Regulating Cofactor Balance In Vivo with a Synthetic Flavin Analogue

Article abstract of DOI:10.1002/anie.201810881

A novel strategy to regulate cofactor balance in vivo for whole-cell biotransformation using a synthetic flavin analogue is reported. High efficiency, easy operation, and good applicability were observed for this system. Confocal laser scanning microscopy was employed to verify that the synthetic flavin analogue can directly permeate into Escherichia coli cells without modifying the cell membrane. This work provides a promising intracellular redox regulatory approach to construct more efficient cell factories.

Full text of DOI:10.1002/anie.201810881

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Accepted Article
Title: Regulating Cofactor Balance in vivo via Synthetic Flavin
Analogue System
Authors: Zhuotao Tan, Chenjie Zhu, Jingwen Fu, Xiaowang Zhang,
Ming Li, Wei Zhuang, and Hanjie Ying
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201810881
Angew. Chem. 10.1002/ange.201810881
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10.1002/anie.201810881
Angewandte Chemie International Edition
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Regulating Cofactor Balance in vivo via Synthetic Flavin
Analogue System
Zhuotao Tan, Chenjie Zhu*, Jingwen Fu, Xiaowang Zhang, Ming Li, Wei Zhuang, Hanjie Ying*
Abstract: A novel strategy to regulate cofactor balance in vivo for
whole cell biotransformation using synthetic flavin analogue system
was reported. High efficiency, easy operation, and good applicability
were observed for the present system. Confocal laser scanning
microscopy was employed to verify synthetic flavin analogue can
directly permeate into Escherichia coli cells without modifying the
cell membrane. This work provides a promising intracellular redox
regulatory approach to construct more efficient cell factories.
convenient and efficient system that does not require a
transporter or modify cell membrane to manipulate cofactor
balance in vivo is highly attractive.
Recently, we reported the feasibility of using synthetic
bridged flavin analogue (SBFA) for the NAD(P)+ regeneration in
vitro.[12] It appeared that further ionization modification of the
structure of SBFA might enhance its uptake by cells through free
diffusion, establishing a new artificial system to manipulate
cofactor balance in vivo without modifying the cell membrane.
Such a synthetic flavin analogue system, which has not been
reported so far to the best of our knowledge, was examined with
E. coli to enhance the intracellular nicotinamide cofactor
recycling for whole cell biocatalysis.
In vitro experiments were first performed with mannitol-1-
dehydrogenase (MtDH)-catalyzed oxidation of D-mannitol into D-
mannose to check the efficiency of the SBFA. Recently, sugars
like D-mannose was getting increasing attentions for health care
and pharmaceutical interests due to its potential biological
activities.[13] As shown in the Scheme 1, SBFA were efficient for
aerobic NAD+ regeneration in vitro, and the order of catalytic
activity of these SBFA appeared to be F1 > F2 > F4 > F3,
agreed well with our previous reports.[12] In the presence of 0.1
mol% F1 and 2 mol% NAD+, the desired product mannose was
obtained in 81% yield in 24 h. Control experiments ruled out the
possibility of direct oxidation of the substrate through SBFA.
Under this nonoptimized condition, the calculated TOF of F1
was 33.8 h-1, which is 10-fold higher than that catalyzed by the
natural flavin FMN (3.3 h-1), indicating a superior catalytic activity
for the synthetic flavin analogue. Recently, an interesting work
about photoexcited flavins for the in vitro NAD+ regeneration was
reported, which also exhibited very high activities.[14]
Advanced bio-manufacturing has drawn considerable attention
for production of valuable chemicals and biofuels.[1] These
processes generally involve multistep biotransformations that
require cofactors functioning as the energy carrier for carbon
metabolism. In addition, cofactors can also impact reaction
thermodynamics and regulate direction of reaction pathways. It
has been well known that over 1500 microbial biotransformation
reactions need the cofactors NAD(H) or NADP(H), cofactor
balance hence plays an important role in manipulating metabolic
flux in biosynthesis to favor the production of desired products.
