Sortase A-Mediated Metabolic Enzyme Ligation in Escherichia coli

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Sortase A-Mediated Metabolic Enzyme Ligation in Escherichia coli Takuya Matsumoto, Kou Furuta, Tsutomu Tanaka, and Akihiko Kondo ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00194 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 2016

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ACS Synthetic Biology

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Sortase A-Mediated Metabolic Enzyme Ligation in

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Escherichia coli

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Takuya Matsumoto,† Kou Furuta,‡ Tsutomu Tanaka,‡ and Akihiko Kondo†,‡,*

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†

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Nada, Kobe 657-8501, Japan

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‡

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University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan

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*

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email: [email protected]

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Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho,

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe

Corresponding author;

tel:+81-78-803-6196

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ACS Paragon Plus Environment

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ABSTRACT

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We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in

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the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling.

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Here, sortase A expression was controlled by the lac promoter to trigger metabolic channeling by

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the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). We tested covalent linking of

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pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and

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evaluated the production of acetate. The time point of addition of IPTG was not critical for

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facilitating metabolic enzyme ligation, and acetate production increased upon expression of

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sortase A. These results show that sortase A-mediated enzyme ligation enhances an

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acetate-producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation

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offers a metabolic channeling approach to redirect a central flux to a desired flux.

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Keywords: Sortase A, Escherichia coli, Enzyme ligation, Metabolic engineering

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Microbial bioproduction exploits sustainable natural resources, such as lignocellulosic biomass,

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to produce target compounds. Escherichia coli (E. coli) is a well-studied microorganism that has

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significant potential to convert biomass to valuable compounds, including fuels, building-block

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chemicals and pharmaceuticals. The use of strain engineering to achieve sufficient titers, yields

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and productivities through E. coli-based bioproduction has been an important focus for several

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decades.1,2 Enhancing the metabolic flux from substrate to product through pathway engineering

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is an important strategy. Some common methods include the introduction of heterologous

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pathways, overexpression of an endogenous gene and deletion of competing pathways. These

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methods have been successfully applied for the design of desired strains. However, the

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traditional gene deletion technique is limited because it cannot be used for essential genes, which

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are often involved in cell growth or maintenance. To overcome this limitation, more complex

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and systematic methods have been reported in recent years. Several genetic tools are more useful

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than these standard methods and have a number of advantages, such as portability, conditionality

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and tunability.3 For example, the noncoding small RNAs-mediated tool,4,5 riboswitch6 and

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synthetic promoter/terminator7,8 approaches have been employed for metabolic engineering. In

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addition, biosensor-assisted methods have been proposed.9,10 Recently, dynamic metabolic

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engineering has attracted much attention as an approach to redirect metabolic pathways from

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endogenous pathways to targeted pathways. The chemical additive- or stress-induced1 promoter

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is a simple tool for dynamic metabolic engineering, which can redirect fluxes by the addition of

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an exogenous factor.11 Dynamic knockdown of a pathway based on controlled protein

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degradation has also been reported. To dynamically alleviate the central metabolic flux, the

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native Pfk-I coding gene was replaced with the degradation-tag modified Pfk-I. The half-life of

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Pfk-I was controlled by inducing the expression of SspB, which is an adapter protein tethered to


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ClpXP.12 Using this system, E. coli-based myo-inositol production was successfully increased in

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both titer and yield.13 Several synthetic gene circuits have also been demonstrated for the

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redirection of an endogenous pathway into a heterologous pathway. These redirections have been

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elaborately designed for improving bioproduction of compounds, including fatty acids,14

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gluconate,15 isopropanol,16,17 and 3-hydroxypropionic acid.18

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Metabolic channeling is another approach to ameliorate a strain for bioproduction.

