Defluorination of 4-fluorothreonine by threonine deaminase

Article abstract of DOI:10.1039/d0ob01358g

4-Fluorothreonine (4-FT) is the only naturally occurring fluorinated amino acid antibiotic. Although two conserved proteins in the 4-FT pathway have been found to be involved in self-detoxification mechanisms, the 4-FT-producing strains may also require an alternative pathway to degrade the intracellular 4-FT. In this study, we examined the possible degradation role of three enzymes involved in threonine metabolite pathways toward 4-FT as a possible degradation route to avoid in vivo 4-FT accumulation. Among these three enzymes, threonine deaminase was found to catalyse a defluorination reaction to generate 4-hydroxy-α-ketobutyrate, which is supposed to be further metabolised by an aldolase that likely is a unique occurrence in the 4-FT-producing strains. Our finding may constitute a 4-FT degradation pathway as a complementary resistance mechanism.

Full text of DOI:10.1039/d0ob01358g

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: L. Wu and H.
Deng, Org. Biomol. Chem., 2020, DOI: 10.1039/D0OB01358G.
Volume 15
Number 47
21 December 2017
Pages 9945-10124
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
Accepted Manuscripts are published online shortly after acceptance,
rsc.li/obc
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
ISSN 1477-0520
PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
shall the Royal Society of Chemistry be held responsible for any errors
single cell transcriptome in vivo analysis (TIVA)
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
rsc.li/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 6
View Article Online
DOI: 10.1039/D0OB01358G
ARTICLE
Defluorination of 4-fluorothreonine by threonine deaminase
Linrui Wua, Hai Denga
4-fluorothreonine (4-FT) is the only naturally occurring fluorinated amino acid antibiotic. Although two conserved proteins
in the 4-FT pathway have been found to be involved in self-detoxification mechanisms, the 4-FT producing strains may also
require an alternative pathway to degrade the intracellular 4-FT. In this study, we examined the possible degradation role
of three enzymes involving in threonine metabolite pathways toward 4-FT as a possible degradation route to avoid in vivo
4-FT accumulation. Among these three enzymes, threonine deaminase was found to catalyse a defluorination reaction to
generate 4-hydroxy-α-ketobutyrate, which is supposed to be further metabolised by an aldolase that likely is unique
occurrence in the 4-FT producing strains. Our finding may constitute a 4-FT degradation pathway as a complementary
resistance
mechanism.
identified the prolific fluorometabolite producing strain
Streptomyces sp MA37 in Ghana, Africa that also produces a wide
Introduction
For decades, bacteria has been known as the most proliferate
producer of natural products (NPs), which accounts for over 40% of
lead small molecules in drug discovery1. In most cases, in bacteria,
genes responsible for their synthesis are clustered together to form
range
of
fluorinated
NPs
including
5-fluoro-2,3,4-
trihydroxypentanoic acid (FHPA) 3 (Figure 1A) 12, 14. Unlike most of
microbial natural products, many of the genes responsible for
bacterial fluorometabolites are spread in the genomes of the
corresponding fluorometabolite producing strains15. Enzymes from
primary metabolisms, such as 5-methylthio-5-deoxy-ribose
isomerase, may also act on the intermediates in the
fluorometabolite pathway, due to the structural similarity among
the fluorinated intermediates and intermediates from primary
metabolisms10.
biosynthetic gene clusters (BGCs), enabling
a more efficient
expression regulation and functional assembly2. To guard the host
strain from the generated NPs, it is a common strategy for BGCs to
incorporate a resistance gene, conferring detoxification mechanism
for self-protection3. This knowledge in return has facilitated the
genome mining process for new NP discovery4.
Despite the similarities of the steric properties between fluorine
and hydrogen atom, fluorinated drug molecules are often endued
with advantages compared to their non-fluorinated counterparts
such as a less susceptibility towards oxidative metabolism by
cytochrome P450 monooxygenases and degrading hydrolytic
effects5. In contrast to the wide distributed synthetic fluorinated
compounds, naturally occurring fluorometabolites are rare.
