Synthetic Cofactor-Linked Metabolic Circuits for Selective Energy

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Synthetic Cofactor-Linked Metabolic Circuits for Selective Energy Transfer Lei Wang, Debin JI, Yuxue Liu, Qian Wang, Xueying Wang, Yongjin J. Zhou, Yixin Zhang, Wujun Liu, and Zongbao Kent Zhao ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b03579 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Synthetic Cofactor-Linked Metabolic Circuits for Selective Energy Transfer

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Lei Wang,†,‡,§ Debin Ji,† Yuxue Liu,† Qian Wang,†,‡ Xueying Wang,† Yongjin J. Zhou,† Yixin

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Zhang,† Wujun Liu,*,†,‡ Zongbao K. Zhao*,†,‡,#

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†

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China

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‡

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Dalian 116023, China

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§

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of Chemical Engineering, Northeast Electric Power University, Jilin 132012, China

Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023,

Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS,

Institute of Green Conversion of Biological Bioresource and Metabolic Engineering, College

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#

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116023, China

State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, CAS, Dalian

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ACS Paragon Plus Environment

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ABSRACT: Cellular energy transfer process can be analogized as the running of an energy

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carrier (EC)-linked metabolic circuit between an energy supplying module (ESM) and an

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energy utilizing module (EUM). Because natural EC such as the reduced nicotinamide

16

adenosine dinucleotide (NAD) links multiple energy transfer modules and metabolic circuits,

17

and the formation of natural EC is routinely coupled with the transformation of endogenous

18

substances, it is challenging to transfer energy selectively. Here we devise synthetic

19

cofactor-linked circuits for pathway-selective energy transfer. We engineer phosphite

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dehydrogenase as ESM to use the synthetic cofactor nicotinamide cytosine dinucleotide

21

(NCD). We construct diverse circuits in vitro by combining different ESM, EUM and EC, and

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demonstrate that energy transfer process is controllable by tuning the feature of each

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component of the circuit. More specifically, we show that it is possible to drive the

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NCD-linked subsystem while leaving the NAD–linked reaction virtually unaffected. When

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armed with such circuits, Escherichia coli cells used phosphite as the electron source to

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generate reduced NCD that drove reductive carboxylation of pyruvate for improved malate

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production from glucose. Together, this study provides opportunity to establish orthogonal

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energy transfer system for engineering cell factories and may be used to set an additional

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layer of control mechanism for life.

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KEYWORDS energy metabolism, non-natural redox cofactor, metabolic circuit, synthetic

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biology, nicotinamide cytosine dinucleotide, phosphite dehydrogenase

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2


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■ INTRODUCTION

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Energy transfer and substance transformation are essential to all living organisms. To facilitate

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efficient microbial production of valuable metabolites, biofuels and chemicals, tremendous

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progresses have been achieved in designing new substance transformation pathways.1-4

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Efforts have also been devoted to controlling energy transfer for example by alternating redox

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cofactor preference,5-7 but with less success partially due to complicated metabolic

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interactions.8 Conceptually, energy transfer requires three basic components, namely, energy

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carrier (EC), energy utilizing module (EUM) and energy supplying module (ESM) (Figure

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1A), and the process can be conceived as the execution of an EC-linked metabolic circuit

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(designated as the ESM-EUM-EC circuit), to resemble electronic circuit. ESM produces

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charged EC, which is taken by EUM to catalyze a reductive reaction coupled with the release

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of discharged EC. Thus, the performance of such a metabolic circuit will be depending on

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several aspects, for example, the level of EC, the EC preference and catalytic efficiency of

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ESM and EUM.

A

B EC

ESM

EUM

EC 48 49

Figure 1. Metabolic circuit and components for energy transfer. (A) A basic metabolic circuit consisting of

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ESM, EUM and EC. Keys: Arrow, the direction of energy transfer; Filled star, charged EC; Partially empty

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star, discharged EC. (B) Chemical structures of NAD and NCD.

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In most biological systems, natural EC, such as pyridine nucleotide cofactors,

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nicotinamide adenosine dinucleotide (NAD) and its reduced form NADH, link multiple

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energy transfer modules and metabolic circuits,9 and the formation of these natural EC is

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routinely coupled with the transformation of endogenous substances.10 Therefore, it is

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difficult to direct a cellular energy transfer event.

