Development of Escherichia coli Strains That Withstand Membrane

Research Article pubs.acs.org/synthbio

Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production Dimitra Gialama,†,‡ Kalliopi Kostelidou,† Myrsini Michou,† Dafni Chrysanthi Delivoria,†,‡ Fragiskos N. Kolisis,‡ and Georgios Skretas*,† †

Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, Athens 11635, Greece Laboratory of Biotechnology, School of Chemical Engineering, National Technical University of Athens, Athens 15780, Greece

‡

S Supporting Information *

ABSTRACT: Membrane proteins perform critical cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochemical/ structural studies. Bacterial production of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields. In this work, we aimed to rewire the E. coli protein-producing machinery to withstand the toxicity caused by membrane protein overexpression in order to generate engineered bacterial strains with the ability to achieve high-level membrane protein production. To achieve this, we searched for bacterial genes whose coexpression can suppress membrane protein-induced toxicity and identified two highly potent effectors: the membrane-bound DnaK cochaperone DjlA, and the inhibitor of the mRNA-degrading activity of the E. coli RNase E, RraA. E. coli strains coexpressing either djlA or rraA, termed SuptoxD and SuptoxR, respectively, accumulated markedly higher levels of final biomass and produced dramatically enhanced yields for a variety of prokaryotic and eukaryotic recombinant membrane proteins. In all tested cases, either SuptoxD, or SuptoxR, or both, outperformed the capabilities of commercial strains frequently utilized for recombinant membrane protein production purposes. KEYWORDS: Escherichia coli, membrane protein overexpression, toxicity, DjlA, RraA

M

Escherichia coli has historically been the most popular and successful expression host for MP biochemical/structural studies.1 Solved structures of bacterially produced MPs correspond to sequences of both prokaryotic and eukaryotic origin, and include difficult targets, such as mammalian G protein-coupled receptors (GPCRs), examples of which are the CXCR1 chemokine receptor8 and an engineered variant of the neurotensin receptor.9 Despite these success stories, however, bacterial MP production remains notoriously difficult, and its success rate rather low, especially for MPs of eukaryotic origin.10 The problems associated with bacterial MP production are mainly three: (i) there is usually very little membraneincorporated protein per cell, (ii) when accumulation in the cell membrane occurs at appreciable levels, there is typically a very small amount of protein that is produced in a well-folded and functional form, and (iii) overexpression is very frequently associated with high levels of toxicity for the host.1,11

embrane proteins (MPs) are a major structural and functional component of biological membranes that mediate structural integrity, signaling, transport, energy production and other essential functions.1,2 The great importance of MPs is reflected by the fact that 20−30% of all genes encode such proteins in both prokaryotes and eukaryotes.1 Furthermore, the proper folding and function of a wide variety of MPs are involved in devastating human diseases such as cystic fibrosis,3 Alzheimer’s disease,4 cancer,5 hypogonadotropic hypogonadism and many more.3 Remarkably, MPs constitute about half of all current targets for pharmaceutical development.6 The development of novel therapeutic molecules that target MPs relies on the detailed understanding of MP structure and function. This, in turn, requires access to significant amounts of isolated protein for biochemical studies, the development of MP-specific antibodies, and MP structure determination. As MP natural abundance is usually very low,1 these proteins are typically produced and isolated after recombinant overexpression in heterologous hosts, such as bacteria, yeasts, insect cells, mammalian cells or transgenic animals.7 © XXXX American Chemical Society

Received: June 22, 2016 Published: October 31, 2016 A

DOI: 10.1021/acssynbio.6b00174 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology Table 1. Membrane Proteins Studied in This Work membrane protein

organism

function

number of TM helices

topology

MW (kDa)

BR2 CB1 CB2 NKR1 NTR1(D03) SCD MotA SapC GarP TcyL GsiC ArtM MdfA CstA YidC

Homo sapiens H. sapiens H. sapiens H. sapiens Mus musculus H. sapiens E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli

Bradykinin receptor 2 (GPCR) Central cannabinoid receptor (GPCR) Peripheral cannabinoid receptor (GPCR) Neurokinin (substance P) receptor 1 (GPCR) Neurotensin receptor 1 variant D03 (GPCR) Stearoyl-CoA desaturase Stator element of the flagellar motor complex Component of putative ABC transporter SapABCDF Galactarate/glucarate/glycerate transporter Predicted membrane subunit of a L-cystine ABC transporter Glutathione transport system permease protein Arginine ABC transporter permease protein Multidrug efflux transporter Peptide transporter induced by carbon starvation Membrane protein integrase

7 7 7 7 7 4 4 5 11 3 6 5 12 18 6

Nout-Cin Nout-Cin Nout-Cin Nout-Cin Nout-Cin Nin-Cin Nin-Cin Nin-Cin Nin-Cin Nout-Cin Nin-Cin Nout-Cin Nin-Cin Nin-Cin Nin-Cin

44.5 52.9 39.7 46.3 44.6 41.1 32.0 31.5 49.0 24.8 34.1 24.9 44.3 75.1 61.5

Improvements of the expression host via genetic engineering12−18 or of the target MP itself via directed evolution19,20 have provided some solutions to the problem of low cellular productivity. The cytotoxicity caused by MP overexpression, however, is an issue which has not been systematically addressed. This is an important problem as MP-induced toxicity is observed routinely and is severe, oftentimes leading to complete growth arrest,11,21 very low levels of final biomass and significant reduction of total volumetric protein yields. A number of possible explanations have been suggested, such as collapse of the protein biosynthetic machinery as a result of high-level recombinant gene expression22 with concomitant degradation of rRNA and ribosome destruction;23 overloading and jamming of the SecYEG translocon resulting in compromised cell envelope and cytoplasmic proteomes and inefficient energy metabolism;11 or the actual biochemical and biophysical properties of the particular overexpressed MP.21 These hypotheses, however, have not yet been exploited to address the issue and improve recombinant MP production sufficiently. Two decades ago, Walker and co-workers utilized two difficult-to-express MPsthe mitochondrial oxoglutarate malate carrier protein (OGCP) and the b subunit of the E. coli FATPaseto isolate E. coli BL21(DE3) mutant strains carrying spontaneously acquired suppressor mutations that alleviate the toxicity caused by the overxpression of these MPs under the control of the strong T7 promoter.24 Two of the evolved strains, named C41(DE3) and C43(DE3), were found to be resistant to the toxicity caused by the production of a variety of membrane/soluble proteins and to allow increased biomass production, and are widely used for the production of hard-toexpress and toxic proteins (mostly MPs). Many years later, de Gier and co-workers found that the mutations responsible for the suppressed toxicity were located in the sequence of the lacUV5 promoter and that their effect was reduction of the expression levels and, thus, the overall translational efficiency of the T7 RNA polymerase, which in turn produced less mRNA corresponding to the target recombinant protein.22 These results revealed that there appears to be an optimal level of mRNA of the target gene that leads to maximal recombinant MP production, and that further increases in target gene expression beyond that point disrupt cellular physiology and become deleterious for recombinant MP production as they decrease final biomass yields.22,25 In an improvement of the

original strains reported many years later, de Gier and coworkers developed a system termed Lemo21(DE3), where the transcriptional activity of the T7 RNA polymerase is controlled by the cellular abundance of its inhibitor T7 lysozyme, whose expression is in turn placed under the tight control of the rhamnose promoter.22 By varying the concentration of rhamnose, optimal induction conditions can be determined such that MP yields can be maximized. The utility of the C41, C43 and Lemo21 strains, however, is strictly limited to the use of the T7 promoter/T7 RNA polymerase system for expression of the target MP, while their use for the production of certain hard-to-express eukaryotic MPs, such as GPCRs, has been very limited. In the present work, we aimed to rewire the E. coli protein synthesis machinery to be able to withstand the toxicity caused by MP overexpression in order to develop engineered strains, which can be generally utilized for achieving high-level MP production. As the molecular sources of this phenomenon are not well understood, we employed a reverse-engineering approach and looked for bacterial genes whose coexpression can suppress MP-induced toxicity. After carrying out a genomewide screen, we identified two highly potent suppressors: (i) djlA, the gene encoding for the membrane-bound DnaK cochaperone DjlA, and (ii) rraA, the gene encoding for RraA, an inhibitor of the mRNA-degrading activity of the E. coli RNase E. E. coli strains coexpressing djlA or rraA, which we refer to as E. coli SuptoxD and SuptoxR, respectively, were found capable of accumulating markedly higher levels of final biomass and of producing dramatically enhanced yields for a variety of recombinant MPs of both prokaryotic and eukaryotic origin. Importantly, SuptoxD and SuptoxR outperformed the capabilities of commercially available strains frequently utilized for MP production purposes in a number of tested cases.

