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Modular engineering intracellular NADH regeneration boosts extracellular electron transfer of Shewanella oneidensis MR-1 Feng Li, Yuanxiu Li, Li-ming Sun, xiaoli chen, Xingjuan An, Changji Yin, Yingxiu Cao, Hui Wu, and Hao Song ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00390 • Publication Date (Web): 11 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

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ACS Synthetic Biology

Modular engineering intracellular NADH regeneration extracellular electron transfer of Shewanella oneidensis MRMR-1

boosts boosts

Feng Li†, ‡, Yuanxiu Li†, ‡, Liming Sun§, Xiaoli Chen†, Xingjuan An†, Changji Yin†, Yingxiu Cao†, Hui Wu#, Hao Song†, * †

Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, SynBio Research Platform, Collaborative Innovation Centre of Chemical Science and Engineering, Tianjin University, Tianjin, 300072, China. §

Petrochemical Research Institute, PetroChina Company Limited, Beijing 102206, China.

#

State Key Laboratory of Bioreactor Engineering, Key Laboratory of Bio-based Material Engineering of China National Light Industry Council, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.

KEYWORDS: Shewanella oneidensis, extracellular electron transfer, NADH regeneration, reducing equivalents, cofactor engineering, microbial fuel cells

ABSTRACT: Efficient extracellular electron transfer (EET) of exoelectrogens is essentially for practical applications of versatile bio-electrochemical systems. Intracellular electrons flow from NADH to extracellular electron accepters via EET pathways. However, it was yet established how the manipulation of intracellular NADH impacted the EET efficiency. Strengthening NADH regeneration from NAD+, as a feasible approach for cofactor engineering, has been used in regulating the intracellular NADH pool and the redox state (NADH/NAD+ ratio) of cells. Herein, we firstly adopted a modular metabolic engineering strategy to engineer and drive the metabolic flux toward the enhancement of intracellular NADH regeneration. We systematically studied sixteen genes related to the NAD+-dependent oxidation reactions for strengthening NADH regeneration in the four metabolic modules of S. oneidensis MR-1, i.e., glycolysis, C1 metabolism, pyruvate fermentation, and tricarboxylic acid cycle. Among them, three endogenous genes mostly responsible for increasing NADH regeneration were identified, namely gapA2 encoding a NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase in the glycolysis module, mdh encoding a NAD+-dependent malate dehydrogenase in the TCA cycle, and pflB encoding a pyruvate-formate lyase that converted pyruvate to formate in the pyruvate fermentation module. An exogenous gene fdh* from Candida boidinii encoding a NAD+-dependent formate dehydrogenase to increase NADH regeneration in the pyruvate fermentation module was further identified. Upon assembling these four genes in S. oneidensis MR-1, ∼4.3-folds increase in NADH/NAD+ ratio, and ∼1.2-folds increase in intracellular NADH pool were obtained under anaerobic conditions without discharge, which elicited ∼3.0-folds increase in the maximum power output in microbial fuel cells, from 26.2 ± 2.8 (wild-type) to 105.8 ± 4.1 mW/m2 (recombinant S. oneidensis), suggesting a boost in the EET efficiency. This modular engineering method in controlling the intracellular reducing equivalents would be a general approach in tuning the EET efficiency of exoelectrogens.

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Extracellular electron transfer (EET) of exoelectrogens involves the flow of intracellular electrons derived from oxidative metabolism of carbon sources to extracellular electron acceptors (such as minerals and inert electrodes) via c-type cytochromes-based and/or soluble electron shuttle-mediated pathways.1-3 Highly efficient EET is essential for the industrial application of a diverse array of bio-electrochemical systems (BES) in environmental and energy fields,4-7 including, microbial fuel cells (MFC) for simultaneous biodegradation of organic wastes and harvest of bioelectricity energy,8-13 unbalanced electro-fermentation (UBF) for biorefinery of organic wastes and biomass to accomplish the production of chemicals,14-18 microbial electrolysis cells (MEC) for hydrogen production,19-21 microbial desalination cells (MDC) for synchronous wastewater treatment and seawater desalination,22-24 and microbial electrosynthesis (MES) for production of valuable chemicals and biofuels from CO2 bioreduction.25-32 However, low EET efficiency remained a crucial bottleneck that seriously restrained commercial applications of these BES systems. The reduced form of nicotinamide adenine dinucleotide (NADH) serving as the main intracellular reducing equivalents is the major releasable intracellular electron carrier and the source of EET.33-35 It is thus conceivable that manipulating intracellular reducing equivalents through increasing NADH regeneration may lead to an increase in the efficiency of EET. NAD(H/+) are also pivotal cofactors participating in most redox biochemical reactions in the microbial metabolism, in which NADH provides cells with reducing equivalents for reductive reactions, meanwhile, NAD+ is the electron acceptor upon the NAD+-dependent oxidative breakdown of organic substrates.36 In addition, NAD+ is also linked intrinsically to signaling reactions inside and outside cells that control gene expression, Ca2+ mobilization, cell death and aging, etc.37 Cofactor engineering was thus developed to manipulate intracellular NAD(H/+) level via metabolic engineering approaches, which had been proven to enable an efficient control of intracellular NAD(H/+) availability.38-42 Such cofactor engineering strategies mainly included introducing biogenesis of NAD+ to control total NAD(H/+) level,43-44 and manipulating the consumption and regeneration of NADH to regulate the NADH/NAD+ ratio.45-46 The underlying EET mechanisms of Shewanella oneidensis MR-1, one of the most intensively studied metal-reducing exoelectrogens,47-48 were intensively investigated in recent decade49-50. Electrons were generated from central carbon metabolism of lactate (i.e., the carbon source and electron donor to S. oneidensis) and stored in the form of NADH as the intracellular electron carrier, which was converted from NAD+ via oxidative reactions catalyzed by a number of dehydrogenases.51-52 Electrons subsequently transferred from NADH to the outer membranes through the menaquinol pool and a number of conductive c-type cytochromes (c-Cyts) including OmcA-MtrCAB and CymA,53-57 and finally to the extracellular electron accepters such as carbon electrodes or metal minerals through the contacted-based EET by c-Cyts and the solvable electron shuttle-mediated EET by redox active flavins.58-59 Thus, NADH and NAD+ were not only essential cofactors for the metabolism of S. oneidensis, but also the intracellular electron carrier and source for its EET. However, there was barely any cofactor engineering effort in genetic manuplation of intracellular NADH to study its effects on EET