Toward that, various cofactor regulation strategies have been
reported.[2] Generally, there are three major cofactor regulation
strategies: 1) altering endogenous cofactor systems by
knocking-down competitive cofactor consumption pathways,[3]
strengthening the favorable pathway of cofactor generation,[4] or
manipulating global regulatory factors that influence redox
pools;[5] 2) supplementing the host with heterologous cofactor
regeneration systems based on enzyme cascade[6] or substrate
combination;[7] 3) changing cofactor preferences.[8]
More recently a fourth strategy via construction of synthetic
cofactor system has emerged as a breakthrough to regulate
cofactor balance. As a result of their affordability, stability,
designability and sometimes “better than nature” superiority,
synthetic cofactors provide an efficient and cost-effective tool for
the regulation of cofactor-dependent reactions.[9] However, the
applications of these flexible molecules have been restricted to
in vitro reactions. Only one in vivo case was reported in which a
synthetic nicotinamide cofactor analogue - nicotinamide cytosine
dinucleotide (NCD), was uptaken by Escherichia coli (E. coli) to
construct a selective intracellular energy-transfer process.[10]
This unique synthetic cofactor system was realized by co-
expressing an NAD+ transporter (Ndt) to enable the NCD
uptaking and depended on the mutant enzymes, which however
tends to impair cellular activity as overexpressing membrane
proteins can become toxic to the host [11] and heavy workload for
the construction of mutant library. Thus, the development of a
Scheme 1. SBFA-catalyzed in situ aerobic NAD+ regeneration in vitro.
Encouraged by these results, further in vivo experiments
were then conducted with E. coli as a model microorganism. We
first tested with parent E. coli BL21 (DE3) and made sure that it
had no influence on the conversion of mannitol, evident from
that no consumption of mannitol or mannose was detected. The
recombinant E. coli BL21 (DE3) hosting the overexpressed
MtDH was then applied for the conversion of mannitol. The yield
of mannose was however quite low (Table 1, entry 1). We were
[*]
Dr. Z. Tan[+], Prof. Dr. C. Zhu[+], J. Fu, X. Zhang, Dr. M. Li, Dr. W.
Zhuang, Prof. Dr. H. Ying
College of Biotechnology and Pharmaceutical Engineering
Nanjing Tech University
30 S Puzhu Rd, 211816 Nanjing (China)
E-mail: zhucj@njtech.edu.cn; yinghanjie@njtech.edu.cn
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
[+]
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10.1002/anie.201810881
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Table 1. Recombinant E. coli whole cell catalytic conversion of D-mannitol.
Mannitol NAD+
F1
MtDH
MtDH-NOX
Yield
[%][c]
Entry
[mM]
[mM]
[mM] [mg/mL][a] [mg/mL][b]
1
2
3
4
5
6
7
8
9
10
50
-
-
10
10
10
10
10
10
10
-
-
-
5
50
50
50
50
50
50
50
50
50
-
0.05
15
33
45
47
92
87
3
0.5
1
-
-
-
-
2
-
-
0.5
0.5
-
0.1
-
0.05
-
-
-
-
10
10
20
0.5
0.5
-
43
61
-
[a] Wet weight of recombinant E. coli BL21 (DE3) that expressing MtDH. [b]
Wet weight of recombinant E. coli BL21 (DE3) that co-expressed MtDH and
NOX. [c] The data represents the averages standard deviations for three
independent samples.
Figure 1. The cellular NAD(H) level for after 12 h reaction time. The data
represent the averages standard deviations for three independent samples. a:
Only using recombinant cells; b: Using recombinant cells and F1.
pleased to find that in the case of the exogenous addition of 0.1
mol% F1, the yield was boomed up by three-fold (Table 1, entry
2). Based on this inspiring result, we further evaluated the
efficiency under the exogenous addition of NAD+. Numerous
studies have shown that whole cell bioconversions were
improved with extracellular supplement of NAD+ due to the
insufficient cellular NAD+ level.[15] Our results also verified that
the yield of mannose was improved with the addition of NAD+
(Table1, entries 3 and 4). However, further increasing the
amount of NAD+ to 4 mol% did not show much improvement
(Table1, entry 5), that means enhancing cellular NAD+/NADH
ratio merely by exogenous addition of NAD+ has quite limited
effectiveness. Excitingly, the yield was dramatically increased to
92% in the presence of 0.2 mol% F1 when only using 1 mol%
exogenous NAD+ (Table 1, entry 6), which was about 3-fold
higher than that obtained in the absence of F1 under the same
condition (Table 1, entry 3). With regard to the dosage of F1, it
was possible to decrease the amount of F1 to as low as 0.1
mol% without a significant loss in catalytic efficiency (Table 1,
entry 7).