■

RESULTS AND DISCUSSION Genetic Screening for the Identification of Factors That Suppress the Cytotoxicity Caused by MP Overexpression. In order to generate bacterial strains with the ability to withstand the physiological side-effects of MP overexpression on cell growth and biomass accumulation, we searched for E. coli genes, whose overexpression can lead to suppression of the cytotoxicity caused by recombinant MP production. As a model MP, we chose the human bradykinin receptor 2 (BR2) (Table 1), a therapeutically relevant GPCR,

B


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ACS Synthetic Biology Table 2. E. coli Strains Used in This Study strain MC1061 BL21(DE3) C41(DE3) C43(DE3) Lemo21(DE3) SuptoxD SuptoxR

genotype

source

F− λ− Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 e14− mcrA0 relA1 rpsL150(StrR) spoT1 mcrB1 hsdR2(r−m+) − F ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) F− ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) with modifications described by Kwon et al.42 and Schlegel et al.43 F− ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) with modifications described by Kwon et al.42 − F ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) pLemo F− λ− Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 e14− mcrA0 relA1 rpsL150(StrR) spoT1 mcrB1 hsdR2(r−m+) pSuptoxD F− λ− Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 e14− mcrA0 relA1 rpsL150(StrR) spoT1 mcrB1 hsdR2(r−m+) pSuptoxR

Laboratory collection Laboratory collection Lucigen Lucigen New England Biolabs This work This work

Figure 1. Coexpression of djlA or rraA suppresses the toxicity caused by BR2 production. (a) Schematic representation of the utilized BR2-KanR and BR2-GFP fusions. A FLAG tag is added to the N terminus of BR2 in all cases, while KanR and GFP tags are added to the C-terminal tail, respectively. (b) (Top) E. coli MC1061 cells carrying the vector pBAD30BR2-GFP and grown on agar plates without (x) and with (check mark) 0.2% arabinose, the inducer of BR2-GFP production. (Bottom) E. coli MC1061 cells producing BR2-GFP as above, while simultaneously overexpressing djlA (10 μM IPTG), rraA (100 μM IPTG), or a randomly selected gene from the ASKA library, i.e., a gene picked at random from the initial unselected ASKA library (later on determined to be yghS), at 25 °C for 3 days. (c) Effect of djlA (0.01% arabinose) and rraA (0.2% arabinose) coexpression from pBAD33 on the growth of E. coli MC1061 cells upon BR2-GFP overexpression from pASKBR2-GFP (0.2 μg/mL anhydrotetracycline, aTc) for 16 h at 25 °C. OD: optical density. Experiments were carried out in replica triplicates and the error bars represent one standard deviation from the mean value. (d) Effect of djlA (0.01% arabinose) and rraA (0.2% arabinose) coexpression from pBAD33 on the growth of E. coli MC1061 cells for 16 h at 25 °C. OD: optical density. Experiments were carried out in replica triplicates and the error bars represent one standard deviation from the mean value. (e) Comparison of BDKRB2 mRNA levels without/with coexpression of rraA using real-time PCR. Two independent experiments were carried out in replica triplicates and the error bars represent one standard deviation from the mean value.

C


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ACS Synthetic Biology whose overexpression in E. coli has been found to be very toxic despite the fact that it accumulates in membrane-integrated form only at low levels.13 E. coli MC1061 cells (Table 2) were initially transformed with the pBAD30BR2-KanR vector, which encodes a BR2 fusion with an N-terminal FLAG tag and a C-terminal aminoglycoside 3′-phosphotransferase reporter (KanR: the enzyme conferring resistance to the antibiotic kanamycin), under the control of the araBAD promoter (Figure 1a, top; Table 3). The FLAG sequence serves as a tag for immunodetection, while KanR allows facile monitoring of BR2 production levels by recording bacterial growth in the presence of kanamycin.17 E. coli MC1061 cells carrying pBAD30BR2-KanR were plated onto LB agar plates in the absence and presence of L(+)-arabinose, the inducer of the overexpression of the BR2-encoding gene BDKRB2. The size of the transformants was dramatically decreased when arabinose was present in the medium, thus demonstrating BR2-induced cytotoxicity on solid media (data not shown). In order to look for genes that act as suppressors of MPinduced toxicity, electrocompetent MC1061 cells carrying pBAD30BR2-KanR were cotransformed with the ASKA library (A Complete Set of Escherichia coli K-12 ORF Archive), an ordered library of plasmids encoding all known E. coli open reading frames under the control of the T5lac promoter.26 Electrocompetent cells were used to ensure high transformation efficiency. A total of approximately 120 000 transformants were plated onto three different large LB agar plates containing: (i) 100 μg/mL ampicillin and 40 μg/mL chloramphenicol, the appropriate antibiotics to ensure plasmid maintenance, (ii) 0.2% arabinose to induce BR2 production, (iii) a low concentration (10 μg/mL) of kanamycin to ensure that fulllength receptor is produced and that gene, plasmid, chromosomal etc. mutations that block BDKRB2 expression do not accumulate, and (iv) three different isopropyl-β-Dthiogalactoside (IPTG) concentrations to induce overexpression of the ASKA library genes: 0, 0.01, and 0.1 mM. Low to medium concentrations of IPTG were preferred as it has been found that high-level induction from the ASKA plasmids at 1 mM IPTG results in severe growth inhibition for more than half of the genes contained in this library.27 Since the ASKA library consists of about 4000 different gene-encoding vectors,26 the diversity of the library was covered by about 30-fold in our screen. After incubation for two overnights at 30 °C, large colonies on a background of pinprick-sized colonies appeared on all three plates, indicating potential suppressed toxicity. 140 such large colonies were picked and their corresponding ASKA plasmids were isolated. In order to confirm that the observed loss-of-toxicity phenotype is indeed mediated through the isolated ASKA genes, fresh MC1061 cells were transformed with fresh pBAD30BR2-KanR vector and the ASKA plasmids isolated from the selected large colonies. The growth of the resulting transformants was evaluated in liquid LB cultures under conditions where BR2 production is toxic. For approximately 100 of these clones, increased cell densities were recorded, compared to control cultures carrying randomly selected ASKA plasmids (Supplementary Figure S1a), thus demonstrating that increased growth occurs indeed due to the presence of the identified ASKA-encoding genes. Since the selected genes may be ameliorating cytotoxicity because they lower the levels of transcription and/or translation of the BR2-encoding gene, we monitored BR2 accumulation in

