Engineering Bacteria to Catabolize the Carbonaceous Component

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Engineering bacteria to catabolize the carbonaceous component of sarin: teaching E. coli to eat isopropanol Margaret E. Brown, Aindrila Mukhopadhyay, and Jay D Keasling ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00115 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016

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ACS Synthetic Biology

Engineering bacteria to catabolize the carbonaceous component of sarin: teaching E. coli to eat isopropanol Margaret E. Brown,1,2 Aindrila Mukhopadhyay,1,2 Jay D. Keasling1-5* 1

Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. Joint BioEnergy Institute, Emeryville, CA, USA. 3 Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, California, USA. 4 Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA. 5 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Alle, DK2970Hørsholm, Denmark. 2

*Corresponding author: [email protected]

We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component

of

the

extremely

toxic

chemical

warfare

agent

sarin.

Enzymatic

decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion, with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.

Keywords: synthetic biology, bioengineering, sarin, biodegradation, acetone carboxylase, carbon catabolism pathway

ACS Paragon Plus Environment

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As the bioengineering toolkit has expanded, we have successfully engineered pathways to produce high titers of interesting and societally relevant compounds like the anti-malarial precursor artemisinic acid,1 the potential gasoline replacement fuels butanol and isobutanol,2, 3 and polyketides.4 Many heterologous pathways either create toxic intermediates or involve enzymes that interfere with endogenous metabolic pathways, so organisms often attempt to rid themselves of this introduced metabolic burden.5 However, if the introduced pathway benefits the host, positive selection will encourage organisms to retain the pathway. By coupling growth requirements and target compound degradation, we can engineer organisms that biosynthesize relevant molecules of interest from otherwise useless contaminants. The goal of this study was to demonstrate the feasibility of such organisms by bioengineering E. coli to catabolize isopropanol, a carbonaceous byproduct of sarin degradation, into the central metabolite acetyl-CoA (Figure 1). Sarin is first biodegraded into phosphate and isopropanol by use of a phosphotriesterase, a phosphodiesterase, and the phosphonate degradation cluster; these steps in sarin degradation have previously been investigated, enzymatically characterized, and functionally expressed in E. coli (Figure 1A).6-9 Detoxification studies of sarin and sarin analogues using phosphotriesterase mutants have proven effective in increasing their reactivities toward the more toxic enantiomer of sarin.6, 10, 11 E. coli even natively encodes a phosphonate degradation cluster that has been observed to degrade the methyl phosphonate associated with this organophosphonate.12-14 However, the complete degradation of this hazardous contaminant has not been demonstrated. We therefore focused on developing a functional pathway that would allow E. coli to consume the downstream carbonaceous degradation product, isopropanol, and thrive. Further optimization of the upstream phosphotriesterase and phosphodiesterase enzymes may be required for complete transformation of—and growth on—sarin; however, a microbe


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engineered with a complete pathway could be reliant on sarin as a source of both phosphate and carbon for growth. Few enzymes are known to be capable of transforming the small and relatively volatile molecule, isopropanol, and many of them have been functionally characterized in E. coli.15-18 While there are several previously-characterized alcohol dehydrogenases capable of oxidizing isopropanol to acetone, enzymes known to catalyze the breakdown of acetone are significantly less evident in the literature and have proven more difficult to express in E. coli.19 Acetone carboxylase (ACX) is a large (370 kDa) multi-subunit protein complex (α2β2γ2) encountered in only a few sequenced bacteria. Having little identity to any currently characterized proteins, it has only been purified and studied from its native hosts. Perhaps best studied is the ACX complex from Xanthobacter autotrophicus PY2,20, 21 followed by a handful of orthologs from Aromatoleum aromaticum22 and Rhodobacter capsulatus.21, 23 These complexes bind two to three equivalents of metal cations per complex; the identities of these cations differ between species but usually involve Fe, Zn, and/or Mn. The Xanthobacter ACX complex binds 1 atom each of Mn, Fe, and Zn, and follow-up Mn2+-depletion studies have shown the Mn2+ to be essential for enzymatic activity.23 Though biotinylation is required for many carboxylating enzymes,24 ACX complexes lack this biotin functionality;22, 23 however, they do require nucleoside triphosphates, Mg2+, and bicarbonate for reactivity. Basic details of the ACX carboxylation mechanism have been proposed, but the presence or role of cofactors and counter-ions remains unknown. A functional isopropanol consumption system in E. coli required many levels of manipulation. We began with the in vitro activity verification of single enzyme variants before assembling the entire pathway in vivo. Because the native organisms express their ACX complexes very highly when grown on isopropanol or acetone as carbon sources, we assumed


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that efficient acetone carboxylation in our engineered system would require similarly high levels of expression. E. coli was unable to express a codon-modified ACX complex from Xanthobacter autotrophicus at the necessary concentrations; however, the native Xanthobacter acx operon (including intergenic sequences) was expressible in E. coli and could be further manipulated to mirror the expression levels found in isopropanol-fed Xanthobacter. We purified this optimized recombinant ACX complex to verify the mechanistic requirement, or lack thereof, of thiamine analogs for enzymatic activity. We engineered Escherichia coli DH1(DE3) with an alcohol dehydrogenase and the Xanthobacter ACX, taking advantage of the endogenous acetoacetylCoA synthetase and thiolase, for the consumption of isopropanol; decoupling enzyme expression allowed us to probe pathway bottlenecks, and careful optimization of growth and induction conditions resulted in engineered E. coli capable of consuming isopropanol both as a supplemental carbon source as well as a sole carbon source. Cells grown in the presence of low glucose were supplemented or medium-exchanged with 160 mM isopropanol (0.75%), and over 4 days they consumed up to 65% compared to no-cell controls. As the engineered E. coli can grow on exclusively isopropanol, we posit that the ability to use isopropanol as a sole carbon source is a major step forward in engineering E. coli for the bioremediation of sarin. To our knowledge, the global quantity of sarin and other chemical warfare agent stockpiles remains unknown, and such engineered organisms could provide a plausible approach for responsible stockpile depletion.