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of S. oneidensis. Herein, we developed a modular engineering strategy to systematically study how NADH regeneration in the four modules of central carbon metabolism (i.e., glycolysis, serine-glycine C1 metabolism, pyruvate fermentation pathway, and TCA cycle, as shown in Figure 1) contributed to the intracellular NADH/NAD+ ratio, intracellular NADH pool, and EET efficiency of S. oneidensis. By systematic study of the sixteen genes related to the NAD+-dependent oxidation reactions for enhancing NADH regeneration in the four metabolic modules, we identified three most crucial endogenous genes, namely, the gapA2 gene encoding the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH) for the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate in the glycolysis module, the mdh gene encoding the NAD+-dependent malate dehydrogenase for converting malate to oxaloacetate in the TCA cycle, and the pflB gene encoding a pyruvate-formate lyase to convert pyruvate to formate in the pyruvate fermentation module,60 which were most significantly responsible for strengthening NADH regeneration from NAD+ in S. oneidensis,61-62 thus increasing its intracellular NADH/NAD+ ratio, and subsequently intracellular NADH pool. In the pyruvate fermentation module, formate is not only product of pyruvate disproportionation catalyzed by the pyruvate-formate lyase under anaerobic conditions, but also a metabolite that carries electrons.63-64 According to the metabolic flux analyses of S. oneidensis, multiple copies of genes encoding formate dehydrogenase and hydrogenase were identified in the genome of S. oneidensis MR-1 to catalyze the conversion of formate to H2 and CO2, which however could not regenerate NADH.65 To enhance NADH regeneration, we thus constructed an exogenous fdh* gene from Candida boidinii encoding a NAD+-dependent formate dehydrogenase that could convert formate to CO2 with NADH regeneration,46, 66 instead of the native reaction that converted formate to CO2 and H2 in S. oneidensis. Upon assemblage of the above four genes (gapA2, mdh, pflB and fdh*), the metabolic flux was redirected to increase NADH regeneration in S. oneidensis, achieving ∼4.3-folds increase in the NADH/NAD+ ratio and ∼1.2-folds increase in reducing equivalents (i.e., NADH, the pool of intracellular electron carrier) under anaerobic conditions without discharge. Such increase resulted in a ∼3.0 folds increase in the maximum electricity output of MFCs, from 26.2 ± 2.8 mW/m2 by the wild-type S. oneidensis (harboring an empty plasmid) to 105.8 ± 4.1 mW/m2 by the recombinant S. oneidensis. Our work demonstrated that modular engineering intracellular NADH regeneration was a feasible approach to promote the EET efficiency of exoelectrogens.

Results and Discussion NADH is a principal electron donor to the respiratory chains, also the main intracellular reducing equivalents synthesized by the central carbon metabolism.67 To systematically engineer and redirect metabolic flux to increase NADH regeneration thus improving reducing equivalents in S. oneidensis MR-1, a rational modular engineering strategy was adopted in this study to categorize the central carbon metabolism into four metabolic modules, i.e., glycolysis, C1 metabolism, pyruvate fermentation, and TCA cycle (Figure 1). Subsequently, the intracellular NAD+-dependent oxidative reactions and the 2


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ACS Synthetic Biology oneidensis under anaerobic conditions69.

corresponding genes (marked in blue, Figure 1) responsible for the NADH regeneration from NAD+ were systematically investigated.