Table 3. Plasmids Used in This Study plasmid pBAD30BR2-KanR pBADBR2-GFP pASKBR2-GFP pASKBR2 pASKCB1-GFP pASKCB2-GFP pASKNKR1-GFP pASKMotA-GFP pASKSapC-GFP pASKGarP-GFP pASKTcyL-GFP pASKGsiC-GFP pASKArtM-GFP pASKMdfA-GFP pASKCstA-GFP pASKYidC-GFP pASKSCD-GFP pASKNTR1(D03)GFP pASKNTR1(D03)TrxA pETBR2-GFP pETCB2-GFP pETNTR1(D03)GFP pETMotA-GFP pETSapC-GFP pASKBR2-EGFP pASKNTR1(D03)EGFP pASKSapC-EGFP ASKA library pSuptoxR (pBAD33RraA) pSuptoxD (pBAD33DjlA)

protein expressed

marker

origin of replication

source

FLAG-BR2-TEVKanR FLAG-BR2-TEVGFP-His8 FLAG-BR2-TEVGFP-His8 FLAG-BR2-His8 FLAG-CB1-TEVGFP-His8 FLAG-CB2-TEVGFP-His8 FLAG-NKR1-TEVGFP-His8 FLAG-MotA-GFPHis8 FLAG-SapC-GFPHis8 FLAG-GarP-GFPHis8 FLAG-TcyL-GFPHis8 FLAG-GsiC-GFPHis8 FLAG-ArtM-GFPHis8 FLAG-MdfA-GFPHis8 FLAG-CstA-TEVGFP-His8 FLAG-YidC-TEVGFP-His8 FLAG-SCD-GFPHis8 FLAG-NTR1(D03)GFP-His8 FLAG-NTR1(D03)TrxA-His6 FLAG-BR2-GFPHis8 FLAG-CB2-GFPHis8 FLAG-NTR1(D03)GFP-His6 FLAG-MotA-GFPHis8 FLAG-SapC-GFPHis8 FLAG-BR2-TEVEGFP-His6 FLAGNTR1(D03)TEV-EGFP-His6 FLAG-SapC-TEVEGFP-His6 All known E. coli proteins RraA-His8

AmpR

ACYC

This work

AmpR

ACYC

AmpR

ColE1

Skretas et al.14 Link et al.13

AmpR AmpR

ColE1 ColE1

Link et al.13 Link et al.13

AmpR

ColE1

Link et al.13

AmpR

ColE1

Link et al.13

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

DjlA-His8

ColE1

This work

R

Kan

ColE1

This work

KanR

ColE1

This work

KanR

ColE1

This work

R

Kan

ColE1

This work

KanR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

AmpR

ColE1

This work

CmR

ColE1

CmR

ACYC

Kitagawa et al.26 This work

CmR

ACYC

This work

the absence and presence of the selected genes by measuring the fluorescence of MC1061 cells overexpressing a C-terminal BR2 fusion with the FACS-optimized variant mut2 of the green fluorescent protein (GFP)28 under the control of the araBAD promoter (Figure 1a, bottom). It has been previously demonstrated that the fluorescence of E. coli cells expressing MP-GFP fusions correlates well with the amount of membraneintegrated recombinant MP.29,30 Fifteen of the tested clones D


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ACS Synthetic Biology

Figure 2. SuptoxD and SuptoxR accumulate enhanced levels of membrane-embedded BR2. (a) Fluorescence of E. coli MC1061 (WT), SuptoxD and SuptoxR cells producing BR2-GFP from pASKBR2-GFP by the addition of 0.2 μg/mL aTc for 16 h at 25 °C. Bulk fluorescence corresponding to an equal number of cells was measured on a plate reader (left), while levels of individual cell fluorescence were measured by flow cytometry (right). M: mean fluorescence of experiments performed in triplicate. (b) SDS-PAGE/Western blot analysis using an anti-GFP antibody on clarified lysates of E. coli MC1061, SuptoxD and SuptoxR cells producing BR2-GFP as in Figure 2a. An equal number of cells was loaded in each lane. MW: molecular weight. (c) Fluorescence of E. coli MC1061, SuptoxD and SuptoxR cells producing BR2-GFP as in (a) at 25 °C for 16 h, 30 °C for 3 h and 37 °C for 2 h. Measurements correspond to cells derived from an equal volume of bacterial culture for each strain. In all measurements of relative fluorescence, the fluorescence of BR2-producing MC1061 cells was arbitrarily set to one. In all panels, experiments were carried out in replica triplicates and the error bars represent one standard deviation from the mean value.

demonstrated BR2-GFP fluorescence, which was at least as high as that demonstrated by wild-type cells overexpressing randomly selected ASKA genes (Supplementary Figure S1b), thus indicating that suppression of BR2-induced toxicity in these clones does not arise from inhibition of BR2 production. DNA sequencing of the ASKA plasmids for these fifteen clones revealed two genes as potential suppressors of MP toxicity: (i) djlA, the gene encoding for the membrane-bound DnaK cochaperone DjlA,31 and (ii) rraA, the gene encoding for RraA, an inhibitor of the mRNA-degrading activity of the E. coli RNase E.32 djlA was present in 4/15 sequenced clones and was isolated both from the 0 and 0.01 mM IPTG screens, while rraA in 11/15 sequenced clones and was isolated from the 0.1 mM IPTG screen. Coexpression of djlA or rraA in E. coli MC1061 cells producing BR2-GFP on agar plates resulted in the formation of colonies with dramatically increased size compared to cells coexpressing a randomly selected gene (Figure 1b). When BR2GFP overexpression was carried out in liquid cultures, coexpression of djlA or rraA resulted in about 30 and 50% increase in final culture density, respectively (Supplementary

Figures S1c and d). Importantly, the effects of DjlA and RraA were found to be independent of the choice of the promoter and the plasmid utilized for BR2, DjlA and RraA production: when BR2-GFP production was placed under the control of the tet promoter in the vector pASK7533 and effector expression was placed under the control of the araBAD promoter in the low-copy number plasmid pBAD33,34 we observed an even larger increase in final cell density of approximately 150% and 50% in the presence of djlA or rraA coexpression, respectively (Figure 1c). The observed growth phenotypes did not occur due to a general growth-promoting effect of djlA and rraA overexpression; on the contrary, djlA overexpression has been found to be toxic for E. coli,31,35 an effect which was also observed here (Figure 1d). Furthermore, when BR2 was produced in a form where GFP had been replaced with an octahistidine tag, djlA and rraA coexpression resulted in similar effects, thus demonstrating that the observed suppressing properties occur independently of the presence of the Cterminal fusion partner (see below). Finally, suppression of BR2-induced toxicity by DjlA and RraA was found to be independent of the particular E. coli strain selected as the MP E


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Figure 3. SuptoxD and SuptoxR enhance the accumulation of membrane-bound BR2 with the correct Nout topology. (a) Schematic representation of the utilized FLAG-BR2 fusion. A FLAG tag is added to N terminus of BR2, while an octa-histidine tag is added to the C-terminal tail. (b) Growth of E. coli MC1061 (WT), SuptoxD and SuptoxR cells producing FLAG-BR2 from pASKBR2 by the addition of 0.2 μg/mL aTc for 16 h at 25 °C. OD: optical density. (c) SDS-PAGE/Western blot analysis using an anti-FLAG antibody (top) on isolated total membranes of E. coli MC1061, SuptoxD and SuptoxR cells producing FLAG-BR2 as in (b). Each lane corresponds to a sample of total membranes isolated from an equal number of cells, as verified by using an antibody against the E. coli maltose-binding protein (MBP) (bottom). 4-fold (4×) and 8-fold (8×) more total membrane preparation was loaded for better visualization of FLAG-BR2 accumulation in WT cells. (d) Schematic representation of the utilized assay for the detection of BR2 receptor inserted into the E. coli inner membrane with the correct Nout topology by monitoring the labeling of E. coli spheroplasts due to the binding of an Alexa Fluor 647-conjugated anti-FLAG antibody to the N-terminal FLAG tag of FLAG-BR2, which is embedded into the E. coli inner membrane. The N-terminal FLAG tag is denoted by F while the C-terminal octahistidine tag of FLAG-BR2 is denoted by H. (e) Fluorescence of spheroplasted E. coli MC1061, SuptoxD and SuptoxR cells producing FLAG-BR2 as in (b) and labeled with an Alexa Fluor 647conjugated anti-FLAG antibody. Spheroplasted bacterial cells overexpressing an N-terminally FLAG-tagged cytoplasmic protein (the DNA-binding domain of human p53) were used as a negative control. Measurements were carried out on a plate reader and correspond to an equal number of cells. In all panels, experiments were carried out in replica triplicates and the error bars represent one standard deviation from the mean value.