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To reduce the complexity, the energy transferring subsystem of interest should be

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insulated from the metabolic network, which has only been partially achieved by special 3


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orthogonalization via compartmentalization.11-13 For example, an entire cytochrome

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P450-dependent pathway has been relocated from endoplasmic reticulum to the chloroplast

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where the pathway has been driven by reduced cofactors generated by photosystem I using

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water as the primary electron donor12. However, there are still other modules within the

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chloroplast competing for the reduced cofactors. Thus, it is intrinsically challenging to

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manage the level of natural EC for pathway-specific energy transfer.8, 12, 14

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To overcome these challenges, it is crucial to 1) introduce non-natural cofactors as EC into

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the biological system,8, 15 and 2) engineer dedicated ESM for EC regeneration preferably

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without the introduction of any organic substance that is liable to further metabolic reactions.

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Here we devise synthetic cofactor-linked metabolic circuits for selective energy transfer. We

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used a synthetic cofactor nicotinamide cytosine dinucleotide (NCD, Figure 1B), an analog of

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NAD,16 as EC and engineered phosphite dehydrogenase as ESM to use NCD instead of NAD.

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We then constructed diverse circuits consisting of different modules, and demonstrate that

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energy transfer process is controllable by tuning the feature of each component of the circuit.

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When such circuit was incorporated into model microorganism Escherichia coli, cells used

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phosphite as the electron source to generate reduced NCD (NCDH) that drove reductive

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carboxylation of pyruvate for improved malate production from glucose. Together, this study

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provided opportunity to establish orthogonal energy metabolism and could be used to set a

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new layer of control mechanism for life.

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■ RESULTS AND DISCUSSIONS

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Biochemical features of energy transfer modules. For this proof-of-concept study, we

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considered using the synthetic cofactor NCD as EC, and malic enzyme (Mae) and lactate

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dehydrogenase (Ldh) as EUM for energy transfer. Wild-type Mae can catalyze

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NADH-dependent reductive carboxylation of pyruvate to malate, but it in fact favors the

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reverse reaction under physiological conditions. The fact that wild type Mae and Ldh both can

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use NADH provides opportunity to analyze energy transfer selectivity. Our previous study

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showed that Mae and Ldh were barely active in the presence of NCDH, while the Mae

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L310R/Q401C mutant (Mae*) was fully active with NCDH but inactive with NADH (Figure

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2A)16. Therefore, Mae* might be explored for reductive carboxylation of pyruvate even in the

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presence of Ldh if NCDH were generated by a dedicated ESM.

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B

A

10

NAD-linked EUM NCD-linked EUM ESM

NAD NCD Mae

+

—

Ldh

+

—

Mae* Pdh Pdh*

—

+

+

8 6

1/V (mM-1 s)

EC

VPdh* (mM s—1)

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4 2

1.0 0.5 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1/[NCD] (mM-1)

0

+

1.5

0

50

100

150

200

NCD (mM)

89 90

Figure 2. Features of components for metabolic circuit construction. (A) Activity patterns of energy

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transfer module-EC combinations. Keys: +, active; -, inactive; ∆, slightly active. Mae, wild-type malic

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enzyme; Mae*, the Mae L310R/Q401C mutant; Ldh, wild-type lactate dehydrogenase; Pdh, wild-type

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phosphite dehydrogenase; Pdh*, the Pdh I151R mutant. Enzyme activity data are listed in Table S1. (B)

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Kinetic profile of Pdh* with NCD. Assay conditions and kinetic data are listed in Table 1. Kinetic profiles

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of Pdh* with NAD and Pdh with NAD/NCD are shown in Figure S1.

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To maintain the function of the metabolic circuit for energy transfer, it is crucial to have a

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robust ESM for the generation of charged EC. We decided to explore phosphite

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dehydrogenase (Pdh) as an ESM, partially because the equilibrium constant for the oxidation

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of phosphite by NAD was about 1011 at pH 7.0, suggesting an essentially irreversible EC

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generating process.17 Moreover, phosphite and its oxidized product phosphate are inorganic

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compounds, which ensure a clean production of charged EC without the introduction of

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otherwise metabolism-prone organic substances. We engineered the cofactor binding site

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(Figure S2) of the Pdh from a Ralstonia sp.18 and obtained the Pdh–I151R mutant (Pdh*).

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Detailed kinetic analysis indicated that Pdh* had a KM of 13.7 µM for NCD and a kcat of 0.13

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s-1 (Figure 2B) and, that Pdh* had 4.2-fold higher catalytic efficiency in the presence of NCD

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than that in the presence of NAD (Table 1). However, in terms of cofactor preference, Pdh*

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was 530–fold more favorable toward NCD. Therefore, Pdh and Pdh* are advantageous as

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ESM to charge NAD and NCD, respectively.