However, their occurrence has provided a new research interest of
the blooming fluorinated drug discovery industry using fluorine
biocatalysts under mild conditions for stereo- and regio-selective
fluorination6. The most well-studied fluorometabolites, the toxin
fluoroacetate (FAc) 1 and the antibiotic 4-fluorothreonine (4-FT) 2
were first identified in Streptomyces cattleya7. The biosynthetic
pathways of these two fluorometabolites have been well-studied
with the hallmark studies of the first naturally occurring fluorination
enzyme, the fluorinase, the first committed enzyme in the
fluorometabolism8-11. Later on, genome mining excavated several
other putative fluorometabolite producing strains including
Due to the similar steric property between fluorine and hydrogen,
both FAc 1 and 4-FT 2 can enter various primary metabolite
pathways related to acetate and L-Thr, respectively. These
misincorporations can be interruptive and toxic to the producing
strains, which in turn have evolved to counteract the potential
detoxification strategies16. The resistance mechanism of host strains
towards FAc 1 has been characterized. The toxicity of 1 roots from
the transformation of 1, via fluoroacetyl-CoA, to the potent toxin
(2R,3R)-fluorocitrate, which is a potent inhibitor of aconitase, a key
enzyme of the citric acid cycle. In case of 1 in the producing strains,
a specialised fluoroacetyl-CoA thioesterase, FlK, can selectively
hydrolyse the misacylated fluoroacetyl-CoA to prevent the
activation of fluoroacetate (Figure 1B)17-19. The detoxification
mechanism of 4-FT 2 employed by S. cattleya has also been
extensively elaborated by the study of FthB and FthC, the
fluorothreonyl-tRNA deacylase and 4-FT transporter, respectively.
The structure similarity between 4-FT 2 and L-Thr allows 2 to form a
misacylated tRNA, 4-FT-tRNA, hence entering the ribosomal protein
biosynthesis. FthB the fluorothreonyl-tRNA deacylase can hydrolase
the 4-FT misacylated tRNA and reduce the misincorporation of 4-FT
(Figure 1B)20. Interestingly, the genetic knock-out studies on both S.
cattleya and S. MA37 showed that its inactivation doesn’t result in
the fluctuation of fluorometabolite biosynthesis or the strain’s
Norcardia brasiliensis, Actinoplanes sp N902-109, as well as the
13
marine-derived Streptomyces xinghaiensis12,
. In 2012, we
a. Department of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, UK
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 6
ARTICLE
Journal Name
phenotype alteration21. The inactivation of 4-FT transporter FthC, were chosen to be tested on 4-FT 2. LAAO (Sigma-AVldierwicAhrtiPclreoOdnulincet
DOI: 10.1039/D0OB01358G
which led to an increased intracellular 4-FT 2 accumulation, also No. A9253), mostly found in snake venom, catalyses the
doesn’t interfered with the cell growth20, leaving an unanswered deamination of L-amino acids to generate the corresponding α-keto
question of the metabolic fate of these intracellular 4-FT 2.
acid, ammonia and hydrogen peroxide22 . LAAO has relatively broad
substrate tolerance toward different amino acids and was found to
transform L-Thr to pyruvate (Figure 1C), albeit less efficient
compared to amino acids with hydrophobic side chains23. In our
previous studies, we successfully overexpressed the 4-
fluorothreonine transaldolase FTaseMA from Streptomyces sp.
MA37 and characterized its function in the last step of the
transaldol reaction of 4-FT biosynthesis. It utilizes L-Thr and
fluoroacetaldehyde (FAd) as substrates and pyridoxal phosphate
(PLP) as cofactor to generate 4-FT.24. To test whether LAAO acts on
4-FT 2, we performed a coupling reaction. An assay of LAAO with
FTaseMA using L-Thr and fluoroacetaldehyde (FAd) to generate 4-FT
2 in situ was carried out (Figure S1). The reaction was monitored in
19F-NMR analysis. However, there was no apparent new
organofluorine signal appearing in the 19F-NMR spectrum while 4-FT
2 from FTaseMA biochemical reaction accumulated, suggesting that
the fluorine atom at C4 position of 4-FT 2 strongly inhibits the
reactivity of the LAAO.
In different organisms there exist various enzymes in primary
metabolisms taking up L-Thr as substrate for its transformation
(Figure 1C). Considering the structural similarity of 4-FT and L-Thr,
these primary enzymes might also be used for the degradation of
intracellular 4-FT 2, counteracting its poisonous effect towards the
host strain.