BDKRB2 mRNA levels significantly (Figure 1e), thus demonstrating that RraA effects do not occur due to interference with the degradation/stability of the mRNA of the target MP. Having established the toxicity-suppressing effects of DjlA and RraA on MP production, we refer to the utilized djlA- and rraA-encoding pBAD33 vectors as pSuptoxD (suppressor of

production host, and was observed in both E. coli K-12 and B strains (Supplementary Figure S2; also see below). RraA is a protein that acts as a regulator of the mRNAdegrading activity of RNase E, and rraA overexpression has been found to globally increase the levels of more than 2000 different mRNAs in E. coli.32 Quantitative real-time PCR analysis, however, revealed that rraA coexpression did not affect F


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Figure 4. SuptoxD and SuptoxR broadly enhance recombinant production for a variety of homologous and heterologous MPs. (a) Growth of E. coli MC1061 (WT), SuptoxD and SuptoxR cells producing either NKR1-GFP or CB2-GFP from pASKNKR1-GFP or pASKCB2-GFP, respectively, by the addition of 0.2 μg/mL aTc overnight at 25 °C. OD: optical density. (b) Fluorescence of an equal number of E. coli MC1061, SuptoxD and SuptoxR cells producing either NKR1-GFP or CB2-GFP as in (a). The fluorescence of NKR1-producing MC1061 cells was arbitrarily set to one. (c) SDS-PAGE/Western blot analysis using N-terminal anti-FLAG and C-terminal anti-GFP antibodies on clarified lysates (left) and isolated total membranes (right) of equal culture volumes of E. coli MC1061, SuptoxD and SuptoxR cells producing NKR1-GFP (top) or CB2-GFP (bottom) from pASKNKR1-GFP or pASKCB2-GFP, respectively, as in (a). (d) Fluorescence of equal culture volumes of E. coli MC1061, SuptoxD and SuptoxR cells producing different MP-GFP fusions from the pASK75 vector as in (a). The fluorescence of MC1061 cells producing BR2-GFP was arbitrarily set to one. In all panels, experiments were carried out in replica triplicates and the error bars represent one standard deviation from the mean value.

toxicity: DjlA) and pSuptoxR (suppressor of toxicity: RraA), respectively (Table 3), and the E. coli strains coexpressing these effectors as SuptoxD and SuptoxR, respectively (Table 2). SuptoxD and SuptoxR Accumulate Enhanced Levels of Membrane-Embedded BR2 with the Correct Nout-Cin Topology. As mentioned above, it has been demonstrated previously that the fluorescence of E. coli cells expressing MP-

GFP fusions correlates well with the amount of membraneintegrated recombinant MP.29 Furthermore, MP-GFP fusions have been utilized in high-throughput screens as a readout for identifying factors that enhance bacterial MP production.14,16 On the basis of this, we evaluated the levels of cellular BR2 accumulation in the generated strains by comparing the levels of individual cell fluorescence of E. coli MC1061 (wild-type, G


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Figure 5. SuptoxD and SuptoxR enhance the recombinant production of well-folded MPs. (a) Photographs depicting the fluorescence over ultraviolet light of pellets derived from cultures of MC1061 (WT), SuptoxD or SuptoxR cells expressing BR2-EGFP (left), NTR1(D03)-EGFP (middle), and SapC-EGFP (right) from pASKBR2-EGFP, pASKNTR1(D03)-EGFP, and pASKSapC-EGFP, respectively, by the addition of 0.2 μg/ mL aTc at 25 °C overnight. (b) Semidenaturing SDS-PAGE analysis of isolated total membrane fractions of MC1061 and SuptoxD cells producing BR2-EGFP as described in (a) and visualization of the produced fusion by in-gel fluorescence (left) and Western blotting using a C-terminal antiFLAG (middle) and a N-terminal anti-GFP antibody (right), without boiling of the samples prior to loading. (c) Semidenaturing SDS-PAGE analysis of isolated total membrane fractions of MC1061 and SuptoxD cells producing NTR1(D03)-EGFP as described in (a) and visualization of the produced fusion by in-gel fluorescence (left) and Western blotting using a C-terminal anti-FLAG (middle) and a N-terminal anti-GFP antibody (right), without boiling of the samples prior to loading. (d) SDS-PAGE analysis of isolated total membrane fractions of MC1061 and SuptoxD cells producing SapC-EGFP as described in (a) and visualization of the produced fusion by in-gel fluorescence (left) and Western blotting using a Cterminal anti-FLAG (middle) and a N-terminal anti-GFP antibody (right), without (nonboiled) and with (boiled) boiling of the samples prior to loading. For comparison between WT and SuptoxD/SuptoxR cells, samples corresponding to an equal volume of culture (volume) or an equal number of cells (cells) were loaded in each lane. H


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SuptoxD and SuptoxR can be generally applied to other MPs as well, we first tested their effect on the toxicity and per-cell productivity of two additional human GPCRs, the neurokinin receptor 1 (NKR1) and the peripheral cannabinoid receptor (CB2) (Table 1). Overexpression of NKR1-GFP and CB2-GFP fusions in SuptoxD and SuptoxR resulted in marked increases in final culture densities, as well as in the levels of individual cell fluorescence for both receptors compared to WT E. coli (Figures 4a and b), thus demonstrating that the beneficial effects of these strains are not limited to the production of BR2. Combination of the toxicity-suppressing and cellular productivity-promoting effects on NKR1 and CB2 overexpression, resulted in a dramatic 2.6- to 11.5-fold enhancement in volumetric accumulation of both receptors in SuptoxD and SuptoxR, as determined by fluorescence measurements of equal culture volumes and Western blotting (Figures 4c and d). To examine further the ability of SuptoxD and SuptoxR to enhance MP production broadly, we extended the panel of MPs to be tested to include additional integral MPs of bacterial or mammalian origin, characterized by different sizes, number of trans-membrane helices, topologies, and biochemical properties (Table 1). All proteins were expressed as C-terminal fusions with GFP and total MP accumulation was monitored by measuring bulk fluorescence of equal culture volumes. Remarkably, all tested MPs accumulated at greatly increased levels either in SuptoxD, SuptoxR, or both, compared to WT E. coli (Figure 4d), thus demonstrating that our engineered strains can broadly act as general enhancers of recombinant MP production. We have not identified any correlation between features of the tested MPs (e.g., molecular weight, number of trans-membrane helices, topology etc.) and their overexpression efficiency in SuptoxD and SuptoxR. It is important to note, however, that SuptoxD and SuptoxR appear to be particularly efficient for notoriously hard-to-express eukaryotic MPs, such as GPCRs. In order to investigate the folding quality of the overproduced MPs in the engineered strains, we analyzed by SDS− PAGE and in-gel fluorescence the production of three MPs, two eukaryotic (BR2, NTR1(D03)) and one prokaryotic (SapC), as C-terminal fusions with the enhanced green fluorescence protein (EGFP) in WT and either SuptoxD or SuptoxR cells, depending on which strain was found previously to be the most efficient producer for each MP. We observed that the cell pellets corresponding to the SuptoxD or SuptoxR cells exhibited fluorescence, which was dramatically enhanced compared to that of WT E. coli (Figure 5a). Previous studies have shown that in-gel fluorescence provides a quantitative measure of the amount of properly folded protein for bacterially overexpressed MP−EGFP fusions.38 As described by Geertsma et al.,38,39 electrophoresis of bacterially expressed MP-EGFP fusions without prior boiling results in dual band migration of the produced fusion, where both bands are fulllength but only the lower band is well-folded and fluorescent, while the upper one corresponds to misfolded protein and is nonfluorescent. Boiling of the sample results in full denaturation of the fusion, which under these conditions migrates as a single band that is nonfluorescent. Western blot and in-gel fluorescence analysis revealed that all tested MPEGFP fusions embedded in the membrane of SuptoxD and SuptoxR cells migrated as two distinct, full-length species from which only the ones with the higher electrophoretic mobility exhibited high levels of fluorescence, as expected (Figure 5b, c, and d). This indicates that all tested MPs produced in our