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Characterization of in vitro metabolic circuits. We assembled 10 circuits composed of

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different energy transfer modules (Table S2) and a representative configuration is shown in

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Figure 3A. The levels of charged EC, namely NADH and/or NCDH, were determined upon

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the assay mixtures being held at 25 ºC for 1 h and the results are shown in Figure 3B. For

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different circuits with an identical ESM, the concentrations of charged EC were depending on

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the EC preference of EUM. When the corresponding EUM disfavored the charged EC, high 5


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levels of charged EC were obtained. Without any exception, the mismatched circuits

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Pdh–Mae*–NAD, Pdh–Ldh–NCD, Pdh*–Mae*–NAD and Pdh*–Ldh–NCD accumulated

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high levels of NADH or NCDH, because Mae* and Ldh had little activity to consume NADH

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and NCDH, respectively. On the other hand, when the charged EC and the corresponding

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EUM matched, low levels of charged EC were observed, as indicated in the circuits

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Pdh–Ldh–NAD, Pdh–Mae*–NCD and Pdh*–Ldh–NAD. However, a relatively higher level of

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NCDH (28 µM) was observed in the circuit Pdh*–Mae*–NCD. This was likely due to the rate

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of NCDH formation by Pdh* overpowered that of NCDH consumption by Mae*. These data

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indicate that energy transfer can be regulated by tuning the compatibility between EUM and

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EC.

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Table 1. Kinetic parameters of Pdh.a Enzyme

Cofactor KM (µM)

kcat (s-1)

Pdhb

NADd

0.37±0.04

0.26±0.01

702

NCDe

62.2±8.3

0.35±0.02

5.62

NADd

54.1±7.2

0.12±0.00

2.21

NCDe

13.7±1.3

0.13±0.01

9.49

Pdh*c

a

kcat /KM (mM -1 s-1)

NCD preference 0.008

4.27

Assays were done in 50 mM HEPES (pH 7.5) contained 5 mM phosphite, 0.4 mM

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 1 mM phenazine ethosulfate, cofactor and phosphite dehydrogenase. Reaction rates were obtained by monitoring the absorbance at 570 nm at room temperature. Assays were done in triplicate and the data represent the average ± standard deviation. bPdh concentration was 0.064 µM. cPdh* concentration was 0.32 µM. dNAD concentration ranged from 0.01 to 200 µM. eNCD concentration ranged from 0.1 to 1000 µM. 126

We further checked the energy transfer properties in more complicated circuits

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Pdh–Ldh/Mae*–NAD/NCD and Pdh*–Ldh/Mae*–NAD/NCD, which mimicked systems

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integrating both synthetic and natural redox pathways. It was found that NCDH levels of the

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two circuits were essentially identical to those of the circuit Pdh–Mae*–NCD and

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Pdh*–Mae*–NCD, while NADH levels identical to those of the circuit Pdh–Ldh–NAD and

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Pdh*–Ldh–NAD, respectively (Figure 3B). These results demonstrated that energy transfer

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via the subcircuit Pdh–Mae*–NCD was orthogonal to the subcircuit Pdh–Ldh–NAD, in the

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circuit Pdh–Ldh/Mae*–NAD/NCD. Similar phenomena were found for the circuit

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Pdh*–Ldh/Mae*–NAD/NCD. In these two examples, NADH and NCDH were generated

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simultaneously as Pdh and Pdh* had appreciable activity in terms of charging both NAD and

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NCD (Table S3 and Figure S3), however, it remained effective to transfer energy selectively

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due to high EC preference of the EUM.

B

Ldh

Pdh

NADH

Charged EC (mM)

A

Mae*

NCDH Pdh*

Pdh

50 40 30 20 10 0

Mae*

+ + – – + –

+ + +

– – + + + –

+ + – – + –

+ + +

– + +

– + –

Ldh

– +

+

– +

– +

+

–

+

NAD NCD

C

D 0.6

◆

Vlac

Reaction rate (mM/h)

Reaction rate (mM/h)

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■ Vmal

0.5 0.4 0.3 0.2 0.1

0.6

◆

■ Vmal

0.5 0.4 0.3 0.2 0.1 0.0

0.0 0

20

40

60

80 100 120

0

20

NCD (mM)

138

Vlac

40

60

80 100 120

NAD (mM)

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Figure 3. Energy transfer by in vitro metabolic circuits. (A) A model metabolic circuit using Pdh as

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ESM (with 5 mM phosphite as substrate), 50 µM NAD and NCD as EC, and Ldh and Mae* as EUM (with

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50 mM pyruvate as substrate). Filled star, NADH; Partially empty star, NAD; Filled pentagon, NCDH;

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Partially empty pentagon, NCD. Arrow indicates the direction of energy transfer. (B) Charged EC levels of

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circuits consisted of different ESM and EUM. Assay conditions and data are shown in Tables S2. (C)

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Product formation rates of the model circuit in the presence of 10 µM NAD and various amounts of NCD.