Figure 2. Confocal microscopic images of E. coli living cells labbled with F1.
Blue signals represent the fluorescence from F1 (λem. = 500 nm).
found at about 400 nm and 500 nm, respectively (Figure S5).
The CLSM experiment was then processed at 405 nm to get the
fluorescence images of E. coli living cells. It was evident from
Figure 2 that the fluorescence emission signal of F1 (visible as
blue spots) appeared when scanning the F1-labelled cells. We
could even detect the motions of the fluorescent live cells under
the CLSM (See videos in Supporting Information). These
exciting images and videos illustrate that F1 can permeate into E.
Coli cells and enhance intracellular NAD+ recycling.
Since it has been proved that the H2O2-forming F1 could
permeated into the cell, the intracellular ROS (reactive oxygen
species) levels were analyzed during the reaction (Figure 3).
ROS production was measured based on intracellular
deacylation and oxidation of 2’,7’-dichlorodihydrofluorescein
diacetate (DCFH-DA) to the fluorescent molecule 2’,7’-
dichlorofluorescein (DCF). It was observed that ROS level in F1-
addition system was almost two-fold higher than that in only
using recombinant cells at the early stage of the reaction. The
Cellular redox state as indicated by the NAD+/NADH ratio is
important for whole-cell biocatalysis. To confirm the intracellular
function of F1, the intracellular NAD(H) level was analyzed using
fast filtration approach according to the Sauer’s method.[16] As
shown in Figure 1, in the absence of F1, the cellular
NAD+/NADH ratio was 1.49. With 0.1 mM F1 supplementation,
the intracellular NADH concentration was dropped by 31% while
NAD+ concentration increased by 21%, and the corresponding
NAD+/NADH ratio increased to 2.60. We intend to believe that
F1 could enter into E. coli cell and accelerate the NAD+ recycling
in vivo.
To further confirm the functioning mode of F1, confocal
laser scanning microscopy (CLSM) experiments were conducted
to verify that F1 could permeate into the E. coli cells. As natural
flavin has fluorescence activity due to its isoalloxazine structure,
we then determined the auto-fluorescence spectrum of F1.
Maximum excitation and emission wavelengths of F1 could be
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10.1002/anie.201810881
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COMMUNICATION
(Table 1, entry 9). However, this value was only about half of
that achieved with F1 system (Table 1, entries 6 and 9). Even
when the cell concentration was increased to 20 g·L-1, the yield
achieved with the MtDH-NOX system (61%) was still much lower
than that of F1 system (92%) (Table 1, entry 10). It has been
suspected that overexpressed NADH oxidase might hamper cell
growth and affect the metabolism.[21] To check this point, the
growth curves of the E. coli BL21 (DE3) that only expressed
MtDH with that of co-expressed MtDH and NOX were compared.
Figure 4 shows that both strains reached stationary phase in
about 24 h after induction with isopropyl-β-D-thiogalactoside
(IPTG). In the exponential growth phase, the NOX co-expressing
cells grew slower than the cells only expressing MtDH. The
OD600 value of the co-expressed NOX cells stabilized at about 3,
which was about half of that of MtDH-only cells (OD600 value was
about 6). To check the impact of F1 on the cell growth, the OD600
value of the MtDH recombinant E. coli incubated with 0.1 mM F1
was also measured. It is noteworthy that addition of F1 almost
had no influence on cell growth, OD600 value of the F1 system
was nearly the same with that of MtDH-only cells.
Figure 3. ROS levels during the reaction. Values are averages of three
replicate experiments.