WT), SuptoxD, and SuptoxR cells overexpressing BR2-GFP under the same conditions. Very interestingly, a large increase in individual cell fluorescence was recorded when BR2-GFP was produced in SuptoxD and SuptoxR (Figure 2a). This suggests that the generated strains may also have the ability to accumulate increased cellular amounts of recombinant BR2, despite the fact that they were not directly selected for this property. Indeed, Western blot analysis of clarified lysates revealed that SuptoxD and SuptoxR cells can accumulate markedly increased amounts of BR2-GFP on a per-cell basis compared to WT E. coli (Figure 2b; Supplementary Figure S3a). Combination of the two positive effects of BR2 production in SuptoxD and SuptoxR, namely suppression of BR2-induced toxicity and enhancement in cellular BR2 accumulation, resulted in a dramatic enhancement in volumetric BR2 accumulation compared to WT E. coli (Figure 2c). In order to investigate whether SuptoxD and SuptoxR can accumulate increased amounts of BR2 protein that is properly embedded in the bacterial membrane, we first utilized a FLAGBR2 fusion (Figure 3a). Suppression of BR2-induced toxicity, as well as the greatly enhanced per-cell accumulation of membrane-integrated receptor in SuptoxD and SuptoxR, was evident also for this construct, despite the absence of the Cterminal GFP tag (Figures 3b and 3c). To examine whether BR2 is inserted in the bacterial inner membrane with the correct Nout topology, we tested the exposure of the N-terminal BR2 tail to the bacterial periplasmic space. Spheroplasts derived from the same cells were labeled with an Alexa Fluor 647conjugated anti-FLAG antibody (Figure 3d) and their fluorescence was recorded. As shown in Figure 3e, red fluorescence was significantly enhanced in SuptoxD and SuptoxR cells, indicating that more BR2 with the correct Nout topology accumulates in the generated strains compared to WT E. coli. Finally, we tested whether the produced BR2 protein acquires a correct Cin topology. For this, we turned back to the BR2-GFP fusion, since MP-GFP fusions have been used extensively to study the localization of the termini of MPs of unknown topology.36 As GFP is unable to fold properly in the bacterial periplasm where it remains nonfluorescent,37 the fluorescence of E. coli cells expressing MP-GFP fusions indicates (i) a C-terminal localization for the GFP-tagged terminus when fluorescence is recorded or (ii) an N-terminal localization for the GFP-tagged terminus when fluorescence is absent.36 Production of a C-terminal BR2-GFP fusion in SuptoxD and SuptoxR resulted in a large increase in cellular fluorescence compared to WT E. coli (Figure 2a), thus confirming that the C-terminal tail of the receptor is also properly localized in the bacterial cytoplasm. Taken together, these results demonstrate that SuptoxD and SuptoxR produce markedly enhanced amounts of properly membrane-embedded BR2 on a per-cell basis, while the enhancement in the production of properly membrane-embedded BR2 is even more pronounced on a volumetric basis due to the suppression of BR2-induced toxicity and the accumulation of higher levels of final biomass (Figure 2c). It is interesting to note that enhanced MP production in SuptoxD and SuptoxR does not occur only when MP overexpression takes place at room temperature but also at higher temperatures, albeit with more moderate enhancing effects (Figure 2c). SuptoxD and SuptoxR Broadly Enhance Recombinant Production for a Variety of Homologous and Heterologous MPs. To examine whether the beneficial effects of I


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Figure 6. SuptoxD and SuptoxR enhance the recombinant production of functional MPs. (a) SDS-PAGE analysis with (denaturing) or without (semidenaturing) boiling of the protein samples prior to loading, followed by Western blotting using an anti-FLAG antibody on isolated total membranes of E. coli MC1061 (WT), SuptoxD and SuptoxR cells producing NTR1(D03)-GFP from pASKNTR1(D03)-GFP by the addition of 0.2 μg/mL aTc overnight at 25 °C. Each lane corresponds to a sample derived from an equal volume of bacterial culture. (b) Growth of E. coli MC1061, SuptoxD and SuptoxR cells producing NTR1(D03)-TrxA from pASKNTR1(D03)-TrxA by the addition of 0.2 μg/mL aTc for 16 h at 25 °C. OD: optical density. (c) Schematic representation of binding of the fluorescent ligand BODIPY-NT(8−13) to NTR1(D03)-TrxA, which is embedded in the inner membrane of E. coli MC1061 spheroplasts (left). Fluorescence of BODIPY-NT(8−13)-labeled E. coli MC1061, SuptoxD and SuptoxR spheroplasts producing NTR1(D03)-TrxA from pASKNTR1(D03)-TrxA as in (b) and measured by flow cytometry (right). The fluorescence of BODIPY-NT(8−13)-labeled E. coli MC1061 cells was arbitrarily set to one. H denotes the presence of a polyhistidine tag. (d) SDS-PAGE/Western blot analysis using an anti-FLAG antibody (top) on clarified lysates of E. coli MC1061, SuptoxD and SuptoxR cells producing NTR1(D03)-TrxA from pASKNTR1(D03)-TrxA as in (b). Each lane corresponds to a sample of clarified lysates derived from an equal number of cells, as verified by utilizing an anti-MBP antibody (bottom). 4-fold (4×) and 8-fold (8×) more clarified lysate was loaded for better visualization of NTR1(D03)-TrxA accumulation in WT cells. MW: molecular weight. In all panels, experiments were carried out in replica triplicates and the error bars represent one standard deviation from the mean value.

in SuptoxD, while its production in SuptoxR has only a marginal impact. Western blot analysis on isolated membranes verified this indication (Figure 6a; Supplementary Figure S3b). NTR1(D03) production as a fusion with thioredoxin 1 (TrxA) in SuptoxD resulted in a 2-fold enhancement in final biomass and a 5-fold increase in cellular labeling by a fluorescent conjugate of the neurotensin peptide 8−13 (NT(8−13)) with dipyrromethene boron difluoride (BODIPY) (Figures 6b and c; Supplementary Figure S4). Since the fluorescence of E. coli cells overexpressing NTR1 and labeled with BODIPY-NT(8−13) has been found to correlate well with the amount of functional overexpressed receptor,15,19 our results indicate that functional

engineered strains adopt a well-folded conformation. Furthermore, in all cases the production of fluorescent, well-folded MP-GFP fusion was markedly enhanced in SuptoxD or SuptoxR compared to WT E. coli (Figure 5b, c, and d). Thus, SuptoxD and SuptoxR not only enhance the overall MP accumulation, but appear to assist the folding pathway of the recombinantly produced MPs as well. To assess whether these increases coincide with the production of more functional MP, we utilized NTR1(D03), a previously engineered variant of the rat neurotensin receptor 1.19 As shown in Figure 4d, NTR1(D03)-GFP fluorescence indicated that NTR1(D03) accumulation is markedly enhanced J


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Figure 7. continued

K


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Figure 7. Comparison of the volumetric MP production capabilities of SuptoxD and SuptoxR cells with commercial strains frequently utilized for MP production purposes. (a) (Left) L-rhamnose titration for the optimization of BR2-GFP overexpression from pETBR2-GFP in Lemo21(DE3). (Right) Comparison of the fluorescence of SuptoxD and SuptoxR cells producing BR2-GFP from pASKBR2-GFP by the addition of 0.2 μg/mL aTc overnight at 25 °C, with the fluorescence of equal culture volumes of C41(DE3), C43(DE3), and Lemo21(DE3) cells producing the same protein from pETBR2-GFP by the addition of 0.4 mM IPTG after overnight induction at 25 °C. BR2 production in the Lemo21(DE3) strain was carried out both at the optimal rhamnose concentration (1 μM rhamnose; Lemo21(DE3) 1 sample) as determined in the left panel, as well as in the absence of rhamnose (Lemo21(DE3) 0 sample). (b) For CB2 as in (a). (c) For NTR1(D03) as in (a). (d) For SapC as in (a). (e) For MotA as in (a). Bulk fluorescence for equal culture volumes was measured on a plate reader. Experiments were carried out in replica triplicates in at least two independent experiments and the error bars represent one standard deviation from the mean value. In all right panels, the fluorescence of the strain exhibiting the highest level of MP accumulation for each MP-GFP fusion was arbitrarily set to ten.