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(D) Product formation rates of the model circuit in the presence of 10 µM NCD and various amounts of

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NAD.

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We also determined the product formation rates by the complicated circuit

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Pdh*–Ldh/Mae*–NAD/NCD while holding one EC constant but varying the concentration of

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the other. It was found that the malate formation rate increased positively proportional to the

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initial NCD concentration, while the lactate formation rate held constant (Figure 3C). In

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addition, the malate formation rate was almost identical to those of the simple circuit

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Pdh*–Mae*–NCD under identical assay conditions (Figure S4). Vice versa, the lactate 7


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formation rate increased linearly over a wide range of initial NAD levels, while the malate

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formation rate held constant (Figure 3D). These data showed successful controlling reductive

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conversion of pyruvate to either malate or lactate where enzymes were present for both malic

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enzyme activity and lactate dehydrogenase activity. Thus, selective energy transfer may be

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achieved upon the incorporation of synthetic EC, by which crosstalk between the synthetic

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subsystem and the NAD–linked metabolism can be drastically reduced.

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Construction and characterization of NCD-linked metabolic circuit in vivo. To

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demonstrate the usefulness of NCD-linked metabolic circuits for controlling the energy

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transfer in vivo, we assembled the circuit Pdh*–Mae*–NCD into the model microorganism

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Escherichia coli. The idea was to use Pdh* for generation of NCDH, which drives specifically

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the reductive carboxylation of pyruvate to produce malate (Figure 4A). It should be

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emphasized that, under physiological conditions, the oxidative decarboxylation of malate into

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pyruvate is the favored reaction direction.19 Therefore, if the devised circuit works, it will lead

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to the reversion of the direction of the natural metabolic pathway.

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To minimize the consumption of malate and pyruvate by endogenous pathways, we used

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the host strain E. coli XZ654, in which malate is formed from phosphoenolpyruvate (PEP) via

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oxaloacetate and major enzymes including those responsible for the interconversion between

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malate and pyruvate are blocked (Figure S5).20 To enable NCD uptaking by E. coli cells, we

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expressed the nucleotide transporter gene ndt from Arabidopsis thaliana under the control of

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the gapAP1 promoter.21, 22 A simplified metabolism is shown to convert glucose into malate

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by the designed strain WL005 (Figure 4A). At the systems level, it is clear that there were two

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layers of circuits (Figure 4B), namely genetic circuit for the control of the production of three

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enzymes and metabolic circuit for the control of energy transfer. Similarly, we also

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constructed WL010 that harbored the circuit Pdh*–Mae*–NCD but without the ndt gene. The

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function of Ndt was confirmed because substantially higher intracellular NCD concentrations

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were observed in WL005 cells in the presence of extracellular NCD (Figure 4C). Successful

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expression of other genes was confirmed by protein gel electrophoresis and Western blot

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(Figure S6) and enzymatic activity assay (Table S4).

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A

C

Oxaloacetate

Cellular NCD (mM)

Glucose

PEP NADH

NAD

Mae

Malate

Pyruvate

Mae* NCD

80 60 40 20 0

NCDH

Pdh*

Ndt

NCD

Phosphite

B Genetic circuit

Gal lac Mae*

–

Pdh*

Pdh*

Mae*

Mae*

Phosphate

W

D IPTG

Ndt

IPTG ndt

gapA P1

lac

Pdh*

0 L0

5 W

10 L0

Malate (mM)

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Mae* Metabolic circuit

Ndt

NCDH

̶ +

NCD

Pdh * NCD

W

0 L0

̶ +

5 W

1 L0

0

181 182

Figure 4. Malate production by E. coli cells harboring NCD-linked metabolic circuits. (A) E. coli WL005

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harboring the circuit Pdh*–Mae*–NCD and expressing the transporter Ndt. Simplified pathways and

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reactions were shown to illustrate major reactions. Dashed arrows indicated an inactive reaction. (B)

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Different circuits in E. coli WL005 cells. Genetic circuits control gene expression, and the NCD-linked

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metabolic circuit controls energy transfer. (C) Intracellular NCD concentrations. Rest cells were incubated

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at 30 °C for 2 h in MOPS medium supplemented with 2.5 mM phosphite, 100 mM glucose and 0.1 mM

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NCD. (D) Malate production results. Cells were incubated in MOPS medium at 30 °C for 4 h in the

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presence of 100 mM glucose, 10 mM sodium bicarbonate, 2.5 mM phosphite and 0.1 mM NCD. Detailed

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data are listed in Tables S5. Experiments were done in triplicate, repeated in different days and error bars

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indicate standard deviation.