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Generally, small metabolites are either processed by metabolic enzymes or by substrate

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channeling involving multi-enzyme complexes that carry out a series of reactions in a

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step-by-step process. Substrate channeling facilitates the transfer of an intermediate between two

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enzymes, which catalyze sequential metabolic reactions in cells. Substrate channeling prevents

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the diffusion of an intermediate and improves the efficiency of a cascade metabolic reaction.

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Naturally, some multi-enzyme complexes, such as carbamoyl-phosphate synthase and

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phosphoribosylpyrophosphate amidotransferase have been suggested as enzyme complexes

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suitable for substrate channeling.19,20 In recent years, designed metabolic channeling has been

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reported for synthetic proteins or RNA scaffolds, or enzyme clusters that facilitate metabolite

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processing on a metabolic pathway in vivo and in vitro.21-24 These studies indicate that placing

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metabolic enzymes in proximity to each other will achieve substrate channeling of a metabolic

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cascade, which enhances microbial bioproduction of desired chemicals. For instance, Lewicka

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and coworkers demonstrated the metabolic channeling that pyruvate decarboxylase (Pdc) and

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alcohol dehydrogenase (AdhB) were fused in E. coli. The strain expressing the fused Pdc-AdhB

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produced more ethanol than strains co-expressing Pdc and AdhB, suggesting that processing of

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acetaldehyde to ethanol were enhanced by metabolic channeling.25 Tippmann and coworkers

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reported also about the metabolic channeling with affibody-based scaffold. Two metabolic


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enzymes were assembled on anti- idiotypic affibody pair in Saccharomyces cerevisiae for the

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production of farnesene, and three metabolic enzymes were assembled on affibody-scaffold in

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E.coli for the production of polyhydroxybutyrate (PHB). A two- or three-component scaffold

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improved farnesene productino or PHB production, respectively.26 Furthermore, designed

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metabolic channeling has the added advantage of directing metabolic fluxes to desired pathways

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without gene disruption or manipulation.

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Here, we demonstrate the redirection of metabolic flux with metabolic channeling based on

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protein ligation. Staphylococcal sortase A (SrtA) is used for ligation of metabolic enzymes. SrtA

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is a transpeptidase that recognizes Leu-Pro-Xaa-Thr-Gly sequences (LP tag) and cleaves

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between Thr and Gly, and subsequently links amino groups of oligoglycine sequences (G tag) by

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formation of a native peptide bond. SrtA-mediated ligation enables the conjugation of a protein

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with other molecules in a site-specific manner. Minimal modification of the protein with the

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short LP and G tags is required for site-specific ligation. Hence, SrtA-mediated protein ligation

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has been used to prepare a variety of bioconjugations in vitro and in vivo.27 In this study, we

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hypothesize that SrtA-mediated metabolic enzyme ligation in the cytoplasm of E. coli facilitates

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the processing of a metabolic intermediate, and redirects metabolic fluxes to a desired pathway.

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As proof of concept, we have constructed an acetate producing E. coli with an engineered

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endogenous metabolic pathway, which redirects central metabolic fluxes to an acetate producing

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flux by metabolic enzyme ligation (Figure 1A, B and C). The expression of SrtA was controlled

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by the lac promoter. Acetyl-CoA was chosen as the intermediate model because acetyl-CoA is

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one of the most important central metabolic intermediates, which is converted to alcohols, fatty

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acids and mevalonate derivatives.1,2 In this study, pyruvate-formate lyase (PFL) and phosphate

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acetyltransferase (PTA) were successfully linked by SrtA-mediated ligation, and this linking of


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two enzymes increased the accumulation of acetate. Our results demonstrate that SrtA-mediated

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metabolic enzyme ligation enables metabolic channeling to redirect a central flux to a desired

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flux.

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RESULTS AND DISCUSSION

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SrtA-Mediated Metabolic Enzyme Ligation in vivo. Disruption of an endogenous gene

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that regulates cell growth or cell maintenance dramatically decreases cell viability and growth.