For Lemo21(DE3), we first determined the optimal concentration of L-rhamnose that maximized volumetric production of each MP according to the manufacturer’s instructions (Figures 7a, b, c, d and e; left). Volumetric accumulation for each protein in the different strains were compared by measuring cell fluorescence of equal culture volumes. Consistently with previous reports,22 Lemo21(DE3) cells accumulated enhanced levels of recombinant protein compared to C41(DE3) and C43(DE3) for the majority of the tested MPs (Figures 7a, b, c, d and e; right). However, either SuptoxD, or SuptoxR, or both, were found capable of accumulating greatly increased levels of recombinant protein for all tested MPs compared to the three frequently utilized commercial strains (Figures 7a, b, c, d, and e; right). It is interesting to note that the volumetric MP productivity of the BL21(DE3), C41(DE3) and C43(DE3) strains could be increased by coexpression of either djlA, rraA, or both (Supplementary Figure S5). Flow cytometric analysis of MC1061, SuptoxD, SuptoxR, C41(DE3), C43(DE3), and Lemo21(DE3) cells producing BR2-GFP or NTR1(D03)-GFP revealed that the enhanced MP production achieved with SuptoxD and SuptoxR is even more dramatic when the productivity of these strains is compared on a per-cell basis (Figure 8). This analysis also demonstrated that SuptoxD and SuptoxR cultures were as homogeneous as WT E. coli, C41(DE3), C43(DE3), or Lemo21(DE3) cells, despite the fact that they produce hard-to-express and otherwise highly toxic proteins at high levels (Figure 8). Finally, SuptoxD and SuptoxR cells were found to scatter light in the forward and side directions more intensely compared to WT E. coli, C41(DE3), C43(DE3), or Lemo21(DE3) cells, thus indicating that they are slightly enlarged and that they possess increased internal complexity, possibly due to the appearance of internal structures. Similar effects have been observed previously in a number of MP-overexpression studies.11,13,21 In conclusion, in the present work we show that the E. coli protein-producing machinery can be rewired to withstand the toxicity caused by the MP overexpression process. After

NTR1(D03) production on a per-cell basis is increased in SuptoxD by about 5-fold compared to WT E. coli. Analysis of the total accumulation of membrane-embedded protein by Western blotting and scanning densitometry revealed a 5.5-fold increase in membrane-embedded NTR1(D03)-TrxA in SuptoxD compared to WT cells (Figure 6d). This suggests that the observed increase in the cellular production of ligand-binding competent receptor in SuptoxD coincides with that of the total membrane-integrated NTR1(D03) that is achieved in this strain. Combination of these two positive effects on the production of active NTR1(D03), i.e., the 5-fold enhancement in cellular accumulation and the 2-fold increase in final bacterial biomass that is achieved with SuptoxD, results in an approximately 10-fold volumetric enhancement in the accumulation of functional NTR1(D03) receptor compared to WT E. coli. Comparison of SuptoxD and SuptoxR with Commercial Strains Frequently Utilized for Recombinant MP Production. Finally, we went on to compare the MPproducing capabilities of SuptoxD and SuptoxR with those of the commercial E. coli strains C41(DE3), C43(DE3), and Lemo21(DE3), which are frequently utilized for the production of recombinant MPs and other toxic proteins.22,24 To make this comparison, we used five test proteins, BR2, CB2, NTR1(D03), MotA, and SapC, the choice of which was based on the following criteria: (i) they include proteins of both bacterial and mammalian origin, (ii) they are characterized by different sizes, number of trans-membrane helices, topologies, and biochemical properties (Table 1), (iii) they include both more and less characterized MP targets, (iv) they have been found in previous studies to accumulate at different levels and to cause variable levels of cytotoxicity when overexpressed in E. coli,13,15,19,36 and (v) they include MPs which are of interest to our laboratory as targets for biochemical and structural studies. These MPs were cloned as C-terminal fusions with GFP into the T7 promoterbased vector pET-28a(+), and the resulting constructs were transformed into C41(DE3), C43(DE3), and Lemo21(DE3). L


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Figure 8. Comparison of the levels of cellular MP accumulation of SuptoxD and SuptoxR cells with commercial strains frequently utilized for MP production purposes. (a) Forward versus side scatter plots as determined by flow cytometry of E. coli MC1061, SuptoxD and SuptoxR cells producing BR2-GFP from pASKBR2-GFP by the addition of 0.2 μg/mL aTc for 16 h at 25 °C and of C41(DE3), C43(DE3), and Lemo21(DE3) cells producing the same protein from pETBR2-GFP by the addition of 0.4 mM IPTG and the optimal rhamnose concentration (only for Lemo21(DE3) cells and as determined in Figure 7) after overnight induction at 25 °C. (b) Comparison of the levels of individual cell fluorescence of E. coli MC1061, SuptoxD, SuptoxR, C41(DE3), C43(DE3), and Lemo21(DE3) cells producing BR2-GFP as in (a) and measured by flow cytometry. Cells were gated as indicated by the gray line in (a). Fluorescence values correspond to the mean value of experiments performed in triplicate. (c) For NTR1(D03) as in (a). (d) For NTR1(D03) as in (b).

in membrane-integrated and well-folded form compared to WT E. coli. Combination of these two beneficial effects, results in dramatic enhancement in volumetric MP accumulation compared to WT E. coli and commercial bacterial strains, which are frequently utilized for MP production purposes. Importantly, SuptoxD and SuptoxR were found to be particularly efficient for notoriously hard-to-express eukaryotic MPs, such as GPCRs. It is interesting to note that djlA and rraA were not identified as hits in our two earlier genetic screens of an E. coli genomic library15 and the ASKA library,14 respectively, for enhancers of cellular MP production. This can be attributed to the following

performing a genome-wide screen for suppressors of MPinduced toxicity, we identified two highly potent effectors: (i) djlA, the gene encoding for the membrane-bound DnaK cochaperone DjlA,31 and (ii) rraA, the gene encoding for RraA, an inhibitor of the mRNA-degrading activity of the E. coli RNase E.32 On the basis of this, we have generated the strains SuptoxD and SuptoxR, which overexpress djlA or rraA, respectively, and which can be broadly utilized for achieving high-level recombinant MP production. These engineered strains are efficient in (i) accumulating markedly increased levels of final biomass upon MP overexpression, and (ii) enhancing the cellular capacity for recombinant MP production M