Reactions with higher mannitol concentrations were further
examined for considerations in reaction intensity for future
industrial scale production. This was done with mannitol
concentration varied from 50 – 1000 mM while keeping all other
parameters constant. Under this nonoptimized condition, the
reaction was not negatively affected by mannitol concentration
up to 250 mM, the relative and space-time yield were 77% and
71 g·L-1·D -1, respectively (Figure 5). The TON and TOF of F1
over the reaction were calculated to be 1925 and 160 h-1,
respectively.
With the aim to verify the applicability of the present system,
this SBFA system was further examined for whole cell
biotransformation of glycerol into 1,3-dihydroxyacetone (DHA)
using recombinant E. coli BL21 (DE3) that was overexpressed
with glycerol dehydrogenase (GlyDH). Ways to convert glycerol,
an inexpensive and readily available byproduct of biodiesel
industry, to DHA has received considerable interest due to DHA
is widely used as an important building block in the cosmetic,
results also showed that the ROS level in both system displayed
a trend of rise first then fall along with time, which probably due
to the fact that microorganisms create a series of antioxidant
enzymes, including catalases, superoxide dismutases, and
glutathione peroxidase to decrease ROS-generated oxidative
damage.[17] And it has also been reported that E. coli naturally
has multiply intercellular response to remove H2O2.[18]
Previously study showed that co-expressing NADH oxidase
(NOX) was an efficient method to manipulate redox cofactor
level, and has been widely used to enhance NAD+-dependent
biosynthesis in both resting cells[19] and growing cells[20]
.
Therefore, the present SBFA system was then compared with
the classical NOX system. NADH oxidase from
Streptococcus pyogenes was co-expressed with MtDH in E. coli
BL21 (DE3) to convert mannitol to mannose. As shown in Table
1, without exogenous NAD+, extremely low yield was observed
only using the MtDH-NOX system (Table 1, entry 8). After
exogenous addition of 1 mol% NAD+, the yield increased to 43%
Figure 4. Comparison of growth curves of MtDH, MtDH-NOX, and MtDH-F1
system.
Figure 5. Yield of MtDH whole cell coupled with F1 under various substrate
concentrations. Reaction condition: mannitol (50 mM -1 M), NAD+ (0.5 mM),
F1 (0.1 mM), Tris-HCl (100 mM, pH 9.5), 10 mL.
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Acknowledgements
This work was supported by the Program for the National
Natural Science Foundation of China (Grant No.: 21776132); the
Young Elite Scientist Sponsorship Program by CAST; the Major
Research Plan of the National Natural Science Foundation of
China (Grant No.:21390204); the Jiangsu National Synergetic
Innovation Center for Advanced Bio-Manufacture. Thanks for the
strain recombinant E. coli BL21 (DE3) GlyDH-NOX provided by
Zongbao Zhao, and Ping Wang and Christopher B. Eiben for
copyediting the manuscript.
Keywords: cofactors • cofactor regulation • flavin • synthetic
cofactor • whole-cell biotransformation
Figure 6. The time course of DHA formation. Reaction condition: glycerol (109
mM), NAD+, F1, recombinant E. coli BL21 (DE3) that only expressing GlyDH
or recombinant E. coli BL21 (DE3) that co-expressing GlyDH and NOX (10
g·L-1), PBS buffer (100 mM, pH 8.0), 10 mL, 30℃ , 150 rpm. The data
represented the averages standard deviations for three independent samples.
[1]
a) U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C.
Moore, K. Robins, Nature 2012, 485, 185; b) M. T. Reetz, J. Am. Chem.
Soc. 2013, 135, 12480; c) G. W. Huisman, S. J. Collier, Curr. Opin.
Chem. Biol. 2013, 17, 284.
[2]
[3]
[4]
[5]
[6]
a) M. Wang, B. Chen, Y. Fang, T. Tan, Biotechnol. Adv. 2017, 35, 1032;
b) X. Chen, S. Li, L. Liu, Trends Biotechnol. 2014, 32, 337; c) Y. Wang,
K. Y. San, G. N. Bennett, Curr. Opin. Biotechnol. 2013, 24, 994.
a) S. H. Park, H. U. Kim, T. Y. Kim, J. S. Park, S. S. Kim, S. Y. Lee, Nat.