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To solve this limitation the redirection of metabolic fluxes has been reported.11-18 Similarly, the

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goal of our concept is based on the redirection method with SrtA representing a protein stapler

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for ligating metabolic enzymes. An additional goal of this study is metabolic flux control using

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only metabolic enzyme ligation. Recently, scaffold-based metabolic enzyme assemblies have

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been reported that facilitate the processing of metabolic intermediates, giving rise to metabolic

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channeling.21,22,24 Here, we hypothesize that simply tethering of metabolic enzymes should also

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achieve metabolic channeling. To tether metabolic enzymes in the cytosol, SrtA-mediated

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protein ligation was employed. SrtA from S. aureus has been used as a protein stapler for linking

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proteins.29 We also successfully demonstrated protein-protein ligation28 or protein assembly29 in

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vitro. In this work, SrtA and SrtA-recognition sequence modified metabolic enzymes were

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expressed under inducible- or constitutive-promoters in E. coli. Acetyl-CoA is one of the most

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important intermediates in central metabolism, which is converted to the variety of useful

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compounds by endogenous or exogenous pathways. Acetyl-CoA is converted to ethanol, acetate

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or citrate through endogenous pathways in E. coli. As proof of concept, we constructed an

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acetate-producing strain with an engineered endogenous metabolic pathway. Pyruvate is

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converted to acetyl-CoA by pyruvate-formate lyase (PFL) under anaerobic (or microaerobic)


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conditions, and subsequently acetyl-CoA is converted to acetate by phosphate acetyltransferase

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and acetate kinase (PTA-ACK) (Figure 1A).

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We constructed the plasmid for the expression of SrtA under the control of the lac

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promoter (PLac) and the plasmid for the expression of metabolic enzymes under the constitutive

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promoter (PHCE). The expression of SrtA was controlled by PLac, thereby the addition of IPTG

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induced metabolic enzyme ligation. PFL encoded by the pflB gene and PTA encoded by the pta

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gene were selected as the model of metabolic enzyme ligation. The LP tag was C-terminally

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appended to PFL, whereas the G tag was N-terminally placed to PTA. Protein expression was

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complemented in native pflB- and pta-deficient E. coli BW25113 (KT19) cells following

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transformation of the pHLA-PFL-lp_g-PTA plasmid under the control of PHCE. In the absence of

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SrtA, all of expressed PFL was not linked to PTA, as observed by the band of unconjugated

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g-PTA (ca. 80 kDa) using SDS-PAGE and western blotting analysis. Only cells expressing SrtA

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led to the formation of the PFL-PTA conjugate at ca. 150 kDa (Figure 2A and B). This result

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indicates that PFL-lp and g-PTA were successfully conjugated by SrtA-mediated ligation in E.

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coli. However, SrtA-mediated ligation between PFL-lp and g-PTA also occurred without the

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addition of IPTG. This result suggests that the expression of SrtA was not strictly controlled by

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the lac operator. In order to redirect metabolic flux with the addition of chemical additives, a

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more strictly controllable system than the lac operator system for the expression of SrtA is

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required in future work, e.g., tetracycline, arabinose or rhamnose-inducible promoters.30-32

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The Effect on Acetate Production by SrtA-Mediated Metabolic Enzyme Ligation. To

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further investigate the effect on metabolic enzyme ligation in E. coli, engineered strains were

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cultivated with 5 g L−1 glucose containing LB medium under microaerobic conditions and

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particular organic acids were analyzed by HPLC. We hypothesized that the conjugation of PFL


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and PTA would lead to metabolic channeling and facilitate the enhancement of a PFL-PTA flux

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(Figure 1B). In the current study, the POX enzyme encoded by the poxB gene was deficient

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(KT19) to minimize acetate production via another pathway (Figure 1A). We examined the

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accumulation of acetate in three strains: a pflB-complemented strain (KT190); a pflB- and

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pta-complemented strain (KT193) and a pflB- and pta- complemented and SrtA-expressing strain

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(KT195). IPTG was added to KT195 cells after 6 h cultivation to induce the expression of SrtA.