Optimization of the pyruvate fermentation module Pyruvate is a center in the central carbon metabolism of S. oneidensis, and the pyruvate fermentation module is a bridge to connect the glycolysis module and the TCA cycle module,48 through which a percentage of pyruvate derived from lactate is shunted into TCA cycle for the production of ATP, reducing equivalents and metabolites. The gene aceE encodes an essential component of the pyruvate dehydrogenase complex (Pdh), which catalyzes the conversion of pyruvate to acetyl-CoA and carbon dioxide while reducing NAD+ to NADH. We thus constructed a S. oneidensis mutant with overexpressed aceE (Figure 2C, left column), however found that this recombinant S. oneidensis strain with overexpressed native gene aceE also showed a similar voltage output and NADH pool in comparison to the WT strain (Figure 2C, middle and right columns). This may be also due to a previous observation that the enzyme AceE was mostly inactive under anaerobic conditions of MFCs.60, 65 Alternatively, the gene aceE only encodes one of the essential components of Pdh, which may be insufficient to enhance the overall catalytic activity of Pdh. Pyruvate can be transformed into malate by NAD+-dependent malate dehydrogenase encoded by the sfcA gene through anapleurotic reactions that reduced NAD+ to NADH and provided malate for TCA cycle. We thus constructed a recombinant S. oneidensis strain with overexpressed native gene sfcA (Figure 2C, left column), which however showed a similar voltage output in comparison to the WT strain. A previous finding may partially provide a possible clue for explanation, in which the metabolic flux from pyruvate to malate (NADH consumption) overwhelmed over that of the reversible reaction (NADH regeneration).61 Our experiments further showed that the expression of sfcA had a negligible impact on the intracellular NADH level (Figure 2C, right column). Under anaerobic conditions, the gene pflB encoding pyruvate-formate lyase catalyzes the conversion of pyruvate to acetyl-CoA and formate. Acetyl-CoA is further transformed into acetate and accompanied by substrate phosphorylation to produce ATP.72 Formate is a reduced metabolite, which could play as an electron donor to S. oneidensis for power generation. Formate can be converted to CO2 and H+ by the individual native formate dehydrogenase,63-64 or CO2 and H2 by the combined action of the native formate dehydrogenase and hydrogenase enzymes in S. oneidensis,65 however, NADH was not regenerated in these reactions. To enhance the production of formate, we overexpressed the endogenous pflB gene encoding a pyruvate-formate lyase to convert pyruvate to formate. To further release the electrons stored in formate to the anode, we constructed an exogenous NADH regeneration system, i.e., an exogenous fdh* gene from Candida boidinii encoding a NAD+-dependent formate dehydrogenase to convert formate to NADH and CO246, 66, 73 (Figure 1, and Figure 2C, left column). Notably, the profiles of MFC output voltage showed that the S. oneidensis strains with the overexpressed pflB and fdh* produced a maximum voltage of ~243.4 ± 4.4 mV and ~266.4 ± 7.4 mV (n = 3), respectively, which was significantly higher than that of the WT strain (~208 ± 8.2 mV) and the mutant with the overexpressed native fdh gene (~227 ± 10 mV) (Figure 2C, middle column). To

Optimization of the glycolysis module In glycolysis, NADH is mainly regenerated by a few essential redox enzymes, i.e., glyceraldehyde 3-phosphate dehydrogenases encoded by the genes gapA1 (SO_0538), gapA2 (SO_2345) and gapA3 (SO_2347) in the genome of S. oneidensis MR-1, as predicted by the genomic sequence analysis.62, 68 The protein GapA2 and GapA3 (encoded by gapA2 and gapA3) were the two paralogues of the protein GapA1 (encoded by gapA1), sharing 43% and 34% identity with that of GapA1, respectively.62, 68 We thus constructed three distinct S. oneidensis recombinants harboring gapA1, gapA2, and gapA3, respectively (Figure 2A, left column), to study whether the overexpression of these individual genes in the glycolysis module would increase the EET efficiency. Among these three genes, the overexpression of gapA2 induced a significant increase in the MFC’s voltage output (~263 ± 5.8 mV) over that of the wild-type (WT) strain (~208 ± 8.2 mV), whereas the effect of overexpressing gapA1 and gapA3 on the power generation was negligible (Figure 2A, middle column). Our results were in line with a previous study, in which the most significant increase in the activity of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in S. oneidensis was induced by the expression of gapA2, instead of gapA1 and gapA3.60, 62 These results suggested that NADH could be efficiently regenerated from NAD+ by the overexpression of gapA2. To ascertain this hypothesis, we further measured the intracellular reducing equivalents (i.e., NADH) level in the three S. oneidensis mutants harboring gapA1, gapA2, and gapA3, respectively. As expected, the recombinant harboring gapA2 generated higher intracellular NADH pool (Figure 2A, right column), which was in good agreement with that of the output voltage of MFCs. Thus, overexpression of gapA2 resulted in a significant increase in the intracellular reducing equivalents (i.e., NADH), which subsequently enhanced EET efficiency. Optimization of the C1 metabolic module As a branch of glycolysis pathway, C1 metabolic module was responsible for the production of serine and glycine,63, 69-70 in which NADH and formate were synthesized as the intracellular electron carriers.61, 71 To increase the NADH regeneration, we overexpressed the relevant genes encoding NAD+-depended enzymes in S. oneidensis, namely, serA, serB, serC, gylA and gcvT, respectively, which could convert NAD+ to NADH (Figure 2B, left column). However, no significant increase in NADH pool was observed upon the individual overexpression of these genes under anaerobic conditions in the absence of voltage discharge (Figure 2B, right column). These results suggested that NADH regeneration in the C1 metabolic module was insensitive to the overexpression of these NAD+-dependent genes. Consequently, the MFC inoculated with each of these recombinant S. oneidensis strains did not exhibit any significant increase in its voltage output (Figure 2B, middle column), suggesting that the EET efficiency was not enhanced by the overexpression of these NAD+-dependent genes in the C1 metabolic module. The result may be attributed to the inactive amino acid metabolism of S. 3



further verify the hypothesis whether the improved output voltage was resulted from the increase in the level of intracellular reducing equivalents, we examined the intracellular NADH level in these strains. As expected, the results of the output voltage were in accordance with the intracellular level of NADH pool (Figure 2C, middle and right columns). We thus selected the genes pflB and fdh* for the subsequent design of pathway assembly.