Next, we overexpressed the genes, encoding TDH and TDA
from E. coli, in E. coli BL21(DE3) and acquired them to near
homogeneity, respectively (Figure S2A, S3A). TDH is
responsible for catalysing the oxidation of L-Thr to L-2-amino-
3-oxobutanoate 4 in amino acid metabolism25 while TDA
catalyses the initial step from L-Thr to α-ketobutyrate 5 (Figure
1C). TDA is a crucial enzyme leading to the branched pathway
of amino acid degradation and L-isoleucine biosynthesis26
(Figure 1C). The catalytic activities of the recombinant proteins
have been confirmed(Figure S2B, S3B). To examine the
catalytical capacity of TDH toward 4-FT 2, an assay of TDH
coupled with FTaseMA was performed overnight and the
reaction mixture was analysed through 19F-NMR. No new
organofluorine signals were observed, demonstrating that 4-FT
is not the substrate of TDH (Figure S4). We then carried out
the coupling assay of TDA with FTaseMA and monitored the
biotransformation in 19F-NMR. Interestingly, the level of 4-FT 2
in this coupling reaction was reduced compared to the control
reaction in which TDA were inactivated (Figure S5). However,
no new organofluorine signals were observed in the 19F-NMR
spectrum, indicating that no organofluorines were produced.
Figure 1. A. The three fluorometabolites produced by MA37. B.
The detoxification of 4-FT and FAcin S. cattleya. C. The
enzymatic reactions on L-Thr in various organisms.
Here we report the biochemical defluorination reaction of 4-FT
2, catalysed by threonine deaminase (TDA), which may serve
an alternative pathway of 4-FT 2 to prevent from its
intracellular accumulation. The products, resulting from this
defluorination reaction, was determined to be inorganic
fluoride and 4-hydroxy-α-ketobutyrate through 19F-NMR
analysis, chemical derivatization and high resolution mass
spectroscopic analysis, the latter of which is proposed to be
further degraded into pyruvate by the promiscuous enzyme, 2-
keto-4-hydroxyglutarate aldolase, through comparative
genomic analysis.
Instead, we found
a fluorine signal at -121.35 ppm,
corresponding to fluoride ion, was accumulated. To further
demonstrate the defluorination reaction by TDA, we
developed two-step biotransformation in
a time-course
fashion. 4-FT 2 was first synthesised by FTaseMA with PLP and
L-Thr, followed by removing FTaseMA by heat denaturing.
After the depletion of FTaseMA, TDA was supplemented to the
system. As shown in Figure 2A, 4-FT 2 was constantly
consumed as observed throughout the time scale and the
decrease of 4-FT 2 was only accompanied with the emerging of
inorganic fluoride ion in the 19F-NMR spectrum (Figure 2B).
Results and discussion
Three enzymes that can metabolise L-Thr, threonine dehydrogenase
(TDH), L-amino acid oxidase (LAAO) and threonine deaminase (TDA),
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 6
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
View Article Online
DOI: 10.1039/D0OB01358G
Figure 2. The time course of the coupling reaction of FTaseMA and
TDA, showing the consume of 4-FT 2 and the generation of fluoride
ion. FAd 1 and 4-FT 2 were the substrate and the product left in the
FTaseMA biotransformation reaction, respectively.
To examine what products were generated during the
defluorination catalysed by TDA, we applied the derivatization
analytical method we used in our previous study24. 2,4-
dinitrophenylhydrazine (DNPH) was used as a derivatization
agent for monitoring aldehyde and ketone species in the
reaction mixture. The reaction mixture was derivatized by
DNPH, followed by HPLC analysis of keto-DNPH derivatives27.
The derivatized mixtures were also subjected to LC-high
resolution MS (HR-MS) analysis. In the coupling assay (Figure
3A), three expected DNPH derived compounds containing keto
functional groups, such as α-ketobutyrate 5 (Figure S6A),
fluoroacetaldehyde (FAd) 7 (Figure S6B), and acetaldehyde
(Ad) 7 (Figure S6C) were observed. FAd 6 and Ad 7 are the
unreacted components from the FTaseMA biotransformation
system while α-ketobutyrate 5 is the product of TDA which
acts on the residue of L-Thr from the FTaseMA
biotransformation system. Interestingly, we also observed the
presence of a new peak at the retention time (RT) of 9.8 min.