RESULTS

Pathway development for the catabolism of the carbonaceous component of sarin (isopropyl methylphosphonofluoridate) was based on studies of microbial decontamination of propane and isopropanol.15, 17, 20 For the transformation of isopropanol into the central metabolite acetyl-CoA,


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we designed a pathway that begins with the alcohol dehydrogenase-dependent oxidation of isopropanol to acetone. An acetone carboxylase complex (ACX) then condenses acetone and bicarbonate to form acetoacetate (Figure 1B). The endogenous acetoacetyl-CoA synthetase and acetoacetyl-CoA thiolase enzymes in E. coli can then catabolize acetoacetate to produce acetylCoA, completing the degradation of isopropanol.

Expression of the acetone carboxylase. Our engineered system relies on active heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2. The ACX complex from Aromatoleum aromaticum was also considered for this project, but in our hands its heterologous expression yielded primarily insoluble protein (data not shown). For the Xanthobacter ACX, we began by recoding the ACX sequence from the native GC-rich sequence (67%) down to E. coli’s moderate GC content while limiting rare codon usage. The three genes encoding the subunits for the ACX enzyme complex were cloned behind the strong Trc promoter in the pTrc33 plasmid as one synthetic operon with strong ribosome binding sites. Since this protein complex has only been purified and characterized from the native organisms, our first task was to verify that active recombinant protein complex was expressed in our host. E. coli expressing either the ACX complex or an empty plasmid control were lysed, and the soluble fraction of each was assayed either in the presence or absence of acetone. The reactions were analyzed for the presence of acetoacetate using HPLC separation. The soluble fraction produced acetoacetate only in the presence of both acetone and the ACX-expressing lysate, confirming the presence of an active ACX complex in E. coli (Figure S1). In the presence of short-chain ketones, Xanthobacter autotrophicus PY2 expresses its ACX complex as 20 - 25% of its total soluble protein content.21 Previous biochemical characterization


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has shown this enzyme to have very low specific activity (~240 nmolmg-1min-1), supporting the hypothesis that low activity requires high amounts of protein to allow the organism to grow on these short-chain ketones as carbon sources. As the native Xanthobacter species produces high concentrations of ACX to allow for doubling rates of 4-10 hours on acetone as a carbon source, we anticipated that an engineered microbe dependent on this protein would require a similarly high protein content. However, we found the standard technique of promoter modification to be ineffective at increasing the total protein content of the ACX complex (Figure S2A), and, in fact, the final expression plasmid required many revisions (Tables S1, S2). Briefly, we cloned the recoded acx synthetic operon into BglBrick plasmids25 to compare transcription from the lacUV5, arabinose, Trc, and T7 promoters, but there was no significant improvement or even difference in the expression of the acx genes in E. coli DH1(DE3). We subsequently created two much more conservative plasmids that contained the native Xanthobacter sequences in their native operon order for expression from the pBbE7c plasmid (T7 promoter, ColE1 origin of replication): plasmid pBbE7c-XAacxn contains the three individual GC-rich genes from the native Xanthobacter genome with the same synthetic intergenic sequences present in the original codon-modified acx sequence, and plasmid pBbE7c-XAacxnT contains the entire native operon. Cells containing the pBbE7c-XAacxn vector expressed no observable protein save for the smallest subunit (Figure 2A, lane 2), while the pBbE7c-XAacxnT expressed the first gene in the operon highly (Figure 2A, lane 3). We subsequently increased the strength of the ribosome binding sites (plasmid pBbE7c-XAacxnT-rbs), thereby improving expression of the second subunit (Figure S2B). A second T7-lacO site preceding the second subunit (plasmid pBbE7cXAacxnT-dT7) further increased the expression of this subunit; however, none of our XAacxnT modifications showed increased expression of the smallest subunit. A chimeric construct with


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the synthetic intergenic sequence preceding the small subunit (plasmid pBbE7c-XAacxnT-dT7ssC) produced little of the small subunit protein. Our final construct, plasmid pBbE7cXAacxnT-dT7-v3, expressed the small subunit 5’ of the gene encoding the first subunit. As determined by SDS-PAGE analysis, this construct yielded total expression similar to that previously established in Xanthobacter with SDS-PAGE analysis (Figure 2A, lane 4).20, 21 Crude densitometric analysis of the total lysate of this final construct was used to calculate the subunit stoichiometry to be approximately 1.2 : 1.3 : 1 in subunits (acxA : acxB : acxC; Figure S2C). To summarize, for high expression of the ACX complex in E. coli, we found it necessary to use the native genetic sequence, including the native intergenic sequences. Rearranging the order of the genes and other plasmid modifications altering the transcription and translation of the ACX complex then yielded a total expression content in E. coli similar to that of the native organism.

Expression of candidate alcohol dehydrogenases. Four alcohol dehydrogenases proposed to be active in the oxidation of isopropanol were considered for our catabolic pathway. Because the importance of intracellular NAD(P)+ cofactor concentrations in a working system was unclear ([NAD+], 2.6 mM; [NADP+], 2.1 µM26), two of our candidate alcohol dehydrogenases were short-chain NAD+-dependent enzymes (Pseudomonas fluorescens15 and Gordonia sp. str. TY517) and two were medium-chain NADP+-dependent enzymes (Thermoanaerobacter brockii and Clostridium beijerinckii18, 27). We overexpressed these genes with the T7 expression system and analyzed lysate activity using NAD(P)+ cofactors as a measure of isopropanol oxidation. We found that both short-chain enzymes had low activity on isopropanol, likely reflecting the fairly insoluble expression of these proteins (Figure S3). The medium-chain enzymes had extremely high activity (Figure 2B). For growth experiments using isopropanol as the carbon source, we


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chose the more active candidate from each cofactor dependency group: Pseudomonas fluorescens (NAD+-dependent, PFadh) and Clostridium beijerinckii (NADP+-dependent, CBadh).