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3B) in the central carbon metabolism at an optimized expression level (upon 0.8 mM IPTG induction, Figure S1, SI). We found that among these engineered S. oneidensis strains, the strain pYYDT-F4 (i.e., harboring the four gene genes related to NADH regeneration in the central carbon metabolic pathways) was able to generate the highest output voltage of MFC (Figure 3B). The increased expression level of the genes was further quantified by the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) (Figure 3C). To determine whether the enhancement of EET was attribute to the increase in the intracellular releasable electron pool, which was analyzed in all the engineered strains under anaerobic conditions in the absence of discharge and the MFC discharge conditions. We found the intracellular NADH/NAD+ ratio in the engineered strain pYYDT-F4 was augmented by ~4.3 folds than that of the WT strain under anaerobic conditions without discharge (Figure 3D), meanwhile, the intracellular level of reducing equivalents (i.e., NADH) increased ~1.2 folds higher than that of the WT strain (Figure 3E). Similar trends were also observed under MFC conditions after discharge (Figure 3F-3G). We found the ratio of NADH/NAD+ and the NADH pool size in the engineered strain pYYDT-F4 were increased by 2.5-, and 1.0-folds higher than those of the WT strain, respectively (Figure 3F-3G). These results suggested that the enhancement of the MFC output voltage of the S. oneidensis strains was in accordance with the increase in the intracellular level of reducing equivalents in these recombinant strains, indicating that NADH regeneration could result in the escalation of the EET efficiency.

Optimization of the TCA cycle module The TCA cycle is not only responsible for the production of ATP to maintain cellular energy metabolism, but also producing reducing equivalents. Because the TCA cycle of S. oneidensis was incomplete under anaerobic conditions,60 we accordingly incorporated three genes to make the TCA cycle complete in S. oneidensis, including the native gene icd encoding the isocitrate dehydrogenase that catalyzed the reaction from isocitrate to α-ketoglutarate, the native mdh gene encoding the NAD+-dependent malate dehydrogenase that converted malate to oxaloacetate, and a heterogeneous gene sucB from Geobacter sulfurreducens (another well-established exoelectrogen) encoding the α-ketoglutarate dehydrogenase responsible for the conversion of α-ketoglutarate to succinyl-CoA under anaerobic conditions (Figure 1, and Figure 2D, left column). 74 We found that overexpression of the gene mdh could produce a maximum voltage of ~268.4 ± 3.6 mV (n = 3) (Figure 2D, middle column), significantly higher than that of the WT strain (~208 ± 8.2 mV). This suggested that the overexpression of mdh could enhance the EET efficiency of S. oneidensis. The overexpression of mdh increased the oxidation of malate to form oxaloacetate, which enabled NADH regeneration from NAD+ 61, 75 (Figure 2D, right column). This may be caused by the formation of a high amount of malate from pyruvate (Figure 1), thus a further oxidation of malate would enable more NADH regeneration. However, the overexpression of either heterogeneous gene sucB or native gene icd (Figure 1) had a negligible impact on the output voltage (Figure 2D, middle column) and the intracellular NADH pool (Figure 2D, right column). These observations were consistent with previous findings that under anaerobic conditions of MFC, a great deal (~70%) of carbon source (i.e., lactate) taken by Shewanella was to form acetate,61 and the TCA cycle was very limited.60-61, 75

Bio-electrochemistry analyses of the EET efficiency of engineered S. oneidensis The voltage output of MFCs with multiple operation cycles suggested that the recombinant S. oneidensis pYYDT-F4 could generate a maximum voltage of ~414.2 ± 12.4 mV (n = 3), much higher than that of the WT strain ~208 ± 8.2 mV (n = 3) (Figure 4A). Bio-electrochemical analyses were conducted to further study the EET efficiency of the rationally engineered pYYDT-F4 stain in MFCs. Cyclic voltammetry (CV) at 1 mV/s was applied to reveal the redox reaction kinetics at the interfaces of the cell and anode. As shown in Figure 4B, there was a typical redox peak of flavins in the CV curves starting from ~-0.41 V (vs. Ag/AgCl), which showed a flavins-mediated catalytic current.58, 79 Furthermore, another catalytic current arose at ~-0.27 V (vs. Ag/AgCl) was observed in both the WT and the recombinant S. oneidensis, which was caused by the direct involvement of conductive c-type cytochromes. We thus concluded that both the flavins-mediated and contact-based EET were increased owing to enhanced NADH regeneration in our engineered strain. The polarization curves (output voltage vs. current density) and the power output curves (power density vs. current density) were used to further investigate the capability of bioelectricity generation of the MFCs inoculated with the WT and the recombinant S. oneidensis pYYDT-F4, respectively (Figure 4C). Notably, the dropping slope of polarization curves obtained from the recombinant S. oneidensis pYYDT-F4 was smaller than that obtained from the WT strain, implying that the internal charge transfer resistance of the recombinant S. oneidensis pYYDT-F4 was smaller than that of the WT S. oneidensis. The power density output curves showed that the

Modular assembly of multiple essential genes identified from metabolic modules to enhance NADH regeneration and promote EET Upon the above optimization of the redox reactions and the corresponding reducing equivalents in the individual four metabolic modules of lactate metabolism in Shewanella, we identified four genes that were mostly responsible for NADH regeneration, namely the pflB and fdh* genes in the pyruvate fermentation module, the gapA2 gene in the glycolysis module, and the mdh gene in the TCA cycle. We then assembled these genes in a step-by-step manner by the Biobrick ligation technology,76-78 and constructed four engineered strains (namely pYYDT-F1, -F2, -F3 and -F4) harboring each of these four plasmids (as shown in Figure 3A). The EET efficiency of each strain was examined in MFC, and the output voltage was found to increase with the increased number of genes (Figure 4