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A typical malate production process with WL005 resting cells in the presence of NCD

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produced 3.91 mM malate, about 38% more compared to that (2.82 mM) in the absence of

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NCD (Figure 4D). Malate yield was 0.11 g/g in the absence of NCD but was improved to 0.15

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g/g in the presence of NCD (Table S5). These results indicated that cells imported NCD and

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generated NCDH to drive the reductive carboxylation of pyruvate. That is, the function of the

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circuit Pdh*–Mae*–NCD created additional flux for malate formation by hijacking pyruvate 9


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that is otherwise converted into other metabolites. However, when phosphite was absent,

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WL005 cells produced 2.4 mM malate in either the presence or absence of NCD (Table S5),

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indicating that endogenous redox enzymes were insufficient to reduce NCD for Mae* activity.

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It was noteworthy mentioning that phosphite consumption by WL005 cells was 1.14 and 1.00

202

mM, respectively, for the reaction in the presence and absence of NCD. The amount of

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consumed phosphite matched well with that of increased malate when NCD was present. The

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reason that cells consumed 1.00 mM phosphite in the absence of NCD was most likely

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because Pdh* reserved obvious activity with NAD (Table 1). Compared to malate level (2.41

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mM) of the experiment in the absence of phosphite, it was apparent that cells failed to transfer

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energy for malate production but wasted energy through NADH-linked metabolism. It should

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be noted that cells remained alive during the incubation process (Figure S7). Under typical

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malate production conditions, WL010 cells, which had no cofactor importer, produced only

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2.72 mM malate in the presence of NCD (Figure 4D). The result together with those of

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WL005 cells indicated that the nucleotide transporter Ndt was key to deliver NCD to facilitate

212

a functional in vivo energy transfer circuit. Taken together, the energy carrier NCDH

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generated from phosphite was used selectively for malate formation, which was

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fundamentally different from those conventional approaches whose reduced cofactor

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formation was linked to and/or competed by endogenous metabolic reactions and was

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involving other organic substances.5-7, 9, 10, 14

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■ CONCLUSION

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Energy transfer is a particularly important aspect for metabolic engineering. In this respect,

219

tremendous efforts have been devoted to regulate the energy transfer process by shifting

220

EC-preference, for example from NADPH to NADH and visa verse, of the corresponding

221

modules or by enhancing natural EC supply.5, 7, 9 Because natural EC is shared by multiple

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energy transfer modules, metabolic pathways and biological processes, it remains inaccessible

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to selectively transfer energy to the reaction-of-interest by increasing cellular EC level.8, 23

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In this study, we introduced non-natural cofactor NCD as EC and engineered Pdh as ESM

225

to favor NCD. We then used Mae and Ldh as EUM to assemble complex metabolic circuits in

226

vitro and demonstrated the effectiveness for selective energy transfer. Finally, we expressed

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Pdh*, Mae* and Ndt in E. coli XZ654 and found cells could import NCD and generate NCDH

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in the presence of phosphite to drive reductive carboxylation of pyruvate for improved malate

229

production and yield from glucose. These in vivo data also suggested that the NCD-linked

230

subsystem was largely orthogonal at the EC level to natural metabolism in E. coli as 1) the 10


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Pdh* module generated NCDH was mainly utilized by Mae* but not other redox enzymes,

232

and 2) NCD reduction by endogenous redox enzymes was negligible. Although this work

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used NCD as non-natural EC, it is also conceivable to explore other NAD analogs such as

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nicotinamide flucytosine dinucleotide (NFCD) and nicotinamide methylcytosine dinucleotide

235

(NMeCD). Indeed, our early study showed that Mae and Ldh could be engineered to favor

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other synthetic cofactors, including NFCD and NMeCD.16, 24-25

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Our capacity on designing synthetic life has been advancing rapidly. Microorganisms that

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use synthetic nucleotides for genetic information storage26 and synthetic amino acids for

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essential protein biosynthesis27 have been established recently. In this scenario, we expect that

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the incorporation of synthetic cofactor-linked circuits into the biological systems should

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provide a new layer of control mechanism for life. To be more practical, synthetic

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cofactor-linked circuit can provide a unique tool to enable pathway-specific energy transfer,

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which should find immediate applications for engineering cell factories to produce valuable

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metabolites.