Purification and in vitro activity determination of the recombinant ACX complex. A recent study of a Desulfococcus biacutus acetone carbonylation reaction established that adding thiamine pyrophosphate (TPP) increased lysate activity by 7-fold.28, 29 Peptide mass fingerprinting showed acetone-induced upregulation of a TPP-requiring enzyme; however, the identity of the proteins responsible for the carbonylation reaction remains unknown.30 To ensure that the ACX mechanism does not rely on any thiamine analog, we purified the ACX complex for kinetic evaluation. We strongly induced ACX complex expression from the plasmid pBbE7c-XAacxnTdT7-v3 in E. coli DH1(DE3) grown in either LB or MOPS-M9 minimal medium. Cells were lysed, and the ACX complex was purified to relative homogeneity using, first, ion exchange chromatography and, subsequently, size-exclusion chromatography (Figure S4). Subunit stoichiometry for the purified products was approximately 1 : 1 : 1 as expected from the total lysate analysis (Figure S4D). We were able to monitor the activity of the purified ACX complex with a kinetic assay that couples the NADH-dependent 3-hydroxybutyrate dehydrogenase activity to acetoacetate production (Figures S5A, S5B). Using sealed reactions to prevent the dissipation of acetone, we found reactivity only in the presence of both purified ACX complex and acetone. We calculated the specific activities to be 40.6 nmolmg-1min-1 and 29.4 nmolmg-1min-1 for the ACX complex purified from cells grown in minimal medium and LB, respectively. Both purifications yielded between 4 and 5 mg of ACX protein complex. The lower reactivity of the ACX complex purified


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from LB culture probably results from protein impurities in the final preparation (Figure S4C). Recombinant ACX complex purified from minimal medium had 6-fold lower activity than ACX purified from Xanthobacter autotrophicus PY2.21 Because the purified complexes from both heterologous expressions had a lower activity than the native enzyme, we believe that reduced activity in E. coli is dependent on expression host instead of growth medium. We posit that the lower activity could reflect a difference in 1) protein folding and complex formation dependent on a chaperone available only in Xanthobacter, 2) the redox state of incorporated metals, or 3) a limited cofactor availability in E. coli. We tested the acetone reactivity of purified protein in the presence of thiamine analogs and found no activity stimulation, suggesting they are likely not involved in the mechanism (Figure S5C). We cannot not dismiss the possibility that these cofactors are required at the time of protein folding to ensure proper cofactor integration, however, so we opted to supplement the growth medium for our engineered cells with thiamine.

Growth of engineered strains on isopropanol. We co-transformed a low-copy, lacUV5-driven, alcohol dehydrogenase plasmid (pBbS5a-ADH) and a high-copy, T7-driven, ACX plasmid (pBbE7c-XAacxnT-dT7-v3) into E. coli DH1(DE3) to create our isopropanol degradation system (Figure S6). Biological quadruplicates were grown in 0.2% glucose-MOPS-M9 medium supplemented with Fe2+, Mn2+, Zn2+, and extra thiamine. Control strains contained the same ACX plasmid accompanied by a second plasmid expressing the gene for red fluorescent protein instead of alcohol dehydrogenase. We induced the cultures at mid-log phase, added isopropanol after two hours of protein expression, and then measured microbial growth over four to six days and final isopropanol concentrations to evaluate consumption.


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The strain harboring Clostridium beijerinckii’s NADP+-dependent medium-chain alcohol dehydrogenase had the best isopropanol-related growth (Figure 3A). Surprisingly, our fluorescent control strain also showed some isopropanol-dependent growth, suggesting the existence of a promiscuous endogenous alcohol dehydrogenase in E. coli that can convert isopropanol to acetone. We found that this promiscuous enzyme was not specific to DH1(DE3); E. coli BW25113(DE3) showed similar growth (Figure S8A). HPLC analysis of growth medium verified a significant decrease in isopropanol for our engineered strains (Figures 3B, S8B). The growth of our engineered system was drastically reduced when inducer concentration differed from the optimal concentration (10 µM IPTG; Figure S9). To ensure that the system was not limited by the intracellular concentrations of bicarbonate, a necessary component in the ACX reaction, we overexpressed the endogenous E. coli carbonic anhydrase in an operon with the alcohol dehydrogenases and saw no impact on growth or isopropanol usage (Figure S10). We therefore chose the C. beijerinckii alcohol dehydrogenase for all other studies.

Further analysis of pathway limitations. As only basic mechanistic details have been proposed for ACX catalysis, and the protein complex itself has only been characterized from native hosts,20-23 it was unclear why overinduction did not improve growth on isopropanol. IPAresponsive growth relied heavily on supplementation with Fe2+, Mn2+, and Zn2+ but not additional thiamine (Figure 3C-D). Similarly, thiamine supplementation in systems that were induced with higher concentrations of IPTG did not result in increased growth (Figure S11A) nor did further metal supplementation (Figure S11B). To ensure that the ACX reaction was not product inhibited by acetoacetate and that intracellular concentrations of acetone were not toxic, we expanded our alcohol dehydrogenase operon to include the acetoacetyl-CoA synthetase from


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Rattus norvegicus, previously reported to express functionally in E. coli,31 as well as the endogenous E. coli acetoacetyl-CoA thiolase.32 We decoupled alcohol dehydrogenase operon expression from ACX expression by placing the former operon under the control of the anhydrotetracycline-inducible Tet promoter; this circumvents reliance on endogenous ability to degrade acetoacetate, which can decarboxylate to re-form acetone, creating a futile cycle. Overexpression of the non-ACX portions of the pathway partially bypassed the 20 µM induction bottleneck (Figure 4AB), but overall growth was lower than cultures induced with 10 µM IPTG, suggesting that the ACX complex itself is the limiting enzyme. We found that the ACX complex was very subtly expressed at our optimal induction concentration (10 µM) and that its solubility was not compromised at higher concentrations (Figure S12), suggesting that the limitation is either in folding (producing soluble but inactive aggregate), incorrect metal redox state(s), or cofactor availability or incorporation; at this stage of the study, the identity of a potential limiting factor remains unknown. Nonetheless, even with low induction, the engineered E. coli was capable of using isopropanol as a sole carbon source (Figure 4CD).