Commun. 2014, 5, 4618; b) J. Liu, S. H. J. Chan, T. Brock-Nannestad,
C. Jun, S. Y. Lee, C. Solem, P. R. Jensen, Metab. Eng. 2016, 36, 57.
a) X. Wang, Y. J. Zhou, L. Wang, W. Liu, Y. Liu, C. Peng, Z. K. Zhao,
Appl. Environ. Microbiol. 2017, 83, 692; b) G. N. Vemuri, M. A. Eiteman,
E. Altman, Appl. Environ. Microbiol. 2002, 68, 1715.
medical, food and agriculture area.[22] To compare the present
method with those of previously reported systems, we typically
applied the reaction conditions analogous to those reported in
previous studies.[15b] Similar to the above study, the catalytic
activity of the GlyDH recombinant cells was generally low when
no F1 and NAD+ were added (Figure 6, Line a). After exogenous
addition of 5 mM NAD+, the reaction rate was slightly improved
(Figure 6, Line b). With further addition of 0.1 mM F1, the
reaction rate was sharply increased, and DHA production was
burst to 41.8 mM within 1 h (Figure 6, Line c), which was about
6.1-fold higher than that only adding 5 mM NAD+. Under the
same reaction conditions, using whole cell E. coli co-expressed
GlyDH and NOX system, only 17.2 mM yield of DHA was
achieved within 1 h (Figure 6, Line d), which was about 2.4-fold
lower than that of F1 system, and the highest DHA concentration
reached to 25.3 mM after 2.25 h in the GlyDH-NOX system. As
also can be seen from the Figure 6, DHA concentration dropped
slowly over time in all cases, suggesting that DHA may be
further consumed due to the conversion glycerol to DHA is a
reversible reaction.[21b]
a) T. S. Reynolds, C. M. Courtney, K. E. Erickson, L. M. Wolfe, A.
Chatterjee, P. Nagpal, R. T. Gill, Biotechnol. Bioeng. 2017, 114, 2685;
b) D. Brekasis, M. S. B. Paget, EMBO J. 2003, 22, 4856.
a) H. Gröger, F. Chamouleau, N. Orologas, C. Rollmann, K. Drauz, W.
Hummel, A. Weckbecker, O. May, Angew. Chem. Int. Ed. 2006, 45,
5677; Angew. Chem. 2006, 114, 5806; b) S. Kim, J. S. Hahn, Metab.
Eng. 2015, 31, 94.
[7]
[8]
M. Ask, M. Bettiga, V. Mapelli, L. Olsson, Biotechnol. Biofuels 2013, 6,
22.
a) S. Brinkmann-Chen, T. Flock, J. K. B. Cahn, C. D. Snow, E. M.
Brustad, J. A. McIntosh, P. Meinhold, L. Liang, F. H. Arnold, Proc. Natl.
Acad. Sci. USA 2013, 110, 10946; b) T. W. Johannes, R. D. Woodyer,
H. Zhao, Biotechnol. Bioeng. 2007, 96, 18.
[9]
a) C. E. Paul, I. W. C. E. Arends, F. Hollmann, ACS Catal. 2014, 4, 788;
b) T. Knaus, C. E. Paul, C. W. Levy, S. Vries, F. G. Mutti, F. Hollmann,
N. S. Scrutton, J. Am. Chem. Soc. 2016, 138, 1033; c) A. Geddes, C. E.
Paul, S. Hay, F. Hollmann, N. S. Scrutton, J. Am. Chem. Soc. 2016,
138, 11089; d) C. E. Paul, F. Hollmann, Appl. Microbiol. Biotechnol.
2016, 100, 4773; e) C. Nowak, A. Pick, P. Lommes, V. Sieber, ACS
Catal. 2017, 7, 5202; f) D. Ji, L. Wang, S. Hou, W. Liu, J. Wang, Q.
Wang, Z. K. Zhao, J. Am. Chem. Soc. 2011, 133, 20857.