HR-MS analysis indicated that an ion with an m/z value of
Figure 3. LC-MS analysis of the reaction mixture after DNPH
derivatization, showing the inactivated TDA and FTaseMA/TDA as
control.
Taken together, a catalytic mechanism of the defluorination on 4-
FT, catalysed by TDA based on the current knowledge of PLP-
dependent biochemistry, was shown in Figure 4. Both L-Thr and 4-
FT 2 will undergo the same pathway of β-dehydration mediated by
TDA to generate the key intermediate 9. In the case of L-Thr,
hydrolysis of 9 would yield the imine molecule 10 which is further
hydrolysed into α-keto-butyrate 5 (Figure 4, blue curly arrow). In
the case of 4-FT 2, it is likely that the hydrolysis, however, would
result in using fluoride ion as the leaving group to produce 2-
iminobut-3-enoic acid 11. Subsequent Michael addition by water
results in 2-keto-4-hydroxybutyric acid 8.
+
299.0629 (calcd. m/z = 299.0622; calcd. MF = C10H11O7N4 ,
Δ=0.675 ppm) corresponds to this DNPH-derived compound 8.
In the control experiments where either a combination of both
denatured FTaseMA and TDA (Figure 3B,C) or a combination of
active FTaseMA and denatured TDA was used in the presence
of substrates and co-factors, this new peak was absent,
demonstrating that 9 is generated by the coupling reaction in
which both FTaseMA and TDA were active. As a result, it is
likely that TDA catalyses a defluorination reaction on 4-FT 2 to
yield inorganic fluoride ion and 2-keto-4-hydroxybutyric acid 8.
The observed defluorination catalysed by TDA is considerably
interestingly as it unusually cleaves the C-F bond, the strongest
covalent bond in organic chemistry. Very few enzymes discovered
thus far have such capacity, one of which is fluoroacetate
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 6
ARTICLE
Journal Name
dehalogenases from Burkholderia sp. and Delftia acidovorans28, 29
.
fluorometabolites was not assessed12. Streptomyces xinghaiensis,
View Article Online
DOI: 10.1039/D0OB01358G
These enzymes recruit Asp in their active sites to displace fluoride on the other hand, is the fluorometabolite producing strain
ion in an SN2 manner to generate an enzyme-glycolate ester originated from marine environments which only produces FAc 1.
intermediate, followed by subsequent hydrolysis of this ester by Bioinformatic analysis of the fluorometabolite biosynthetic gene
water to yield glycolate and recover the enzymes to their original cluster in S. xinghaiensis suggested that the putative FTase gene is
states30.
truncated, meaning that the strain has no capacity of producing 4-
FT 216. Coincidently, S. xinghaiensis lacks 2-keto-4-hydroxyglutarate
aldolase gene in its genome as well. Taken together, TDA and 2-
keto-4-hydroxyglutarate aldolase may constitute a 4-FT degradation
pathway in the producing strains to recycle formaldehyde and
X
X
H
-
-
CO2
CO2
HO
HO
N+
NH+
OH
H
O-
-
CO2
O-
pyruvate into primary metabolisms. It may serve as
a
2-
2-
OPO3
OPO3
+
X
NH3
PLP
H2O
complementary mechanism in addition to FthB to maintain a
minimal level of intracellular 4-FT 2 for a normal state of ribosomal
protein biosynthesis. The potential 4-FT degradation pathway of 4-
FT catalysed by putative TDA and 2-keto-4-hydroxyglutarate
aldolase from the 2 producing strains will be the focus of our future
studies.
N+
H
CH3
N
H
CH3
X = H or F (2)
X
X
H2O
-
CO2
H+
-
CO2
H2O
X = H
NH+
-
CO2
NH
O
Conclusions
-
CO2
H
O-
O
O-
2-
OPO3
2-
NH
10
In this study, we tested several L-Thr metabolism related enzymes
and conducted biochemical assays to determine their activity
against 4-FT 2. Incubation of 4-FT 2 generated by FTaseMA with the
recombinant TDA leads to the identification of an interesting 4-FT
defluorination mechanism. Bioinformatics analysis found a copy of
2-keto-4-hydroxyglutarate aldolase gene in both genomes of S.