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ACS Synthetic Biology NAD+-dependent formate dehydrogenase in the pyruvate fermentation module. Upon assemblage of these four genes for their simultaneous overexpression in the recombinant S. oneidensis, the NADH/NAD+ ratio and intracellular reducing equivalents (i.e., NADH) were increased by ~4.3 and ~1.2-folds, respectively, which consequently enabled a ∼3.0 times’ increase in the maximum power output in MFCs, from ~26.2 ± 2.8 mW/m2 (the WT S. oneidensis MR-1) to ~105.8 ± 4.1 mW/m2 (the recombinant S. oneidensis with enhanced NADH regeneration). This study provided a systematic approach in tuning the intracellular reducing equivalents and EET efficiency of exoelectrogens by modular engineering of central carbon metabolism.

recombinant S. oneidensis pYYDT-F4 obtained a maximum power density of ~105.8 ± 4.1 mW/m2 (n = 3), which is ∼3.0 times higher than that of the WT strain (~26.2 ± 2.8 mW/m2, n = 3) (Figure 4C). A typical example was selected from triplicate experiments (Figure S2). In addition, we found that the recombinant S. oneidensis pYYDT-F4 had a slight growth advantage over that of the WT S. oneidensis under anaerobic conditions, which may be attributed to the increased metabolic flux by electrode respiration, thus facilitating cell growth (Figure 5A). In additional, the decrease in the formate and succinate synthesis and the increase in the acetate synthesis in the anolytes of MFCs also indicated that the programmed cofactor regeneration system redirected the metabolism of S. oneidensis toward the enhancement of intracellular NADH regeneration (Figure 5B). We further found the recombinant S. oneidensis pYYDT-F4 could synthesize a slightly higher level of riboflavin than that of the WT S. oneidensis (Figure 5C), which may be resulted from the enhanced intracellular reducing equivalents. Such increase in the synthesis of riboflavin (i.e., the electron shuttle) could also contribute to the increase in the power generation of the recombinant S. oneidensis pYYDT-F4. To clarify whether the improved EET could be also attributed to more attached Shewanella cells on the anodes or the higher electro-activity of single cell enabled by riboflavin, the CV signals were further normalized to the colonized biomass on electrodes (Figure 5D). The recombinant S. oneidensis pYYDT-F4 with the enhanced NADH regeneration was found to have a total ~285.6 ± 8.7 µg protein (n= 3) attached on the anodes, while the WT S. oneidensis had ~232 ± 12.4 µg protein (n = 3) attached on the anodes. The CV (at 1mV/s) normalized by the attached biomass showed a higher EET rate of the single recombinant cell of S. oneidensis over that of the WT S. oneidensis (Figure 5D). These results indicated that the overall elevated EET was not only due to the higher bio-electrochemical activity of each cell enabled by the increased intracellular NADH and electron shuttle (riboflavin), but also due to more colonized cells on the anodes.

Materials and methods In vitro gene synthesis. The information and coding sequences of genes (Table 1) were extracted from NCBI database and adapted for expression in S. oneidensis MR-1 by a Java codon adaption tool (JCAT) in order to prevent blocked translation due to shortage of tRNAs for rare codons. Each gene component was synthesized as a Biobrick,76-78, 80 and restriction enzyme sites of EcoRI, XbaI, SpeI, and SbfI were avoided in the codon-optimized sequences. The optimized gene sequence was flanked by an upstream prefix (containing EcoRI and XbaI), a RBS site (BBa_B0034, iGEM) 6 bp ahead of the start codon, and a downstream suffix (containing SpeI and SbfI) (Table S1). The designed gene sequences were synthesized in vitro, verified by Sanger sequencing (AuGCT, China). Plasmid construction, transformation and culture conditions. All plasmid constructions were performed in Escherichia coli Trans T1. E. coli strains were cultured in the LB (Luria-Bertani) medium at 37°C with 200 rpm. Whenever needed, 50 µg/ml kanamycin was added in the culture medium for plasmid maintenance. To benefit the multi-gene assembly in S. oneidensis, a Biobrick compatible expression vector pYYDT was adopted, which allowed various levels of gene expression upon differential IPTG induction, being previously constructed in our laboratory. 76 With an increased number of genes from central carbon metabolism being programmed, the constructed plasmids were named as pYYDT-F1, pYYDT-F2, pYYDT-F3 and pYYDT-F4, respectively (Figure 3A). One additional Ptac promoter was placed after the gene gapA2 to achieve coordinated and efficient expression of all genes in the plasmid. The plasmid to be transformed into S. oneidensis MR-1 (ATCC 700550) was firstly transformed into the plasmid donor strain E. coli WM3064 (auxotroph), and transferred into S. oneidensis by conjugation. Then, 100 µg/ml 2, 6-diaminopimelic acid (DAP) was added for the growth of E. coli WM3064. For anaerobic growth, 0.6 ml S. oneidensis culture suspension was inoculated into 15 ml Shewanella basal medium (SBM), including K2HPO4, 0.225 g/l; KH2PO4, 0.225 g/l; NaCl, 0.46g/l; (NH4)2SO4, 0.225 g/l; MgSO4 0.117 g/l, casamino acids 0.2g/l; amino acid mix 10 ml/l; trace mineral solution 10 ml/l, supplemented with 15 mM lactate as electron donor and carbon source in a sealed 15 ml test tube.60, 76 When needed, 30 mM sodium fumarate was supplemented as the electron acceptor.