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■ EXPERIMENTAL SECTION

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Chemicals. NCD was chemically synthesized.16 Chemicals were purchased from Sigma.

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Culture conditions. E. coli cultures were grown for protein induction in Luria–Bertani

248

(LB) broth with 1 mM of IPTG and 50 µg/mL of kanamycin at 25 °C at 200 rpm for 2 d. For

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anaerobic reactions with rest cell, E. coli cultures were harvested by centrifuging at 8,000×g

250

for 2 min, washed and re–suspended to an OD600 of 9 in MOPS medium. Supplemented the

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rest cell suspension with 10 mM sodium bicarbonate, 2.5 mM phosphite, 100 mM glucose

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and when added 0.1 mM NCD, then filled 0.6 mL-tubes with 0.5 mL of the mixture and

253

incubated at 30 °C, 200 rpm for 4 h, and quenched by adding 9 volume of quenching buffer

254

(40:40:10 acetonitrile/methanol/water).28 Intracellular NCD or NAD was extracted as

255

previously reported.29 Cell extract for enzyme activity assay was prepared by treating the cells

256

with lysozyme and thawed twice by liquid nitrogen.

257

Plasmid construction and mutagenesis. Plasmids construction and screening of

258

site–directed mutation were carried out in E. coli DH10B, strains for rest cell reactions using

259

E. coli XZ654 as the host.20 Construction of plasmids and site–directed mutations were

260

carried out according to the PCR–based strategy using pUC18 as vector backbone.30 Ndt gene

261

was amplified from plasmid pBluescript–AtNDT2.21 Template for Mae gene was plasmid

262

carrying the gene on pET24b.16 Template genes for Pdh were synthesized. All the genes

263

carried a His6 tag at the N–terminal. Strains and plasmids used were listed in Table S6. 11


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Primers used were listed in Table S7. Strategies for plasmids construction were listed in Table

265

S8. Plasmids and mutants were validated by sequencing carried out by Invitrogen.

266

Protein purification. Recombinant Pdh, Mae, Ldh and their mutants were produced by E.

267

coli cells harboring the corresponding plasmids (Table S6) and purified by using the Ni–NTA

268

kit (Invitrogen). Purified proteins (Figure S8) were stored at –80 °C in 50 mM Tris–HCl (pH

269

7.5) supplemented with 20% glycerol. Protein concentration was measured by using the

270

Bio–Rad protein assay kit.

271

Analytic methods. NAD(H) and NCD(H) were assayed by enzymatic cycling assays.

272

NAD(H) concentrations were assayed by ADH as described previously.31 NCD(H)

273

concentrations were assayed by Mae*, the mixture contained 50 mM HEPES (pH 7.5), 0.4

274

mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 1 mM phenazine

275

ethosulfate (PES), 5 mM malate, 10 mM MgCl2 and 1.7 U/L Mae*. The reaction was started

276

by mixing 0.1 volume of sample with 0.9 volume of above mixture. Reaction rates were

277

determined by monitoring the increase of absorbance at 570 nm.

278

Activities of purified enzymes and cell extracts were assayed in a mixture contained 50

279

mM HEPES (pH 7.5), 0.4 mM MTT, 1 mM PES, 50 µM NCD/NAD (or gradient

280

concentration for kinetic parameters) and 5 mM substrate (phosphite for Pdh, malate for Mae,

281

lactate for Ldh), 10 mM MgCl2 was added for Mae. The reaction was started by addition of

282

0.1 volume of enzyme or cell extract. Reaction rates were obtained by monitoring the

283

absorbance at 570 nm at room temperature.32 One unit of enzymatic activity was defined as 1

284

µmol NADH produced per minute.

285

Lactate, phosphite and malate in mixture of in vitro circuits and rest cell reactions were

286

determined using an ICS–2500 ion chromatography system (Dionex, Sunnyvale, California),

287

equipped with a guard column IonPac AG11–HC (50 mm × 4 mm), an IonPac AS11–HC

288

analytic column (250 mm × 4 mm, Dionex), and an ED50A conductivity detector. The column

289

and guard column were maintained at 30 °C. A mobile phase of 18 mM NaOH was used for

290

the analysis at a rate of 1 mL/min for 30 min. Glucose was determined by biosensor analyzer.