Purification of recombinant ACX from cells grown in supplemented minimal medium. To investigate a potential difference in activity of protein purified from high inducer concentrations in minimal media and protein purified from optimal inducer concentrations in media containing the necessary metal supplements, the ACX was purified from the optimized growth conditions. ACX complex was expressed using E. coli DH1(DE3) carrying pBbE7c-XAacxnT-dT7-v3 grown in 1 L of metals-supplemented minimal medium at low concentrations of inducer (10 µM). The protein was purified as previously noted using ion-exchange and size-exclusion chromatography. The purity of the resultant ACX product was lower than the ACX products


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from the previous purifications (Figure S13) but still adequate for a kinetic assessment; its specific activity against acetone was 34 nmolmg-1min-1 – approximately the same as ACX complexes purified from cultures grown in the presence of high concentrations of inducer (Figure S13D). Again, thiamine analogs failed to stimulate activity (Figure S13E). All purifications yielded ~4-5 mg of purified protein regardless of the induction concentration, although there is a distinct increase in total soluble ACX complex with higher induction. We posit that the enzyme’s activity is limited by E. coli’s intracellular environment.

DISCUSSION

Modern bioengineering limits us to the use of proteins that are heterologously expressed in an active – which frequently equates to soluble – form. A more intricate understanding of protein expression and catalytic mechanism would allow bioengineers to optimize intracellular environments to support expression of functional protein; previous successful engineering feats have included modifying the reducing nature of the cytoplasm,33 overexpressing chaperones to assist in protein folding,34 or overexpressing auxiliary machineries required for enzymatic function or cofactor incorporation.35,

36

Using heterologous expression platforms can allow

probing of requirements for functional protein expression. To our knowledge, this is the first example of heterologous expression of a functional ACX. Even though the ACX complex proved to be a difficult enzyme complex to incorporate into our de novo isopropanol catabolism pathway, we were successful in engineering an IPA consumption pathway in E. coli. We first faced an unexpected challenge in increasing overall expression of this complex but managed to overcome this; much research surrounds engineering optimization of protein production in a heterologous environment,37-40 and we found that not only did we need to use native coding sequences, but we also had to preserve the operon


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structure for efficient expression of the entire complex. These results suggest that codon modification introduced unwanted regulatory factors, potentially as internal ribosome binding sites. Recognizing this, we were able to achieve high production of the complex with subunit stoichiometries similar to those of the enzyme from its native host. A conservative estimate for ATP yield per reduced cofactor using the electron transport chain during bacterial aerobic growth is 1 ATP/2 e-.41 As this pathway transforms 1 IPA molecule into 2 acetyl-CoA and consumes 2 ATP, a low-end energy estimate we can expect from this transformation is 8 ATP/IPA molecule. This rough analysis supports our hypothesis that IPA should be a sufficient substrate for bacterial growth. Growth of E. coli DH1(DE3) containing our optimized ACX-encoding plasmid pBbE7c-XAacxnT-dT7-v3 and an alcohol dehydrogenase biochemically characterized to oxidize isopropanol grew on isopropanol at low inducer concentrations. Even though the intracellular concentration of NADP+ has been measured to be significantly lower than that of NAD+ in E. coli,26 we found NADP+ dependency to be no hindrance for our pathway; the high enzymatic activity and solubility of the alcohol dehydrogenase from Clostridium beijerinckii was clearly more important than cofactor usage. We were successful in optimizing growth conditions as supplementation with ACX-specific metals proved to be critical for observing high growth on isopropanol. By knowing some of the potential biochemical requirements of the protein, we were able to rationally modify the growth medium resulting in a direct and positive response from our engineered organism. We purified the ACX complex from cells grown under three different conditions and found similar activity as well as total purified protein yield among them; the activity of our purified product was consistently found to be 6-fold lower than that purified from the native Xanthobacter host. As isopropanol-dependent growth was only observed at low induction, this


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suggests that protein expressed at IPTG inducer concentrations higher than 10 µM was simply not active and, thus, was only metabolically burdensome on the cells. Furthermore, the subunit stoichiometry in the purified recombinant protein was comparable to that of the native protein. Thus, it is unlikely that the lower activity is due to low production of one or more of the ACX subunits. Further increasing metal availability did not lead to increased growth, which superficially appears to suggest that metal loading is not the issue; nonetheless, it does not exclude the possibility that the redox state of the metals could be pertinent to either their incorporation or their activity. Other possible explanations for the lower activity include a cofactor or cofactor analog that is limited or unavailable in E. coli which may be essential to either the function, stability, or folding of the ACX complex, as well as a folding issue requiring specific chaperones from Xanthobacter that assist in ACX folding. As expression experiments from both low and high induction regimes yielded the same total protein, the remaining soluble protein from the high induction expressions must purify in different fractions, removed from our active fraction. With increased expression of the ACX complex but an inability to produce it functionally, the GTP and ATP requirement associated with protein manufacturing is likely higher than the energy provided by isopropanol consumption; therefore, we saw less growth under higher induction conditions. However, as the study of many heterologous pathway assemblies is inhibited by protein produced in an insoluble form, making them impossible to study or even understand the protein requirements for solubility, the optimized overexpressed ACX in this system is largely soluble and part of a functional system that can actually be investigated. Taking advantage of this, we were able to optimize growth conditions, and we found that, by including the rest of the pathway enzymes, we were able to partially alleviate our bottleneck suggesting that either the cells show some acetone toxicity or that the ACX shows


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product-inhibition by acetoacetate. Even with this pathway bottleneck, we were able to engineer E. coli to grow on isopropanol as a sole carbon source for the first time. Reconstitution of enzymes in a naïve system allows us to build a system to probe the biochemical requirements for enzymatic activity, allowing us to engineer more efficient biological systems for compound synthesis and toxin degradation. We moved a large and complex acetone carboxylase protein complex into E. coli, removed from both native regulation mechanisms as well as cellular context, and, in combination with proteins from other organisms, engineered a system that consumes 50 - 65% of the isopropanol over a 4-day period compared to no-cell controls. This shifts the focus from engineering organisms for toxin degradation to engineering organisms for toxin consumption.