Conclusions To the best of our knowledge, this research is the first to adopt a modular engineering approach to systematically engineer NADH regeneration and redistribute the metabolic flux of S. oneidensis MR-1 toward the generation of intracellular reducing equivalents (i.e., NADH), thus increasing its EET efficiency. The central carbon metabolism of S. oneidensis from the carbon source (lactate) was categorized into four modules based on the pathway architecture, namely glycolysis, C1 metabolism, pyruvate fermentation, and TCA cycle. Sixteen genes related to NADH regeneration in the four metabolic modules were individually overexpressed to study their effect on the EET efficiency, respectively. We then identified four most crucial genes in determining the intracellular NADH regeneration and voltage output in MFCs, namely, an endogenous gapA2 gene encoding a NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH) in the glycolysis module, an endogenous mdh gene encoding a NAD+-dependent malate dehydrogenase in the TCA cycle, an endogenous pflB gene encoding a pyruvate-formate lyase to convert pyruvate to formate, and an exogenous gene fdh* from Candida boidinii encoding a 5



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BES setup. To evaluate the efficiency of bacterial outward electron transfer, overnight Shewanella culture suspension (1.5 ml) was inoculated into 150 ml fresh LB broth, and incubated at 30 °C with shaking (200 rpm) till the optical density (OD600) of the cell culture reached 0.6~0.8, which was measured by an ultraviolet and visible spectrophotometer (TU-1810, Beijing, China). Then, the cells were harvested by centrifugation and washed 3 times with fresh M9 buffer (Na2HPO4, 6 g/l; KH2PO4, 3 g/l; NaCl, 0.5 g/l; NH4Cl, 1 g/l; MgSO4, 1 mM; CaCl2, 0.1 mM). The cell pellets were subsequently re-suspended in 140 ml electrolyte (5% LB broth plus 95% M9 buffer supplemented with 18 mM lactate). The cell suspension was transferred into the anodic chamber of microbial fuel cells (MFCs) to evaluate the efficiency of EET. In additional, previous experiments in our laboratory have proved that IPTG has no effect on cell physiology and EET of Shewanella.79 The medium was supplemented with 0.8 mM IPTG (as optimized in Figure S1, Supporting Information (SI)) and 50 µg/ml kanamycin to ensure consistent culture condition. The dual-chamber MFCs with a working volume of 140 ml separated by the nafion 117 membrane (DuPont Inc., USA) were used. Carbon cloth was used as the electrodes for both anode (2.5 × 2.5 cm, i.e., the geometric area is 6.25 cm2) and cathode (2.5 × 3 cm). The cathodic electrolyte was made of 50 mM K3[Fe(CN)6] in 50 mM K2HPO4 and 50 mM KH2PO4 solution. To measure voltage generation, a 2 KΩ external resistor was connected into the external circuit of MFCs, and the output voltage (V) across this external loading resistor was measured by a digital multimeter (DT9205A).

performed using SsoAdvanced SYBR Green supermix (Bio-Rad, USA).80 The expression level of the target genes were normalized with respect to the expression level of gyrB. The data were analyzed using the 2−∆∆CT method.81 The primers listed in Table S2 were used to amplify small parts of the genes.

Electrochemical analysis. Cyclic voltammetry (CV) analysis was performed in a three-electrode configuration with an Ag/AgCl reference electrode on a CHI 1000C multichannel potentiostat (CH Instrument, Shanghai, China). Linear sweep voltammetry (LSV) analysis with a slow scan rate (0.1 mV/s) was conducted on a two-electrode mode to obtain the polarization curves to estimate the maximum power density (the potential decreased from the open circuit potential (OCP) to -0.1V). Power density (P) was calculated as P = V (output voltage) × I (current density). Both I and P were normalized to the projected area of the anode surface.

ASSOCIATED CONTENT

Quantification of intracellular NAD(H/+). The cells (10 mL) were firstly cooled in ice bath for 20 minutes to retard cell metabolism, then collected by centrifugation (10,000 rpm at 4°C for 5 min) and immediately re-suspended in 300 µl of 0.2 M HCl (for NAD+) or 0.2 M NaOH (for NADH). The suspensions were boiled for 7 min, rapidly quenched in an ice bath, and added with 300 µl of 0.1 M NaOH (for NAD+) or 0.1 M HCl (for NADH)82. Cell debris was removed by centrifugation at 10,000 rpm for 10 min, and the supernatant was used in a cycling assay to determine the amounts of NAD+ and NADH.33, 83 Meanwhile, the cell concentration of pYYDT, -F1, -F2, -F3, and -F4 for the detection of NAD(H/+) concentration was detected by plate counts on LB agar. Measurement of electrode-attached biomass The electrode was placed in a 50 ml tube containing 5 ml NaOH (0.2 mol/l), then vortexed for 2 min and incubated in a water bath to lyse cells at 96 °C for 30 min. The extracts were tested by bicinchoninic acid protein assay kit (Solarbio, China) after being cooled to 25 °C.

Supporting Information The following file is available free of charge on the ACS Publications website at DOI. The output voltage of recombinant S. oneidensis pYYDT-F4 upon induction by different IPTG concentrations; the independent triplicates of polarization discharge curves and power density output curves of the MFCs, the synthesized gene sequences in this study and the primers used in RT-qPCR. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Riboflavin and metabolite quantification. For the quantification of riboflavin, the samples in the MFC supernatant were firstly centrifuged (35,000 rpm for 20 min) and filtered (0.22 µm), and the eluted medium were detected by a liquid chromatograph-tandem mass spectrometer (LC-MS) (Agilent LCMS-1290-6460) in positive ion mode using a Waters XBridge C8 column (2.1 × 100 mm; particle size: 3.5 µm). The metabolites including formate, succinate and acetate in the anolytes were analyzed using a high-performance liquid chromatography (HPLC) system equipped with a diode array detector. Sulfuric acid (5 mM) was used as the mobile phase flowing at 0.6 ml/min through the Aminex HPX-87H column (Bio-Rad, U.S.A.), which was incubated at 50°C. Signals at 210 nm were used to quantify organic acids.