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As expected, the KT190 cells showed minimal accumulation of acetate (Figure 3B), whereas

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both KT193 and KT195 cells were found to accumulate acetate. There were a number of

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observable differences in the accumulation of acetate after the addition of IPTG to KT193 and

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KT195 cells. KT193 no longer accumulated acetate after 12 h cultivation. Conversely, the

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accumulation of acetate using KT195 cells increased after the addition of IPTG at 6 h cultivation.

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These results suggest that the induction of the expression of SrtA at 6 h cultivation facilitated an

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enhancement of the PFL-PTA flux. Contrastingly, KT190 and KT193 accumulated lactate during

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the early phase (Figure 3C). With regard to pyruvate accumulation, only KT190 and KT193 cells

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released this metabolite into the culture supernatant (Figure 3D) because neither cells was able to

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convert pyruvate to acetate. On the other hand, KT195 cells efficiently processed pyruvate to

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acetate through acetyl-CoA. Hence, KT190 and KT193 cells were likely to convert pyruvate into

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lactate during the early phase; however, both cells steadily accumulated and effused pyruvate

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because the strains could not efficiently process pyruvate and acetyl-CoA. In addition, although

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KT195 grew significantly slower but exhibited higher glucose uptake rate than KT190 and

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KT193 cells (Figure 3A, E). This result complements the observation that KT195 cells did not

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accumulate pyruvate and produced more acetate than KT193 cells. We also evaluated the effect

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on an acetate producing flux by the burden of the expression of SrtA using a pflB- and pta-


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complemented and deactivated SrtA mutant-expressing strain (KT198). SrtA was deactivated by

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the replacement of active cysteine in the active site of sortase A to alanine. From the metabolite

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analysis results (Figure S2), the KT198 exhibited closely similar performance with KT193.

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These results suggest that PFL-PTA ligation catalyzed by active sortase A was required for

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enhancing acetate-producing flux. As shown in Figure 2, PFL-lp and g-PTA were conjugated

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even without the addition of IPTG and the time point of IPTG addition has no effect on the

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efficiency of PFL-lp and g-PTA ligation. The time point of addition of IPTG also had minimal

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effect on the accumulation of organic acids (Supporting Information Figure 1). In addition,

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western blotting analysis showed that PFL-lp and g-PTA were not completely conjugated by

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SrtA-mediated ligation (Figure 2). Therefore, insufficient metabolic ligation might have

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decreased the effect of metabolic channeling. Previous results have shown that the insertion of a

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proper linker facilitated the efficiency of SrtA-mediated protein ligation.33 In addition, other

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works have shown that the concentration of Ca2+ was insufficient for SrtA-mediated protein

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ligation in the E. coli cytosol, and have also demonstrated that a mutation of SrtA dramatically

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improved its catalytic activity under conditions of depleted Ca2+.34,35 Thus, more effective

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metabolic channeling with our system may be achieved by considering these results.

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CONCLUSIONS

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We succeeded in metabolic channeling triggered by enzyme mediated-transpeptidation in E. coli

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cells. An acetate-producing strain was successfully produced by the metabolic enzyme ligation.

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IPTG-induced expression of SrtA facilitated the conjugation of PFL-lp and g-PTA in E. coli,

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leading to an enhancement in acetate production. These results imply that SrtA-mediated

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metabolic ligation facilitated an enhancement of the PFL-PTA flux. To harness this system,

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microbial bioproduction of chemicals can be improved without disrupting genes.


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METHODS

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Bacterial Strains and Growth Conditions. The bacterial strains used in this study are

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listed in Table 1. E. coli NovaBlue cells (Novagen Inc., Madison, WI, USA) were used for DNA

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manipulations. E. coli BW25113 cells were used as the base strain for acetate production. Cells

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were pre-cultivated with 100 µg mL−1 ampicillin and 20 µg mL−1 kanamycin containing 5 mL

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Luria-Bertani (LB) medium in a test tube overnight. Cells were grown in 10 mL 0.5% glucose

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containing LB medium at 37 °C, 190 rpm in 15 mL sealed test tubes (initial optical density at

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600 nm = 0.1). SrtA expression was induced by the addition of IPTG (final conc. 0.5 mM).