291

Construction of in vitro metabolic circuits. In vitro metabolic circuits were constructed

292

in a 0.1 mL of mixture of 50 mM HEPES (pH 7.5), 1 mM MnCl2, 50 mM pyruvate, 10 mM

293

NaHCO3, when needed supplemented with 5 mM phosphite, 20 mM malate, 8.8 U/L Pdh or

294

8.6 U/L Pdh*, 144 U/L Mae or 165 U/L Mae*, 0.24 U/L Ldh, and 50 µM NCD/NAD (or a

295

gradient concentration to tune the circuits). The 0.1 mL of reaction mixtures were incubated at

296

30 °C for 1 h and quenched by adding 0.9 mL of quenching buffer.

12


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In vitro metabolic circuits for assay cofactor concentration were constructed in a 0.2 mL

298

of mixture as described above except substituting 1 mM MnCl2 with 10 mM MgCl2 to avoid

299

influence on MTT–PES assay. The 0.1 mL of reaction mixtures were incubated at 30 °C for 1

300

h in 1.5 mL tubes and quenched by adding 90 µL of the mixture to 10 µL 2 M HCl (for assay

301

of NAD/NCD) or 2 M NaOH (for assay of NADH/NCDH) followed a 50 °C water bath for

302

10 min. The samples were neutralized by 100 µL of 0.1 M NaOH or HCl, and stored at

303

–80 °C.

304

■ ASSOCIATED CONTENT

305

Supporting Information

306

The Supporting Information is available free of charge on the ACS Publications website.

307

Additional Tables (Table S1-S8) and Figures (Figure S1-S8) supplied as Supporting

308

Information.

309

■ AUTHOR INFORMATION

310

Corresponding Authors

311

*E-mail for W. L.: [email protected]

312

*E-mail for Z.K.Z.: [email protected]

313

ORCID

314

Zongbao K. Zhao: 0000-0003-0654-1193

315

Notes

316

The authors declare no competing financial interest.

317

■ ACKNOWLEDGMENTS

318

We thank L. O. Ingram (University of Florida, USA) for providing E. coli XZ654, H.

319

Ekkehard Neuhaus (Technische Universität Kaiserslautern, Germany) and Ferdinando

320

Palmieri (Università degli Studi di Bari Aldo Moro, Italy) for providing AtNDT2. This work

321

was supported by National Natural Science Foundation of China (Nos. 21325627, 21572227),

322

State Key Laboratory of Catalysis (R201306) and National Basic Research and Development

323

Program of China (No. 2012CB721103).

324

■ REFERENCES

325

(1) Choi, Y. J.; Lee, S. Y. Nature 2013, 502, 571–574.

326

(2) Fossati, E.; Ekins, A.; Narcross, L.; Zhu, Y.; Falgueyret, J.-P.; Beaudoin, G. A. W.; Facchini P. J.;

327

Martin, V. J. J. Nat. Commun. 2014, 5, 3283.

328

(3) Zhou, Y. J.; Gao, W.; Rong, Q.; Jin, G.; Chu, H.; Liu, W.; Yang, W.; Zhu, Z.; Li, G.; Zhu, G.; Huang,

329

L.; Zhao, Z. K. J. Am. Chem. Soc. 2012, 134, 3234–3241.

13


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 16

330

(4) Nakagawa, A.; Matsumura, E.; Koyanagi, T.; Katayama, T.; Kawano, N.; Yoshimatsu, K.; Yamamoto,

331

K.; Kumagai, H.; Sato F.; Minami, H. Nat. Commun. 2016, 7, 10390.

332

(5) Brinkmann-Chen, S.; Flock, T.; Cahn, J. K. B.; Snow, C. D.; Brustad, E. M.; McIntosh, J. A.;

333

Meinhold, P.; Zhang, L.; Arnold, F. H. Proc. Natl. Acad. Sci. USA 2013, 110, 10946–10951.

334

(6) Cardenas, J.; Da Silva, N. A. Metab. Eng. 2016, 36, 80–89.

335

(7) King, Z. A.; Feist, A. M. Metab. Eng. 2014, 24, 117–128.

336

(8) Mampel, J.; Buescher, J. M.; Meurer, G.; Eck, J. Trends Biotechnol. 2013, 31, 52–68.

337

(9) Wang, Y.; San, K. Y.; Bennett, G. N. Curr. Opin. Biotechnol. 2013, 24, 994–999.

338

(10) Chen, X.; Li, S.; Liu, L. Trends Biotechnol 2014, 32, 337–343.

339

(11) Agapakis, C. M.; Ducat, D. C.; Boyle, P. M.; Wintermute, E. H.; Way, J. C.; Silver, P. A. J. Biol. Eng.

340

2010, 4, 3.