OPO3
5
N+
H
CH3
N+
H
CH3
X = F
9 and 9a
9 X = H
9a X = F
H2O
PLP, F-
-
CO2
NH
-
cattleya and S. MA37, indicating there exists
a new 4-FT
CO2
-
CO2
degradation pathway for its detoxification and recycling. Combined
with the fluorothreonyl-tRNA deacylase, they may serve as the 4-FT
resistance mechanism employed by the fluorometabolite producing
strains to avoid homeostasis disruption. This discovery would also
facilitate our understanding on the biochemistry of
fluorometabolites.
OH
NH2
OH
O
11
8
Figure 4. The proposed defluorination mechanism of 4-FT, catalysed
by TDA based on the current knowledge of PLP dependent
biochemistry. The releasing from PLP can branch in two ways
depending on the substitution.
Conflicts of interest
The identified defluorination reaction of 4-FT 2 suggested that the
host strain may recruit TDA from the primary metabolism for the
intracellular 4-FT degeneration, preventing a toxic level of 4-FT 2
accumulation in vivo. The examination of the metabolic fate of 2-
keto-4-hydroxybutyric acid 8 revealed that, although not present as
There are no conflicts to declare.
Acknowledgements
This study was funded by IBioIC PhD studentship (LW) and the
Royal Society-NSFC Newton Mobility Grant Award
(IEC\NSFC\170617 to HD).
a
common primary metabolite, it can be accepted by the
promiscuous 2-keto-4-hydroxyglutarate aldolase to be converted
into formaldehyde and pyruvate as presented by previous studies
31. A copy of gene encoded 2-keto-4-hydroxyglutarate aldolase was
found in the genome of S. cattleya (accession No: AEW93759).
BLAST search using its sequence as a query against the annotated
MA37 draft genome in RAST server revealed the presence of one
gene encoding 2-keto-4-hydroxyglutarate aldolase in MA37.
Interestingly, 2-keto-4-hydroxyglutarate aldolase is rarely found in
Streptomyces including Norcardia brasiliensis and Actinoplanes sp
N902-109 that contain fluorinases12, 32. Only twelve homologous
genes were found among thousands of Streptomyces genomes in
NCBI database. It is worth to note that Norcardia brasiliensis cannot
produce any fluorometabolites in the laboratory conditions12, 32 and
Actinoplanes sp N902-109 is not available in the public domain so
that the capacity of whether this strain is able to produce
Notes and references
1.
2.
G. M. Cragg and D. J. Newman, Biochim. Biophys. Acta,
2013, 1830, 3670-3695.
H.-W. Nützmann, C. Scazzocchio and A. Osbourn, Annu.
Rev. Genet., 2018, 52, 159-183.
3.
4.
Y. Yan, N. Liu and Y. Tang, Nat. Prod. Rep., 2020.
Y. Yan, Q. Liu, X. Zang, S. Yuan, U. Bat-Erdene, C. Nguyen,
J. Gan, J. Zhou, S. E. Jacobsen and Y. Tang, Nature, 2018,
559, 415-418.
5.
6.
B. K. Park, N. R. Kitteringham and P. M. O'Neill, Annu. Rev.
Pharmacol. Toxicol., 2001, 41, 443-470.
L. Wu, F. Maglangit and H. Deng, Curr. Opin. Chem. Biol.,
2020, 55, 119-126.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 6
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
7.
M. Sanada, T. Miyano, S. Iwadare, J. M. Williamson, B. H.
View Article Online
DOI: 10.1039/D0OB01358G
Arison, J. L. Smith, A. W. Douglas, J. M. Liesch and E.
Inamine, J. Antibiot. Res., 1986, 39, 259-265.
D. O'Hagan, C. Schaffrath, S. L. Cobb, J. T. Hamilton and C.
D. Murphy, Nature, 2002, 416, 279-279.
8.
9.
C. Dong, F. Huang, H. Deng, C. Schaffrath, J. B. Spencer, D.
O'hagan and J. H. Naismith, Nature, 2004, 427, 561-565.
