglutarate (2-
KG), 0.25 mg·mL-1 ATs, 0.1 mg·mL-1 LGOX, 0.1 mg·mL-1 catalase and 20 μM PLP. When
L-amino acid was completely consumed, the second cascade reactions were initiated by
adding 0.3 mg·mL-1 StDAPDH W121L/H227I or PfNMAADH, 0.2 mg·mL-1 GDH, 1 mM
NADP+, 1.2 equivalents of glucose and 5 or 10 equivalents of amines.
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Overall, we succeeded in developing a novel enzymatic methodology for the
oxidation of natural L-amino acids into 2-ketoacids that exploits the AT/LGOX/catalase
cascade under mild and aqueous conditions. The strategy of scattering sequences into 2-
dimensional space by subfamily HMM profiles or motif scouting that was used here lead
to the identification of a panel of ATs. In total, 27 out of the 13068 computationally
characterized ATs were overexpressed and 6 soluble forms enabled efficient conversion to
the corresponding 2-ketoacids, which effectively complements the biocatalytic toolbox for
natural L-amino acids. With a catalytic amount of L-glutamate, this AT-based enzyme
system can produce atom efficiency similar to that of LAAO and LAAD. However, the
vast diversity of ATs in nature compromises the system for a much broader substrate
spectrum. It is feasible that their activities can be further improved by directed evolution
or computational engineering.45 Moreover, this amine oxidative cascade can be combined
with other chemo- or biocatalysts for the preparation of a large variety of related useful
building blocks. As such, our work sets the stage for the further development of green,
sustainable and pot-economic synthesis of fine chemicals that starts with amino acids and
inexpensive amines.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Supporting Figures S1-S26; Supporting tables S1-S9; Materials, experimental procedures,
analytic methods, 1H and 13C NMR spectra (PDF)
ACKNOWLEDGMENTS
This work is supported by National Key R&D Program of China (No. 2018YFA0901600)
the National Natural Science Foundation of China (Grant Nos. 31822002), and the
Biological Resources Programme (KFJ-BRP-009) and Key Research Program of Frontier
Sciences (ZDBS-LY-SM014) from the Chinese Academy of Sciences.
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Scheme 1. The enzymatic cascade for the synthesis of 2-ketoacids from L-amino acids
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Figure 1. The bioinformatic procedure for rationally mining novel ATs.
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Figure 2. One-pot, two-step enzymatic cascade for the synthesis of chiral unnatural
amino acids.
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Table of contents
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