Corresponding Authors Hao Song, E-mail: [email protected]. Tel: +86-18722024233. Author Contributions FL and YL contributed equally to the work. FL and YL designed the project, performed experiments, analyzed data, and drafted the manuscript; LS, XC, XA and CY performed a few experiments, collected data, analyzed data, and drafted the manuscript; YC and HW provided some reagents, helped design the experiment and drafted the manuscript; HS designed and supervised the project, analyzed data, and critically revised the manuscript. Notes The authors declare no competing financial interest.

Real-time quantitative PCR. A bacterium total RNA extraction kit (APEXBIO, China) was used to isolate total RNA from mid-log-phase cultures. And cDNA was synthesized using the GoScript reverse transcription system (Promega, USA). Quantitative analyses of target gene expression were

ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (NSFC 21376174, 21621004), and the National Basic Research Program of China (“973” Program: 6


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2014CB745102).

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ABBREVIATIONS MFC, microbial fuel cell; EET, extracellular electron transfer; WT, wild type; IPTG, isopropyl β-D-1-thiogalactopyranoside; CV, cyclic voltammetry; OCP, open circuit potential; LSV, linear sweep voltammetry; MQ, methyl naphthoquinone; IM, inner membrane; OM, outer membrane; CymA, inner membrane tetraheme c-type cytochromes; MtrA, periplasmic decaheme; MtrB, β-barrel trans-OM protein; MtrC and OmcA, two OM decaheme c-type cytochromes; TCA, tricarboxylic acid cycle; FDH, formate dehydrogenase; ndhII, NADH dehydrogenase; P, phosphate; PEP, phosphoenolpyruvate; Q, quinone; QH2, reduced quinone; NAD+, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; CoA, coenzyme A.

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ACS Paragon Plus Environment 9


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Table 1. Genes used in this study. Module

Glycolysis

C1 metabolism

Pyruvate fermentation

Gene

Aliases

gapA1

SO_0538

gapA2

SO_2345

gapA3

SO_23479

serA

SO_0862

serB

SO_1223

serC

SO_2410

gylA

PCC_0517

gcvT

SO_0779

aceE

SO_0424

sfcA

SO_3855

pflB fdh

SO_2912 SO_3922

fdh*

TCA cycle

mdh

SO_0770

sucB

SO_193

icd

SO_2629

Enzyme NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase D-3-phosphoglycerate dehydrogenase Phosphoserine phosphatase Bifunctional 3-phosphoserine aminotransferase Serine hydroxymethyltransferase Aminomethyltransferase Pyruvate dehydrogenase E1 component NAD+-dependent malate dehydrogenase Pyruvate formate-lyase Formate dehydrogenase NAD+-dependent formate dehydrogenase NAD+-dependent malate dehydrogenase 2-oxoglutarate dehydrogenase complex dihydrolipoate succinyltransferase E2 isocitrate dehydrogenase

E.C.

Source

Reference

EC 1.2.1.12

S. oneidensis MR-1

62

EC 1.2.1.12

S. oneidensis MR-1

62

EC 1.2.1.59

S. oneidensis MR-1

62

EC 1.1.1.95

S. oneidensis MR-1

60

EC 3.1.3.3

S. oneidensis MR-1

60

EC 2.6.1.52

S. oneidensis MR-1

60

EC 2.1.2.1

S. oneidensis MR-1

60

EC 2.1.2.10

S. oneidensis MR-1

60

EC 1.2.4.1

S. oneidensis MR-1

65

EC 1.1.1.38

S. oneidensis MR-1

60

EC 2.3.1.54 EC 1.2.1.2

S. oneidensis MR-1 S. oneidensis MR-1

65

EC 1.2.1.2

Candida boidinii

46

EC 1.1.1.37

S. oneidensis MR-1

60

EC 2.3.1.61

Geobacter sulfurreducens

EC 1.1.1.42

S. oneidensis MR-1


65

60

62

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Figure 1. Modular engineering of the intracellular NADH regeneration by overexpressing redox enzymes in the central carbon metabolism of Shewanella oneidensis MR-1. The central carbon metabolism of S. oneidensis was categorized into four metabolic modules, i.e., glycolysis, C1 metabolism, pyruvate fermentation pathway, and TCA cycle. Genes related to the NADH regeneration (marked as blue) were gylA1, gylA2 and gylA3 in the glycolysis module; serA, serB, serC, gylA and gcvT in the C1 metabolism module; sfcA, aceE, pflB, fdh and fdh* in the pyruvate fermentation module; and mdh, icd and sucB in the TCA cycle module. Among them, the heterologous genes fdh* and sucB were from Candida boidinii and Geobacter sulfurreducens (another well-established exoelectrogen), respectively. The genes encoding the respective enzymes in the network referred to their commonly used names.