Materials and Methods

Commercial materials. LB Broth Miller (LB), MgSO4, Tris-HCl, BugBuster, and MgCl2 were

purchased

from

EMD

Biosciences

(Darmstadt,

Germany).

Isopropyl

β-D-1-

thiogalactopyranoside (IPTG), HEPES, boric acid, manganese chloride tetrahydrate, H2SO4, and NH4Cl were purchased from Fisher Scientific (Pittsburgh, PA, USA). Dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), L-arabinose, sodium phosphate dibasic, potassium phosphate monobasic, iron sulfate heptahydrate, calcium chloride dihydrate, thiamine⋅HCl, thiamine monophosphate, thiamine pyrophosphate, ammonium carbonate, ammonium molybdate tetrahydrate, cobalt chloride hexahydrate, cupric sulfate pentahydrate, D-glucose, isopropanol, acetone, acetoacetate, adenosine triphosphate (ATP), NAD+, NADP+, NADH, and ammonium carbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloramphenicol (Cm) was purchased from Acros Organics (Morris Plains, NJ, USA). Carbenicillin (Cb) was purchased


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from Teknova (Hollister, CA, USA). Sodium chloride and glycerol were purchased from VWR Analytical (Radnor, PA, USA). MOPS was purchased from J. T. Baker (Avantor, Center Valley, PA, USA). DifcoTM agar was purchased from BD (Franklin Lakes, New Jersey, USA). Zinc sulfate hexahydrate was purchased from MP Biomedical (Burlingame, CA, USA). 3Hydroxybutyrate Dehydrogenase (3-HBDH) from Rhodobacter sphaeroides was purchased from Roche Applied Science (Penzberg, Germany). All PCR amplifications for cloning were carried out using Phusion® HF DNA polymerase (New England Biolabs; Ipswich, MA, USA). Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). Deoxynucleotides (dNTPs) and Novex® 8–16% Tris-Glycine SDS-PAGE gels were purchased from Invitrogen (Carlsbad, CA, USA). DNA was isolated using the QIAprep Spin Miniprep Kit, QIAquick PCR Purification Kit, and QIAquick Gel Extraction Kit (Qiagen; Valencia, CA, USA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA), resuspended at a stock concentration of 100 µM in 10 mM Tris-HCl pH 8.5, and stored at either 4 °C for immediate usage or -20 °C for longer term usage.

Bacterial strains and genomic DNA template. E. coli DH10B-T1R was used for plasmid

construction and E. coli DH1(DE3) was used for heterologous protein production as well as growth studies on isopropanol. Genomic DNA for Xanthobacter autotrophicus PY2 was purchased from the American Tissue Type Collection (Manassas, VA, USA). Genomic DNA from E. coli MG1655 was prepared using the Promega Wizard® Genomic DNA Purification Kit (Madison, WI, USA). Genes for alcohol dehydrogenases (pfadh, gsadh2, tbadh, and cbadh), acetoacetyl-CoA synthetase (rnaacs), and acetone carboxylase (acx) were codon-modified using


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the Integrated DNA Technologies (IDT) codon-optimization platform and purchased from IDT (Coralville, IA, USA) as g-blocks (see Table S4) for Gibson assembly into expression plasmids.

Plasmid construction. All strains and plasmids used in this work can be obtained from

http://public-registry.jbei.org42 and are summarized in Table S1. All expression plasmids were constructed using Gibson cloning for assembly except when noted.43 Genes for pfadh, gsadh2, tbadh, cbadh, and rnaacs were ordered as g-blocks and inserted into the pET16b plasmid using Gibson assembly (Tables S3 and S4 for oligonucleotide and g-block sequences). For the genes encoding the four ADH proteins, all were amplified from two g-blocks (*-G1, *-G2) with their respective primers (*-F1/R2 and *-F2/R2) using Phusion DNA polymerase (New England BioLabs; Ipswich, MA). They were then inserted into the NcoI and NdeI sites (to allow expression of genes with no His-tag) using Gibson assembly. For rnaacs, three sets of primers (RNaas-F1/R2, -F2/R2, and -F3/R3) were used to amplify their respective g-blocks (RNaas-G1, G2, and -G3). This similarly was inserted in the same manner into pET16b. The gene encoding the ECaact was amplified from E. coli MG1655 genomic DNA with ECaat-F1/R2 and inserted into pET16b in the same manner. The sequences encoding the ACX proteins from Aromatoleum aromaticum and Xanthobacter autotrophicus PY2 were also originally ordered as g-blocks and amplified with their respective primers: acxA was amplified from three g-blocks (*acxA-G1, G2, and -G3), acxB was amplified from four g-blocks (*acxB-G1, -G2, -G3, and -G4), and acxC was amplified from one g-block (*acxC-G1) using their respective primers. All were then assembled and inserted into the KpnI-SalI sites of pTrc33 using Gibson cloning making the pTrc33-XAacx and pTrc33-AAacx constructs. The resulting constructs were verified by sequencing (Quintara Biosciences; Berkeley, CA, USA).


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ACX next-generation plasmids. To compare the effects of promoter substitution on acx expression, the codon-modified acx was amplified and inserted into BglBrick vectors (pBbE*c, where * = 1, Trc; 5, lacUV5; 7, T7; 8, arabinose).25 To do this, the LCR cloning technique was used.44 Both the acx synthetic operon as well as the pBbE*c vectors were amplified using phosphorylated primers. The operon was amplified using XAacx_F/_R and the vectors were amplified using the pBbEXc_F/_R primers. Finally, the LCR was completed using the appropriate bridging primers (Table S1).