341

(12) Nielsen, A. Z.; Ziersen, B.; Jensen, K.; Lassen, L. M.; Olsen, C. E.; Moller, B. L.; Jensen, P. E. ACS

342

Synth. Biol. 2013, 2, 308–315.

343

(13) Tan, C. Y.; Hirakawa, H.; Nagamune, T. Sci Rep 2015, 5, 8648.

344

(14) Opgenorth, P. H.; Korman, T. P.; Bowie, J. U. Nat. Commun. 2014, 5, 4113.

345

(15) Way, J. C.; Collins, J. J.; Keasling, J. D.; Silver, P. A. Cell 2014, 157, 151–161.

346

(16) Ji, D.; Wang, L.; Hou, S.; Liu, W.; Wang, J.; Wang, Q.; Zhao, Z. K. J. Am. Chem. Soc. 2011, 133,

347

20857–20862.

348

(17) Vrtis, J. M.; White, A. K.; Metcalf, W. W.; van der Donk, W. A. Angew. Chem. Int. Ed. 2002, 41,

349

3257–3259.

350

(18) Hirota, R.; Yamane, S.-t.; Fujibuchi, T.; Motomura, K.; Ishida, T.; Ikeda, T.; Kuroda, A. J. Biosci.

351

Bioeng. 2012, 113, 445–550.

352

(19) Stols, L.; Donnelly, M. I. Appl. Environ. Microbiol. 1997, 63, 2695–2701.

353

(20) Zhang, X.; Wang, X.; Shanmugam, K. T.; Ingram, L. O. Appl. Environ. Microbiol. 2011, 77, 427–434.

354

(21) Palmieri, F.; Rieder, B.; Ventrella, A.; Blanco, E.; Do, P. T.; Nunes-Nesi, A.; Trauth, A. U.; Fiermonte,

355

G.; Tjaden, J.; Agrimi, G.; Kirchberger, S.; Paradies, E.; Fernie, A. R.; Neuhaus, H. E. J. Biol. Chem. 2009,

356

284, 31249–31259.

357

(22) Charpentier, B.; Branlant, C. J. Bacteriol. 1994, 176, 830–839.

358

(23) Hou, J.; Scalcinati, G.; Oldiges, M.; Vemuri, G. N. Appl. Environ. Microbiol. 2010, 76, 851–859.

359

(24) Ji, D.; Wang, L.; Liu, W.; Hou, S.; Zhao, Z. K. Sci. China Chem. 2013, 56, 296–300.

360

(25) Ji, D.; Wang, L.; Zhou, Y. J.; Yang, W.; Wang, Q.; Zhao, Z. K. Chin. J. Catal. 2012, 33, 530–535.

361

(26) Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Correa, I. R., Jr.;

362

Romesberg, F. E. Nature 2014, 509, 385–388.

363

(27) Rovner, A. J.; Haimovich, A. D.; Katz, S. R.; Li, Z.; Grome, M. W.; Gassaway, B. M.; Amiram, M.;

364

Patel, J. R.; Gallagher, R. R.; Rinehart, J.; Isaacs, F. J. Nature 2015, 518, 89–93.

365

(28) Bennett, B. D.; Yuan, J.; Kimball, E. H.; Rabinowitz, J. D., Nat. Protoc. 2008, 3, 1299–1311.

366

(29) Zhou, Y. J.; Wang, L.; Yang, F.; Lin, X.; Zhang, S.; Zhao, Z. K., Appl. Environ. Microbiol. 2011, 77,

367

6133–6140.

14


Page 15 of 16

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ACS Catalysis

368

(30) Wang, J.; Zhang, S.; Tan, H.; Zhao, Z. K. J. Microbiol. Methods 2007, 71, 225–230.

369

(31) Wang, L., Zhou, Y.; Ji, D.; Lin, X.; Liu, Y.; Zhang, Y.; Liu, W.; Zhao, Z. K. Enzyme Microb. Technol.

370

2014, 58-59, 75–79.

371

(32) San, K. Y.; Bennett, G. N.; Berrios-Rivera, S. J.; Vadali, R. V.; Yang, Y. T.; Horton, E.; Rudolph, F. B.;

372

Sariyar, B.; Blackwood, K., Metab. Eng. 2002, 4, 182–192.

373 374

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TOC graphic Glucose Oxaloacetate

PEP

NAD

Malate

Mae

NADH

Pyruvate

Mae * NCD

Pdh*

NCDH

Ndt

376

NCD

Phosphite

e

Phosphate

16


Synthetic Cofactor-Linked Metabolic Circuits for Selective Energy

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