300

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**

200 100 0 --

WT apA1 apA2 apA3 g g g

--

NADH (µg/109 cells)

(A)

4 **

3 2 1 0 WT apA1 apA2 apA3 g g g

100 0 WT serA serB serC gylA gcvT

Voltage (mV)

(C)

300 **

**

200 100 0 WT aceE sfcA pflB fdh* fdh

(D)

300

**

200 100 0 WT mdh sucB

icd

--

NADH (ug/109 cells)

200


300


Voltage (mV)

(B)

Voltage(mV)

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

Voltage (mV)


--

--

4 3 2 1 0 WT serA serB serC gylA gcvT

4

**

**

3 2 1 0 * WT aceE sfcA pflB fdh fdh

4

**

3 2 1 0 WT mdh sucB

icd

--

--

Figure 2. Optimization of the four modules of the central carbon metabolism in S. oneidensis MR-1. (A) Optimization of the glycolysis module; (B) Optimization of the C1 metabolism module; (C) Optimization of the pyruvate fermentation module; and (D) Optimization of the TCA cycle. The left column was the gene constructs in the plasmid harboring a critical gene in each of the four modules. For example, pYYDT-gapA1 denoted a plasmid pYYDT harboring a synthesized gene gapA1 under the control of the Plac promoter. The other plasmids were denoted in the same manner. The middle column was the output voltage of microbial fuel cells (MFCs) inoculated with the recombinant S. oneidensis harboring the plasmid construct shown in the left column. The right column was the measured intracellular NADH level in each of the recombinant S. oneidensis under anaerobic conditions in the absence of discharge. The error bars were calculated from triplicate experiments.


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(A)

(B) Out voltage (mV)

Relative expression levels

(C)

400

pYYDT pYYDT-F1 pYYDT-F2 pYYDT-F3 pYYDT-F4

300 200 100 0

0

2

4

6

8

10

12

14

Time (days)

(D)

14 12 10 8

0

fdh*

gapA2

mdh

pYYDT-F4 + 0.8mM IPTG

6

2

1

0

4

2

0

1 2 3 4 WT T-F DT-F DT-F DT-F YD Y Y Y Y Y Y Y p p p p

(F)

pflB

pYYDT-F4


NADH/NAD+

16

(E)

3

1 2 3 4 WT T-F DT-F DT-F DT-F YD Y Y Y Y Y Y Y p p p p

(G) 1.0 NADH (µg/109 cells)

2.0 1.5 NADH/NAD+

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


1.0 0.5 0.0

0.8 0.6 0.4 0.2 0.0

4 3 2 1 T YD DT-F DT-F DT-F DT-F pY Y Y Y Y Y Y Y Y p p p p

4 3 2 1 WT T-F DT-F DT-F DT-F Y Y Y YD Y Y Y Y p p p p

Figure 3. Modular assemblage of the four genes identified from the above four metabolic modules, and bio-electrochemical and biochemical analyses of the recombinant S. oneidensis. (A) The construction of four plasmids by a step-by-step assemblage manner of the four critical genes (mdh, gapA2, pflB, and fdh*) identified from the four metabolic modules. (B) Voltage output of these recombinant mutants (pYYDT-F1, -F2, -F3 and -F4) and the WT strain in MFCs, respectively. (C) The relative expression levels of the four genes in the recombinant S. oneidensis pYYDT-F4, upon induction by 0.8 mM IPTG. The gyrB gene was used as the internal control in the RT-qPCR. The control is the gyrB cDNA’s basal expression in the strain pYYDT-F4 (in the absence of IPTG). (D-E) Quantitative measurements of



Page 14 of 17

the ratio of NADH/NAD+ and NADH in these recombinant S. oneidensis strains under anaerobic conditions in the absence of discharge. (F-G) Quantitative measurements of the ratio of NADH/NAD+ and NADH in these recombinant S. oneidensis strains under MFC discharge conditions. The error bars were calculated from triplicate experiments.


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Output voltage (mV)

(A)

WT pYYDT-F4

400 300 200 100 0 0

(B)

10

20

30

40

50

Time (days)

Current density (mA/m2)

500

WT pYYDT-F4

400

Outer-membrance cytochromesmediated catalytic current

300 200

Flavins-mediated catalytic current

100 0 -0.8

-0.6

-0.4

-0.2

0.0

0.2

Potential (V vs. Ag/AgCl) 800

120

WT pYYDT-F4

600

90

400

60

200

30 0

0 0

100

200

300

400

Power density (mW/m2)

(C) Output voltage (mV)

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


Current density (mA/m2) Figure 4. Bio-electrochemical characterization of the extracellular electron transfer (EET) and electricity output capability. (A) Voltage output of WT (black square) and recombinant S. oneidensis strain pYYDT-F4 (red circle) in the multiple operational cycles of microbial fuel cells (MFCs). (B) Turnover cyclic voltammetry (CV) at a scan rate of 1 mV/s. (C) Polarization discharge curves and power density output curves of the MFCs. The error bars were calculated from triplicate experiments.



(B)

(A)

10.0

WT pYYDT-F4

Concentration (mM)

OD(600nm)

0.3

0.2

0.1

0.0

0

5

10

15

20

25

30

WT pYYDT-F4

7.5 5.0 2.5 0.0

m For

Time (h)

e e ate inat Acetat c c Su

0.8

300

Riboflavin Attached biomass

200

0.6 0.4

100

0.2 0.0

0

WT

pYYDT-F4

Attached biomass (µg)

1.0

Current to protein (mA/µg)

(D)

(C) Riboflavin(µM)

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

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WT pYYDT-F4

1.5 1.0 0.5 0.0 -0.8

-0.6

-0.4

-0.2

0.0

0.2

Potential (V vs. Ag/AgCl)

Figure 5. Biochemical and bio-electrochemical analyses of the wild-type (WT) S. oneidensis and the recombinant S. oneidensis strain pYYDT-F4. (A) Anaerobic growth curves (OD600 ~ t) in the Shewanella basal medium (SBM). (B) The level of critical metabolites in the anolytes of MFCs, including formate, succinate and acetate. (C) Riboflavin concentration in the anolytes of MFCs and the attached biomass of each strain on the anode surfaces were measured from three independent MFC anode chambers. (D) Standardized CV curves normalized by the attached biomass on the anodes. The scan rate of CV was 1 mV/s. The error bars were calculated from triplicate experiments.


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