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Plasmid Construction and Gene Disruption. All of the plasmids and primers used in this

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study are listed in Table 1 and Supplementary Table 1, respectively. The polymerase chain

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reaction (PCR) was performed with KOD plus polymerase (TOYOBO CO., LTD., Osaka, Japan).

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Vectors and inserts were ligated with the In-Fusion® HD Cloning Kit following the

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manufacturer’s protocol (TAKARA BIO INC., Shiga, Japan). The pHLA vector36 was employed

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as the base vector for the expression of tagged metabolic enzymes. Each amplified fragment was

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inserted into the BglII/XhoI restriction sites of the pHLA vector. The resulting plasmids were

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pHLA-pflBlp and pHLA-pflBlp-gpta, and named pHKT1 and pHKT2, respectively. To confirm

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the expression of PFL and PTA, the FLAG peptide and cmyc peptide were genetically

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introduced at the C-terminus, respectively. To construct vectors expressing SrtA, the

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pZA23MCS (Expressys, Bammental, Germany) was employed as the backbone plasmid. To

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suppress the expression of genes under the lac promoter, the lacI gene was inserted under the

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constitutive promoter of pZA23MCS. The resulting plasmid was named pZA23LMCS. The gene

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of sortase A from Staphylococcus aureus was amplified using pET30b-SrtA37 as the template.


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The amplified sortase A gene was inserted into pZA23MCSL, and the resulting plasmid was

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named pZA23LSrtA. In addition, pZA23LSrtAc184a was constructed by quick-change PCR. 38

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E. coli BW25113 genes, pflB, pta and poxB were deleted, as described in a previous

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report.39 Confirmation of gene deletions was carried out by colony PCR. The resulting strain was

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BW25113 ∆pflB, ∆pta and ∆poxB, and was named KT19. pHKT1 and pZA23LMCS, pHKT2

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and pZA23LMCS, pHKT2 and pZALSrtA, pHKT2 and pZA23LSrtAc184a, were transformed

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into KT19 cells, which were named KT190, KT193, KT195 and KT198, respectively.

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SDS-PAGE Analysis and Western Blotting Analysis. To evaluate the ligation between

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PFL and PTA using SrtA in vivo, extracts of cells were analyzed by SDS-PAGE or western

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blotting analysis. After cultivation for 24 h at 37 °C, samples were centrifuged and the

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supernatant removed. Cells were gently mixed with B-PER™ Bacterial Protein Extraction

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Reagent (Thermo Fisher Scientific Inc., Kanagawa, Japan) at RT for 30 min. The reaction

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mixture was then mixed with the SDS-PAGE sample buffer (50 mM Tris-HCl, 2% SDS, 6%

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2-mercaptoethanol) followed by boiling. The samples were then subjected to SDS-PAGE and the

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gels were stained with Coomassie brilliant blue R-250, or analyzed by western blotting with

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rabbit anti-c-myc (Bethyl Laboratories, Inc., Montgomery, TX) and anti-rabbit IgG (Fc) AP

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conjugate (Promega KK, Tokyo, Japan) and BCIP/NBT, according to the manufacturer’s

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procedure.

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Metabolite Analysis. The concentrations of acetic acid, pyruvate and lactic acid were

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determined by high-performance liquid chromatography (Shimadzu Co., Kyoto, Japan; solvent

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delivery system, LC-10ADvp; column, Shim-pack SPR-H; column temperature, 50 °C; detector,

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CDD-10A). A 5 mM concentration of p-toluenesulfonic acid was used as the mobile phase, and


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20 mM bis-Tris containing 5 mM p-toluenesulfonic acid and 100 µM EDTA was mixed just

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before detection to enhance sensitivity. Chromatography was carried out at 50 °C at a flow rate

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of 1.4 mL/min.