H. Deng, S. M. Cross, R. P. McGlinchey, J. T. Hamilton and
D. O'Hagan, Chem. Biol., 2008, 15, 1268-1276.
M. Onega, R. P. McGlinchey, H. Deng, J. T. Hamilton and
D. O’Hagan, Bioorg. Chem., 2007, 35, 375-385.
H. Deng, L. Ma, N. Bandaranayaka, Z. Qin, G. Mann, K.
Kyeremeh, Y. Yu, T. Shepherd, J. H. Naismith and D.
O'Hagan, ChemBioChem, 2014, 15, 364-368.
S. Huang, L. Ma, M. H. Tong, Y. Yu, D. O'Hagan and H.
Deng, Org. Biomol. Chem., 2014, 12, 4828-4831.
L. Ma, A. Bartholome, M. H. Tong, Z. Qin, Y. Yu, T.
Shepherd, K. Kyeremeh, H. Deng and D. O'Hagan, Chem.
Sci., 2015, 6, 1414-1419.
10.
11.
12.
13.
14.
15.
F. Huang, S. F. Haydock, D. Spiteller, T. Mironenko, T.-L. Li,
D. O'Hagan, P. F. Leadlay and J. B. Spencer, Chem. Biol.,
2006, 13, 475-484.
16.
17.
D. O’Hagan and H. Deng, Chem. Rev., 2015, 115, 634-649.
A. M. Weeks, N. S. Keddie, R. D. Wadoux, D. O’Hagan and
M. C. Chang, Biochemistry, 2014, 53, 2053-2063.
M. V. Dias, F. Huang, D. Y. Chirgadze, M. Tosin, D.
Spiteller, E. F. Dry, P. F. Leadlay, J. B. Spencer and T. L.
Blundell, J. Biol. Chem., 2010, 285, 22495-22504.
A. M. Weeks, N. Wang, J. G. Pelton and M. C. Chang, Proc.
Natl. Acad. Sci. U. S. A., 2018, 115, E2193-E2201.
J. L. McMurry and M. C. Chang, Proc. Natl. Acad. Sci. U. S.
A., 2017, 114, 11920-11925.
L. Wu, M. H. Tong, K. Kyeremeh and H. Deng,
Biomolecules, 2020, 10, 1023.
K. K. Tan, B. H. Bay and P. Gopalakrishnakone, Toxicon,
2018, 144, 7-13.
18.
19.
20.
21.
22.
23.
D. H. Souza, L. M. Eugenio, J. E. Fletcher, M.-S. Jiang, R. C.
Garratt, G. Oliva and H. S. Selistre-de-Araujo, Arch.
Biochem. Biophys., 1999, 368, 285-290.
24.
L. Wu, M. H. Tong, A. Raab, Q. Fang, S. Wang, K.
Kyeremeh, Y. Yu and H. Deng, Appl. Microbiol. Biotechnol.,
2020, 1-12.
25.
26.
27.
28.
29.
30.
31.
32.
K. Ishikawa, N. Higashi, T. Nakamura, T. Matsuura and A.
Nakagawa, J. Mol. Biol., 2007, 366, 857-867.
L. Chen, Z. Chen, P. Zheng, J. Sun and A.-P. Zeng, Appl.
Microbiol. Biotechnol., 2013, 97, 2939-2949.
X. Guan, E. Rubin and H. Anni, Alcohol.: Clin. Exp. Res.,
2012, 36, 398-405.
C. Donnelly and C. D. Murphy, Biotechnol. Lett., 2009, 31,
245.
T. Kurihara, T. Yamauchi, S. Ichiyama, H. Takahata and N.
Esaki, J. Mol. Catal. B: Enzym., 2003, 23, 347-355.
J. Jin and U. Hanefeld, Chem. Commun., 2011, 47, 2502-
2510.
C. Wang, J. Ren, L. Zhou, Z. Li, L. Chen and A.-P. Zeng, ACS
Synth. Biol., 2019, 8, 2483-2493.
Y. Wang, Z. Deng and X. Qu, F1000Research, 2014, 3.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Organic & Biomolecular Chemistry
Page 6 of 6
View Article Online
DOI: 10.1039/D0OB01358G
181x160mm (150 x 150 DPI)