The native genes for X. autotrophicus acx were amplified from genomic DNA for two construct assemblies: 1) XAacxn, using the native gene sequences with the same synthetic intergenic regions as previously used in the XAacx construct; and 2) XAacxnT, using the total acx operon as a single amplicon from X. autotrophicus. All amplifications of native sequences were performed with Phusion DNA polymerase in the presence of 8% DMSO and inserted into the pBbE7c vector at the NdeI/BamHI sites using Gibson cloning. Thus, for pBbE7c-XAacxn, the three genes were amplified from X. autotrophicus PY2 genomic DNA using the XAacxAn-F6/R4, XAacxBn-F5/-R5, and XAacxCn-F2/-R4 primers. For pBbE7c-XAacxnT, the operon was amplified from genomic DNA using XAacxAn-F6/XAacxCn-R4. For the pBbE7c-XAacxnT-rbs construct, the ribosomal binding site strengths were increased by amplifying the subunits from pBbE7c-XAacxnT using the XAacxAn-F6/-R5, XAacxBn-F6/-R6, and XAacxCn-F3/-R4 primers. For the pBbE7c-XAacxnT-dT7 construct, the second T7 promoter was inserted within the native operon before acxBn by extension PCR of acxAn by using, first, XAacxAn-F6/-R6, followed by XAacxAn-F6/-R7, and finally XAacxAn-F6/-R8; acxBn was amplified using XAacxBn-F7/-R6; acxCn amplicon from the pBbE7c-XAacxnT-rbs construct was used. To investigate the impact of the synthetic vs. native intergenic sequence before subunit acxCn,


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constructing the pBbE7c-XAacxnT-dT7-ssC vector, the acxAn amplicon was that which was used in the pBbE7c-XAacxnT-dT7 construct; acxBn was amplified using acxBn-F7/R5; acxCn was amplified with the XAacxCn-F2/-R4 primers. The final construct, pBbE7c-XAacxnT-dT7v3, was constructed by Gibson cloning from three amplicons from the pBbE7c-XAacxnT-dT7 vector using the following primer sets: XAacxAn-F8/-R9, XAacxBn-F8/-R9, and XAacxCnF4/-R5. All constructs were sequence-verified.

The plasmids for expression of the rest of the pathway enzymes were constructed as follows. The cbadh and pfadh genes were amplified from their respective pET16b vectors with their respective primer sets. For pfadh amplification, PFadh-F5/-R5 primers amplified the gene which was then extended using PFadh-F6/-R5 for insertion into the BglII/BamHI sites of pBbS5a. The cbadh gene was amplified using the CBadh-F3/R3 primers for insertion into the BglII/BamHI sites of pBbS5a. The pBbS5a-PFadh-CA and pBbS5a-CBadh-CA vectors were constructed by amplifying CA from E. coli MG1655 genomic DNA first with CA-F1/-R4 and then with either CA-F4/-R4 (for insertion behind the pfadh) or CA-F5/-R4 (for insertion behind the cbadh) for insertion in the BamHI site of the two pBbS5a-adh constructs. The pBbS2a-CB-CA vector was constructed by first amplifying the cbadh from pBbS5a-CB vector using the pTet-F1/CBadh-R3 primers and inserting this into the BglII/BamHI sites of pBbS2a; CA was inserted in the same way as pBbS5a-CBadh-CA. Finally, the two vectors pBbS2a-CB-CA-RN-EC and pBbS5a-CBCA-RN-EC were constructed by amplifying rnaacs and ecaact from their respective pET16b plasmid constructs using RNaas-F7/-R6 and ECaat-F3/-R8 and inserted into the XhoI site of the pBbS*a-CB-CA plasmids.


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Protein expression studies. E. coli DH1(DE3) was transformed with the appropriate plasmids

and plated onto LB agar with corresponding antibiotics. Colonies were picked into LB supplemented with antibiotic and allowed to grow with shaking at 37 °C and 200 rpm for 12-16 hours. Cultures were diluted to an OD600 of 0.05 into LB-antibiotic for expression. They were then allowed to shake at 37 °C and 200 rpm until induction after which they were allowed to shake overnight at 30 °C.

Alcohol dehydrogenase expression for lysate analysis. In LB-Cb (100 µg/mL), cells carrying the respective pET16b-ADH plasmids were induced with 500 µM IPTG once the culture OD600 reached 0.5 - 0.6. Cultures were shaken for 21 hours at 30 °C after which cells were pelleted by centrifugation (8 min; 9,000 × g) and frozen at -80 °C. Total and soluble lysates were prepared by resuspending the cells in BugBuster (supplemented with 50 mM NaCl, 1 mM DTT, and 0.5 mM PMSF) at 5 mL/g cell paste. The resuspensions were lysed at room temperature on an orbital shaker for 20 minutes. Insoluble lysate was removed by centrifugation (20 min; 20,817 × g) at 4 °C. Soluble lysate was used immediately for lysate activity assays as well as protein solubility analysis by SDS-PAGE.

Acetone carboxylase expression for lysate activity analysis. In LB-Cm (30 µg/mL), E. coli cells carrying either the pTrc33-empty or pTrc33-XAacx were induced 500 µM IPTG once the culture OD600 reached 0.5 - 0.6. Cultures were then shaken for 17 hours at 30 °C and 200 rpm after which cells were pelleted by centrifugation (8 min; 9,000 × g) and frozen at -80 °C. Total and soluble lysates were prepared by resuspending the pellets in buffer (150 mM HEPES pH 8; 50 mM NaCl; 1 mM DTT) at 5 mL/g cell paste, lysing the cells using sonication, and removing the


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insoluble lysates by centrifugation (20 min; 20,817 × g) at 4 °C. Soluble lysates were used immediately for HPLC activity assays.

Acetone carboxylase expression comparison. In LB-Cm (30 µg/mL), E. coli DH1(DE3) cells carrying the pBbE(x)C-XAacx plasmids were grown at 37 °C and 200 rpm until culture OD600 reached between 0.5 and 0.6 at which cells were induced with either 500 µM IPTG or 20 mM arabinose (for the pBbE8c-XAacx vector) and allowed to shake for 20 hours at 30 °C. Cells were pelleted by centrifugation (8 min; 9,000 × g) and frozen at -80 °C. Total cell protein was analyzed using SDS-PAGE.