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digested pASKBR2-GFP.13 For the construction of pASKYidCGFP and pASKCstA-GFP, the MP-GFP-encoding DNA fragments were digested from pBADYidC-GFP and pBADCstA-GFP,14 respectively, using XbaI and HindIII and were subsequently ligated into similarly digested pASK75.33 For the amplification of the genes encoding the E. coli MPs SapC, GarP (YhaU), TcyL (YecS), GsiC (YliC), ArtM, MdfA (Cmr) and MotA, the primer pairs SapCEcoRIfor-SapCPstIrev, YhaUEcoRIfor-YhaUPstIrev, YecSEcoRIfor-YecSPstIrev, YliCEcoRIforYliCPstIrev, ArtMEcoRIfor-ArtMPstIrev, MdfAEcoRIforMdfAPstIrev and MotAEcoRIfor-MotAPstIrev were used, respectively, while genomic E. coli DNA was utilized as the template. These PCR products were cloned into a modified pASK75 vector, which was generated by PCR amplification of pASKBR2-GFP using the primers FLAGpASKEcoRIrev and GFPm2PstIfor to insert the optimized Shine-Dalgarno sequence AGGAGGAAACG, a start codon, a FLAG tag and an EcoRI site after the XbaI site. The PCR product of the modified vector pASK75-GFP and the PCR product encoding the MP of interest were both digested with EcoRI and PstI and then ligated to generate pASKSapC-GFP, pASKGarP-GFP, pASKTcyL-GFP, pASKGsiC-GFP, pASKArtM-GFP, pASKMdfA-GFP and pASKMotA-GFP. For the construction of pASKNTR1(D03)-TrxA, the NTR1(D03)-TrxA-His6-encoding PCR product was amplified from pBADSmRNTR1(D03)15 using the primers D03XbaIfor and TrxAHisHindIIIrev, digested with XbaI and HindIII, and inserted into similarly digested pASK75. For the construction of pASKNTR1(D03)-GFP, the NTR1(D03)-encoding DNA fragment was digested from pASKNTR1(D03)-TrxA using XbaI and PstI and used to replace the similarly digested DNA sequence of TcyL in pASKTcyL-GFP. pETBR2-GFP, pETCB2-GFP, pETMotAGFP, pETNTR1(D03)-GFP and pETSapC-GFP were constructed by subcloning the DNA fragments encoding BR2-GFP, CB2-GFP, MotA-GFP, NTR1(D03)-GFP and SapC-GFP from pASK BR2-GFP, pASK CB2-GFP, pASK MotA-GFP, pASKNTR1(D03)-GFP and pASKSapC-GFP, respectively, into pET-28a(+) using the enzymes XbaI and HindIII. For the construction of the vectors pASKBR2-EGFP, pASKNTR1(D03)-EGFP and pASKSapC-EGFP, the sequence encoding EGFP was amplified by PCR from the vector pETAβ-GFP41 (a kind gift from Prof. Michael Hecht, Princeton University) and cloned in place of the gene encoding GFPmut2 of pASKBR2GFP, pASKNTR1(D03)-GFP, and pASKSapC-GFP, respectively, using the restriction enzymes PstI and HindIII. For the construction of the vectors pSuptoxD and pSuptoxR the genes djlA and rraA were amplified from E. coli genomic DNA using the primers DjlAFLfor-DjlAFLrev and RraAfor-RraArev, respectively. The resulting PCR products contained an optimized Shine-Dalgarno sequence and were inserted into the XbaI and HindIII sites of similarly digested pBAD33.34 The correct sequences for all constructs were verified by DNA sequencing. Membrane Protein Overexpression in Liquid Cultures. E. coli cells freshly transformed with the appropriate expression vector(s) were used for all protein production experiments. Single bacterial colonies were used to inoculate liquid LB cultures containing the appropriate combination of antibiotics (100 μg/mL ampicillin, 40 μg/mL chloramphenicol or 50 μg/mL kanamycin (Sigma)). These cultures were used with a 1:50 dilution to inoculate fresh LB cultures with 0.01 (MC1061 and SuptoxD cells) or 0.2% arabinose (SuptoxR cells), which were grown at 30 °C to an optical density at 600

reasons: (i) these systems were not designed to screen directly for suppression of MP-induced toxicity; (ii) strong overexpression of either djlA or rraA has been shown to be toxic for E. coli;31,35,40 (iii) FatI, the restriction endonuclease, which was used for the construction of the genomic DNA library in one of the aforementioned screens,15 cleaves the sequence of the djlA gene at two different sites and, thus, a clone with the intact gene may not have been present in that library; and (iv) it is a wellknown characteristic of genetic screening that different systems with different designs and experimental conditions can result in the identification of different hits. Compared to our previously identified multicopy enhancers of bacterial recombinant MP production FtsH,13 YbaB, YciQ, GlpQ,14 NagD, PtsN−YhbJ− Npr, and NlpDΔ(349−380),15 DjlA and RraA perform as well as the most efficient of those enhancers or even better, while at the same time they exhibit a beneficial effect on the production of a wider range of recombinant MPs. Elsewhere, we show that (i) DjlA and RraA are unique among similar E. coli proteins in their ability to facilitate bacterial recombinant MP production; (ii) DjlA and RraA act independently, i.e., the beneficial effects of each protein on MP production occurs through a mechanism that does not involve the other, and in a nonadditive manner; (iii) the beneficial effects of RraA on MP overexpression occur through a mechanism that involves the ribonuclease RNase E but not its paralog, RNase G; (iv) full-length and membrane-bound DjlA is required for exerting the observed beneficial effects on MP production; and that (v) the beneficial effects of DjlA on MP overexpression are mediated by the action of the molecular chaperone DnaK (D.G., D.C.D. and G.S., unpublished results). On the basis of these observations, we have generated a model about the mechanism with which MP-induced toxicity is suppressed and cellular MP accumulation is enhanced in the engineered strains SuptoxD and SuptoxR (D.G., D.C.D. and G.S., unpublished results). We anticipate that SuptoxD and SuptoxR will be utilized broadly in future studies involving overexpression of recombinant MPs.

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METHODS Bacterial Strains. E. coli MC1061 was used unless otherwise stated. C41(DE3) and C43(DE3) were purchased from Lucigen, while Lemo21(DE3) was purchased from New England Biolabs. All utilized E. coli strains and their genotypes are shown in Table 2. Plasmid Constructions. DNA primers utilized for cloning of recombinant DNA are listed in Supplementary Table S1. All enzymes for cloning of recombinant DNA were purchased from New England Biolabs. The plasmid pBAD30BR2-KanR was generated by amplifying the sequence encoding for the enzyme aminoglycoside 3′-phosphotransferase (KanR) by PCR from the vector pET-28a(+) (Novagen) with the primers Kanfor and Kanrev, digesting with PstI and HindIII, cloning into similarly digested vector pBAD33BR2-GFP15 by replacing the GFPencoding gene, and then subcloning of the entire FLAG-BR2TEV-KanR fusion into pBAD3034 after digesting with XbaIHindIII. For the amplification of the SCD gene (encoding for the human Stearoyl-CoA desaturase amino acids 1−355), overlap extension PCR was used to insert a silent mutation in order to remove an internal PstI site. The primers SCDAafor, SCDAaPstImutrev, SCDAaPstImutfor and SCDAarev were used to amplify and mutate the gene encoding SCD from pBADSCD-GFP.14 Then, the insert was digested with XbaI and PstI and ligated in place of the gene encoding BR2 into similarly N