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ASSOCIATED CONTENT

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Supporting Information

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

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・Supporting figure, primer list

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AUTHOR INFORMATION

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Corresponding Author

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Akihiko Kondo

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[email protected]

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Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe

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University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS


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This work was supported in part by a Grant-in-Aid for Young Scientists B (15K18276) from the

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Japan Society for the Promotion of Science (JSPS), and by Special Coordination Funds for

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Promoting Science and Technology, Creation of Innovation Centers for Advanced

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Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan

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Figure Captions

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Figure 1. An integrated shceme of sortase A-mediated metabolic enzyme ligation in E. coli. (A)

357

Engineered E. coli metabolism of acetate producing flux used in this study. (B) Schematic

358

illustration of metabolic enzyme ligation between PFL and PTA by SrtA as a stapler for enzyme

359

conjugation. (C) The strategy for PFL-PTA ligation catalyzed by SrtA. The expression of SrtA,

360

PFL and PTA were controlled under Lac promoter and constitutive HCE promoter, respectively.

361

PFL-PTA ligation was triggered by the expression of SrtA.

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Figure 2. SDS-PAGE analysis (A) or Western blotting analysis (B) of metabolic enzyme ligation

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in E. coli extracts after 24 h cultivation. Lane 1, KT193; Lane 2, KT195 without the addition of

364

IPTG; Lanes 3−6, KT195 with the addition of IPTG at 0, 3, 6, 9 h cultivation.

365

Figure 3. The cell growth (optical density at 600 nm) (A), the production of acetate (B), lactate

366

(C), pyruvate (D) and the consumption of glucose (E). The cells were inoculated at initial OD600

367

= 0.1 and IPTG was added after 6 h cultivation. Symbols represent: KT190: black; KT193: blue;

368

and KT195: red. Data are presented as the average of three independent experiments, and error

369

bars represent the standard deviation.

370


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Table 1. Strains and plasmids used in this study

Strain

Characteristics

Source

Nova blue

endA1 hsdR17(rK12− mK12+) supE44 thi-1 recA1 gyrA96 relA1 lac[F′ proAB+ lacIqZΔ M15::Tn10 (Tetr)]; host for DNA manipulation

Novagen

BW25113

Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) λ− rph-1 Δ(rhaD-rhaB)568 hsdR514

National Institute of Genetics, Japan

KT19

As BW25113, but ΔpflB, ΔpoxB, Δpta

This study

KT190

KT19 harboring pHKT1 and pZA23LMCS

This study

KT193

KT19 harboring pHKT2 and pZA23LMCS

This study

KT195

KT19 harboring pHKT2 and pZA23LSrtA

This study

KT198

KT19 harboring pHKT2 and pZA23LSrtAc184a

This study

Plasmid

Characteristics

Source

pHLA

ColE1 ori; AmpR; PHCE::pgsA

36

pHKT1

ColE1 ori; AmpR; PHCE::pflBlp

This study

pHKT2

ColE1 ori; AmpR; PHCE::pflBlp-gpta

This study

pZA23MCS

p15A ori; KanR; PA1lacO-1::MCS1

Expressys, Bammental, Germany

pZA23LMCS

p15A ori; KanR; Placiq-::lacI; PA1lacO-1::MCS1

This study

pZA23LSrtA

p15A ori; KanR; Placiq-::lacI; PA1lacO-1::SrtA

This study

pZA23LSrtAc184a

p15A ori; KanR; Placiq-::lacI; PA1lacO-1::SrtAc184a

This study


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Figures

373

Figure 1

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374 375 376

Figure 2

377 378


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Figure 3

380 381 382 383


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TOC

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Sortase A-Mediated Metabolic Enzyme Ligation in Escherichia coli

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