Acetone carboxylase expression and purification for activity studies. E. coli DH1(DE3) cells were transformed with the pBbE7c-XAnT-dT7-v3 plasmid and plated onto LBCm agar. Colonies were inoculated into LB-Cm and allowed to grow at 37 °C overnight at 200 rpm. 1 mL of this culture was frozen back at -80 °C in 15% glycerol. This culture was then acclimated into MMCm-1%glc using same method as the cultures in the isopropanol growth studies (see “Isopropanol growth studies” section below) after which the acclimated culture was frozen back in 15% glycerol at -80 °C. Both cultures, LB- and MM-, were inoculated into their respective media supplemented with antibiotic and grown overnight at 37 °C and 200 rpm. These were back-diluted to an OD600 of 0.05 in either LBCm or MMCm-1%glc and allowed to grow at 37 °C and 200 rpm until OD600 reached 0.5 at which both cultures were induced with 500 µM IPTG (or 10 µM IPTG in the case of the supplemented MM-grown ACX) and allowed to shake at 30 °C for 17 hours before cell collection by centrifugation (8 min; 9,000 × g) and storage at -80 °C.


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Cells were thawed and resuspended in buffer A (50 mM Tris pH 7.5, 25 mM NaCl, 1 mM DTT, 5 % glycerol) at 5 mL/g cell paste. After lysing the cells using sonication, the insoluble lysate was removed by centrifugation (20 min; 20,817 × g) at 4 °C. Soluble lysate was immediately passed over HiTrap DEAE Sepharose FF resin (5 mL; GE Healthcare Life Sciences; Piscataway, NJ, USA) at a flow rate of 2 mL/min on an ÄKTApurifier FPLC (GE Healthcare Life Sciences). The protein-loaded column was washed with 5 column volumes (CVs) of buffer A followed by 2 CV gradient to 30% Buffer B (50 mM Tris pH 7.5, 250 mM NaCl, 1 mM DTT, 5 % glycerol) at which the column was isocratically held for 3 CVs before a 15 CV gradient to 100% B; the column was washed with 5 CVs of Buffer B. Previous purifications of natively expressed ACX have found the complex to elute around 160 mM NaCl, so the fractions neighboring this elution concentration were analyzed by SDS-PAGE for the presence of the protein complex. The complex was found the elute in the range of 40% to 76% B, so the fractions of enriched protein were pooled and concentrated to ~2 mL with an Amicon filtration device using an Ultracel®100k membrane (Millipore Corporation; Billerica, MA, USA). The complex was further purified by size exclusion chromatography using a HiPrep 26/60 SephacrylTM S-300 HR size exclusion chromatography column (GE Healthcare Life Sciences; Piscataway, NJ, USA) equilibrated with Buffer C (50 mM Tris pH 7.5, 100 mM NaCl, 5 % glycerol). Fractions were analyzed by SDSPAGE for presence and purity of the complex. These fractions were pooled, concentrated to a concentration of ~4 mg/mL with an Amicon filtration device using an Ultracel®-100k membrane membrane, and stored at -80 °C after the addition of glycerol (10% (v/v) final concentration). Protein concentrations were calculated using the calculated extinction coefficients at 280 nm for the ACX amino acid sequence (MW= 369.2 kDa, ε280 = 613,760 M-1cm-1).


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Isopropanol growth studies. DH1(DE3) E. coli transformants carrying plasmids for IPA

consumption were picked in quadruplicate and inoculated first into LB with appropriate antibiotic (100 µg/mL carbenicillin and 15 µg/mL chloramphenicol). After growth at 37 °C for 1 day, the cultures were then acclimated to minimal medium by diluting the cells 1:50 into MOPSM9 minimal medium (MM; M9 salts, 100 mM MOPS pH 7.4, 2 mM MgSO4, 10 mM ferrous sulfate, 100 µM CaCl2, 100 ng/mL thiamine, 30 nM ammonium molybdate tetrahydrate, 4 µM boric acid, 300 nM cobalt chloride hexahydrate, 150 nM cupric sulfate pentahydrate, 800 nM manganese chloride tetrahydrate, and 100 nM zinc sulfate heptahydrate) supplemented with the appropriate antibiotic and 1% glucose. The cultures were then diluted twice more daily at 1:100 dilution into MM-1%glc with antibiotics once the overnight OD600 reached above 1.0. At this point, 15% glycerol stocks were made for strain storage at -80 °C.

Growth studies. Overnight cultures of MM-acclimated DH1(DE3) were diluted to an OD600 of 0.05 into 10 mL of MM or MMS (MM supplemented with extra 50 ng/mL thiamine, 10 mM iron sulfate, 5 µM zinc sulfate, and 5 µM manganese chloride) with 0.2% glucose as an initial carbon source. MM was prepared the evening before growth studies and supplements were added immediately prior to cell dilution. The quadruplicate cultures were then grown by shaking at 37 °C until OD600 reached approximately 0.5. After induction with IPTG (and anhydrotetracycline if required), the cultures were shaken at 30 °C for 4 days. Isopropanol consumption control included triplicate or quadruplicate isopropanol-doped medium with no cells; this allowed for control of isopropanol evaporation over the four-day growth at 30 °C. Daily OD660 values were taken; OD660 was used instead of OD600 to avoid RFP fluorescence that occurs in this range at high RFP expression levels. Samples to analyze the extracellular presence of isopropanol and


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acetone were collected by removing cellular debris from 1 mL of cell growth using centrifugation (5 min; 20,817 × g), freezing the supernatants at -80 °C followed by thawing and centrifugation (5 min; 20,817 × g) to remove any remaining particulate matter that was found to precipitate in cooler temperatures. These samples were then analyzed by high-performance liquid chromatography (HPLC).