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ACS Synthetic Biology nm (OD600) of ∼0.3−0.5 with shaking. The temperature was then decreased to 25 °C and after a temperature equilibration period of 10−20 min, MP expression was induced by the addition of 0.2 μg/mL anhydrotetracycline (aTc) (Sigma) overnight. As described also in previous studies,21,25 in certain cases MP-producing cultures exhibited abnormally high levels of growth after overnight induction of MP overexpression, a phenomenon which occurs due to chromosomal and/or plasmid mutations that inhibit the otherwise toxic process of MP production. This phenomenon was more frequently observed when MP overexpression occurred in the absence of our effectors, thus indicating that genetic stability during recombinant MP overexpression is increased in SuptoxD and SuptoxR compared to WT E. coli. In the cases where nonexpressing bacterial cells managed to outcompete the expressing cells and abnormal growth was recorded, the samples were discarded. Membrane Isolation. Total membrane fractions were isolated from 500 mL LB cultures in all cases with the exception of in-gel fluorescence experiments, where 2 L cultures were utilized in order to acquire higher amounts of recombinant proteins to allow for better visualization of the fluorescent bands, especially for WT E. coli. Cells were harvested by centrifugation and resuspended in 10 mL of cold lysis buffer (20 mL for in-gel fluorescence experiments) (300 mM NaCl, 50 mM NaH2PO4, 15% glycerol, 5 mM dithiothreitol, pH 7.5). The cells were lysed by brief sonication steps on ice and the resulting lysates were clarified by centrifugation at 10 000g for 15 min. The supernatant was then subjected to ultracentrifugation on a Beckman 70Ti rotor at 42 000 rpm (130 000g) for 1 h at 4 °C. The resulting pellet was finally resuspended in 10 mL of cold lysis buffer (1−5 mL for in-gel fluorescence experiments) and homogenized. Western Blot and In-Gel Fluorescence Analysis. Proteins samples were analyzed by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) on 10 or 15% gels. In-gel fluorescence was analyzed on a UVP ChemiDoc-It2 Imaging System equipped with a CCD camera and a GFP filter, after exposure for about 3 s. For Western blotting, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck) for 50 min at 12 V on a semidry blotter. Membranes were blocked with 5% nonfat dried milk in Trisbuffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. After washing with TBST three times, membranes were incubated with the appropriate antibody dilution in TBST containing 0.5% nonfat dried milk at room temperature for 1 h. The utilized antibodies were a mouse monoclonal antipolyhistidine antibody (Sigma) at 1:2500 dilution, a mouse monoclonal anti-FLAG antibody (Sigma) at 1:1000 dilution, a mouse anti-GFP antibody at 1:20 000 dilution (Clontech) and a mouse anti-MBP antibody (New England Biolabs) at 1:2500 dilution, all conjugated with horseradish peroxidase. After washing with TBST three times, the proteins were visualized on X-ray film with SuperSignal West Pico chemiluminescent substrate (Pierce). Spheroplast Generation and Labeling. Cells corresponding to 3 mL of a culture with OD600 equal to 1 were resuspended in 350 μL of an aqueous solution containing 0.75 M sucrose, 0.1 M Tris, pH 8. The samples were mixed by vortexing while 700 μL of 1 mM EDTA, pH 8 were being added dropwise, and the resulting cell suspension was incubated for 4 min at room temperature. 35 μL of 20 mg/ mL lysozyme in PBS were then added and the samples were

mixed by slow rotation on a rotating wheel for 20 min. After the addition of 50 μL MgCl2 0.5 M, the samples were kept on ice for 10 min. Spheroplasts were pelleted at 10 000g for 10 min at 4 °C and resuspended in 0.5 mL PBS. 167 μL of sample were spun down at 10 000g for 10 min at 4 °C, the supernatant was discarded and the pellet was resuspended in a 200 μL PBS solution containing a 1:400 dilution of an Alexa Fluor 647conjugated anti-FLAG antibody (Cell Signaling Technology) and rotated on a rotating wheel for 3 h. The spheroplasts were then washed with 500 μL PBS, spun down at 10 000g for 10 min at 4 °C and resuspended in 100 μL PBS. Bulk Fluorescence Measurements. Cells corresponding to 0.25 OD600 units were harvested and resuspended in 100 μL PBS. The cell suspension was then transferred to a black 96well plate and after fluorophore excitation at 488 nm for GFP or 647 nm for Alexa Fluor 647, fluorescence was measured at 510 nm for GFP and 670 nm for Alexa Fluor 647 using a TECAN SAFIRE2 plate reader. GFP Fluorescence Analysis by Flow Cytometry. 107 cells were resuspended in 1 mL PBS and after fluorophore excitation at 488 nm, the fluorescence of 50 000 cells was measured at 530/30 nm using a Partec CyFlow ML flow cytometer and analyzed statistically using FlowJo 7.6.2. Neurotensin Labeling. Spheroplasts were prepared as described above using about one-third of the cells and buffer quantities. For spheroplasting, lysozyme (20 mg/mL) was prepared in Tris−KCl buffer (50 mM Tris−HCl, pH 7.4, 150 mM KCl) instead of PBS. Spheroplasts corresponding to about 107 cells were labeled with 500 nM BODIPY-labeled neurotensin(8−13) (NT(8−13); Innovagen) in 20 μL cold Tris−KCl buffer for 1 h with shaking at room temperature in the dark. Then, the samples were diluted in 300 μL PBS and their fluorescence was measured by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences). The background BODIPY-NT(8−13) fluorescence corresponding to MC1061 cells carrying pASKNTR1(D03)-TrxA in the absence of aTc was subtracted from all measurements (Supplementary Figure S4). Real-Time PCR. Membrane protein overexpression with or without effector coexpression was performed as described above with cells grown at 30 °C and a 4-h induction period at 25 °C. For total RNA isolation, one OD600 unit of bacterial culture was mixed with twice the volume of RNAprotect Bacteria Reagent (QIAGEN). The sample was incubated for 5 min at room temperature and the cells were collected by centrifugation at 19 000g for 10 min. Bacteria were lysed and total RNA was extracted using the NucleoSpin RNA kit (Macherey Nagel) according to the manufacturer’s instructions. Nucleic acid concentration and purity was measured by spectrophotometry. Residual DNA was digested by incubation of the samples with two units of TURBO DNase (Ambion). After DNase removal from the samples using the NucleoSpin RNA kit (Macherey Nagel), RNA was quantified and 250 ng were reverse-transcribed using Superscript III reverse transcriptase (Invitrogen) and random hexamer primers according to the manufacturer’s instructions. Real-time PCR was performed using appropriate primer pairs (BR2RTfor, BR200RTrev or S1for, S1200rev; Supplementary Table S1) and quantified using SYBR Green I (iQ SYBR Green Supermix, Biorad) on an iQ5 Real-Time PCR detection System (Biorad) using the ribosomal protein S1 mRNA as internal control for comparative quantitative analysis. S1 was used as a reference as it has been shown that its expression levels remain steady upon O



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rraA overexpression.32 All assays were performed in 11 μLreactions. To assess the specificity of the PCR product, the melting curve was analyzed and a small aliquot of the reaction was visualized by agarose gel electrophoresis and stained with ethidium bromide. Serial dilutions of total RNA were prepared and a standard curve was generated for each qPCR reaction set. The results were analyzed using the Relative Standard Curve Method described in the technical notes provided by Applied Biosystems.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00174. Figure S1, Screening of the ASKA library for the identification of suppressors of the toxicity caused by membrane protein overexpression; Figure S2, djlA and rraA coexpression have beneficial effects on the growth and protein-accumulating capacity of E. coli BL21(DE3) producing BR2-GFP; Figure S3, SuptoxD and SuptoxR accumulate enhanced cellular levels of recombinant MPs; Figure S4, NTR1(D03) ligand-binding activity; Figure S5, Effects of djlA and rraA coexpression on the proteinaccumulating capacity of E. coli BL21(DE3), C41(DE3), and C43(DE3) cells producing BR2-GFP from the bacteriophage T7 promoter; Table S1, PCR Primers used in this study (PDF)

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Research Article

AUTHOR INFORMATION

Corresponding Author

*Tel: +302107273736. Fax: +302107273677. E-mail: [email protected]. Author Contributions

GS conceived and supervised the project. GS, DG, and KK designed the research. DG carried out the genetic screens and the development and characterization of the new strains; KK, MM, and DG carried out the comparison of SuptoxD and SuptoxR with wild-type and commercial E. coli strains. DCD carried out the in-gel fluorescence experiments and part of the flow cytometry experiments. DG, KK, MM, DCD, FK, and GS analyzed the data. GS and DG wrote the paper with contributions from KK and FK. All authors read and approved the final manuscript. Notes

The authors declare the following competing financial interest(s): GS and DG are inventors on a patent application for SuptoxD and SuptoxR.

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ACKNOWLEDGMENTS We would like to thank Michael H. Hecht (Princeton University) for plasmids, Dr Alexander Pintzas for facilitating the RT-PCR experiments, and Athina Stavridou, Konstantinos Asimakopoulos and Cleopatra Avrampou for technical support. This work was supported by the research grant “Synergasia” 09ΣYN-21-1078 and the Synthetic Biology research infrastructure OMIC-ENGINE, financed by the Hellenic General Secretariat of Research and Technology and the National Strategic Reference Framework (NSRF). KK was the recipient of the Hellenic State Scholarships Foundation (IKY-Idryma Kratikon Ypotrofion) “Excellence” award for postdoctoral research. P


Research Article

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Development of Escherichia coli Strains That Withstand Membrane

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