In conclusion, we have demonstrated
a convenient,
practical, and efficient method to regulate cofactor balance in
vivo based on synthetic flavin analogue system, which does not
rely on the modification of the cell membrane through
overexpressing channel proteins or increasing cell membrane
permeability due to F1 could straightforward permeated into the
cell. This work not only provides a promising green strategy to
manipulate redox balance in vivo, but also offers new
opportunities to enable pathway-specific energy transfer for
engineering cell factories to more efficient produce valuable
metabolites. We believe this system is not limited to E. coli and
can be extended to other eukaryotic or prokaryotic cells. More
interesting works towards demonstrating this system in yeast
and other organisms are currently under investigation in our
laboratory.
[10] L. Wang, D. Ji, Y. Liu, Q. Wang, X. Wang, Y. J. Zhou, Y. Zhang, W. Liu,
Z. K. Zhao, ACS Catal. 2017, 7, 1977.
[11]
a) H. Oshikane, M. Watabe, T. Nakaki, Protein Expression Purif. 2018,
148, 40; b) B. Miroux, J. E. Walker, J. Mol. Biol. 1996, 260, 289.
[12] C. Zhu, Q. Li, L. Pu, Z. Tan, K. Guo, H. Ying, P. Ouyang, ACS
Catal. 2016, 6, 4989.
[13] X. Hu, Y. Shi, P. Zhang, M. Miao, T. Zhang, B. Jiang, Compr. Rev. Food
Sci. Food Saf. 2016, 15, 773.
[14]
M. Rauch, S. Schmidt, I. W. C. E. Arends, K. Oppelt, S. Kara, F.
Hollmann, Green Chem. 2017, 19, 376.
[15] a) B. Kaup, S. Bringer-Meyer, H. Sahm, Appl. Microbiol. Biotechnol.
2005, 69, 397; b) W. Yang, Y. Zhou, Z. K. Zhao, Afr. J. Biotechnol.
2013, 12, 4387.
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COMMUNICATION
[16] H. Link, J. M. Buescher, U. Sauer, Methods Microbiol. 2012, 39, 127.
[17] a) W. Chin, K. Lin, C. Liu, K. Tsuge, C. Huang, BMC Biotechnol. 2017,
17, 36; b) J. A. Imlay, Annu. Rev. Microbiol. 2003, 57, 395; c) I.
Fridovich, Annu. Rev. Biochem. 1995, 64, 97.
[18]
J. L. Brumaghim, Y. Li, E. Henle, S. Linn, J. Biol. Chem. 2003, 278,
42495.
[19]
a) B. R. Riebel, P. R. Gibbs, W. B. Wellborn, A. S. Bommarius, Adv.
Synth. Catal. 2002, 344, 1156; b) Z. Xiao, C. Lv, C. Gao, J. Qin, C. Ma,
Z. Liu, P. Liu, L. Li, P. Xu, PloS one 2010, 5, 8860; c) Q. Han, M. A.
Eiteman, Enzyme Microb. Technol. 2017, 106, 106.
G. N. Vemuri, M. A. Eiteman, E. Altman, Biotechnol. Bioeng. 2006, 94,
538.
[20]
[21]
a) A. K. Holm, L. M. Blank, M. Oldiges, A. Schmid, C. Solem, P. R.
Jensen, G. N. Vemuri, J. Biol. Chem. 2010, 285, 17498; b) Y. J. Zhou,
W. Yang, L. Wang, Z. Zhu, S. Zhang, Z. K. Zhao, Microb. Cell Fact.
2013, 12, 103.
[22]
R. Ciriminna, A. Fidalgo, L. M. Ilharco, M. Pagliaro, ChemistryOpen
2018, 7, 233.
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Entry for the Table of Contents
COMMUNICATION
Zhuotao Tan, Chenjie Zhu*, Jingwen Fu,
Xiaowang Zhang, Ming Li, Wei Zhuang,
Hanjie Ying*
Page No. – Page No.
Regulating Cofactor Balance in vivo
via Synthetic Flavin Analogue System
A novel strategy to manipulate intracellular cofactor balance for whole cell
biotransformation based on synthetic flavin analogue is constructed. The synthetic
flavin analogue can directly permeate into E. coli cells accelerating cellular NAD+
regeneration without requiring the cell membrane modification.
This article is protected by copyright. All rights reserved.