HPLC analysis of growth medium. Cleared sample supernatants were analyzed for the presence of acetone and isopropanol by an organic acids analysis column on an Agilent 1200 Series HPLC coupled to a refractive index detector. Samples (10 µL) were chromatographed on an Aminex® HPX-87H ion exclusion column (300 × 7.8 mm; Bio-Rad Laboratories; Hercules, CA, USA) in a column compartment held at 65 °C. Separation was performed isocratically using 4 mM sulfuric acid for 30 min.

Solubility analysis of ACXnT. MM-acclimated E. coli DH1(DE3) overnight cultures carrying the pBbE7c-XAacxnT-dT7 + pBbS5a-RFP plasmid combination were diluted into 4 x 50 mL of MMS-CbCm-1%glc and allowed to grow until OD600 reached 0.55, at which the cultures were induced with increasing concentrations of IPTG (2.5, 10, 20, and 100 µM) after which the cells were allowed to shake at 30 °C and 200 rpm for 16 hours. Cells were pelleted by centrifugation (8 min; 9,000 × g) and frozen at -80 °C until solubility analysis. Cells were resuspended in buffer (150 mM HEPES pH 7.9, 50 mM NaCl, 1 mM DTT, 0.5 mM PMSF) at 5 mL/g cell paste and lysed using sonication. Insoluble lysate was removed by centrifugation (20 min; 20,817 × g) at 4 °C and soluble lysates were analyzed using SDS-PAGE.


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HPLC analysis of ACX reaction with acetone. Cleared lysates of cells expressing from either

the pTrc33-empty or XAacx plasmid were used in the following reaction mixture. Reactions (500 µL total) comprised of HEPES buffer (50 mM, pH 8), acetone (2.5 mM), ATP (5 mM), MgCl2 (10 mM), NH4Cl (20 mM), (NH4)2CO3 (20 mM), and 40 µL of soluble lysate. Reactions were initiated with the addition of acetone, capped, and stirred overnight at room temperature. Samples were centrifuged to remove any precipitate (5 min; 20,817 × g). Acetoacetate production was monitored using the HPLC isopropanol analysis method described above.

Spectrophotometric activity assays. Kinetic assays were performed at 25 °C on a SpectraMax

M2 platereader (Molecular Devices; Sunnyvale, CA, USA). Data are reported as the mean ± s.d. (n = 3-6). Total volume used was 200 µL.

Alcohol dehydrogenase activity in isopropanol oxidation. Cleared lysates of E. coli expressing either RFP or ADHs from the pET16b vector were used immediately after cell lysis. Assays were comprised of HEPES (50 mM, pH 8), NAD(P)+ (3 mM), and clarified lysate (6 µL). Reactions were initiated by the addition of isopropanol (30 mM) and monitored by the production of reduced NAD(P)H cofactors at 340 nm (ε340 = 6220 M-1cm-1).

Acetone carboxylase kinetic activity assay. Kinetic assay of ACX activity was performed using literature protocol.22 Comprised of the same reaction mixture as that from the HPLC lysate activity assay monitoring the production of acetoacetate, the supplementation of 3hydroxybutyrate dehydrogenase (14 mU) and NADH (0.4 mM) allowed for the monitoring of


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acetoacetate production. This coupled analysis monitored the oxidation rate of NADH at 340 nm (ε340 = 6220 M-1cm-1) as the rate of acetoacetate production by the purified enzyme. Supporting information is available for supplemental figures and tables. Supporting information Abbreviations Figure S1. Lysate reactivity of E. coli expressing ACX complex Figure S2. Promoter and sequence modification for ACX protein complex expression Table S1. Plasmids used in this study Table S2. Plasmid depictions for ACX operon construction Figure S3. SDS-PAGE analysis of alcohol dehydrogenase expression and solubility Figure S4. Purification of recombinant ACX complex from E. coli DH1(DE3) Figure S5. Kinetic assays surveying activity of the recombinant ACX Figure S6. Plasmid system for growth of E. coli on IPA Figure S7. HPLC analysis of linearity in IPA and acetone detection Figure S8. Growth of plasmid-bearing E. coli BW25113(DE3) on IPA Figure S9. Increasing IPTG induction concentrations leads to lower IPA-related growth Figure S10. Expanding the IPA degradation system to include over-expression of the carbonic anhydrase Figure S11. Supplementation with thiamine or extra metals show no growth benefit Figure S12. Solubility of ACX protein as induced with increasing concentrations of IPTG inducer Figure S13. Purification of MMS-grown ACX Table S3. Oligonucleotides used for plasmid construction


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Table S4. G-blocks used for gene assembly

Abbreviations ADH, alcohol dehydrogenase; ACX, acetone carboxylase; AACS, acetoacetyl-CoA synthetase; AACT, acetoacetyl-CoA thiolase; PTE, phosphotriesterase; PDE, phosphodiesterase; PHN, phosphonate degradation cluster; CA, carbonic anhydrase; XAacx, acetone carboxylase from Xanthobacter autotrophicus PY2; AAacx, acetone carboxylase from Aromatoleum aromaticum PY2; PFadh (PF), alcohol dehydrogenase from Pseudomonas fluorescens; CBadh (CB), alcohol dehydrogenase from Clostridium beijerinckii; GSadh2 (GS), alcohol dehydrogenase from Gordonia sp.; TBadh (TB), alcohol dehydrogenase from Thermoanaerobacter brockii; RNaacs (RN), acetoacetyl-CoA synthetase from Rattus norvegicus; ECaact (EC), acetoacetyl-CoA thiolase from E. coli; IPA, isopropanol; IPTG, isopropyl β-D-1-thiogalactopyranoside; HPLC, high-performance liquid chromatography; FPLC, fast protein liquid chromatography; TPP, thiamine pyrophosphate; TMP, thiamine monophosphate; T, thiamine; MM, minimal media; MMS, minimal media with supplements; LB, lysogeny broth; RFP, red fluorescent protein; glc, glucose; aTet, anhydrotetracycline

Authors’ contributions MEB performed experiments, analyzed data, and drafted the manuscript. JDK supervised the work. The manuscript was read, revised, and approved by all authors.

Acknowledgements


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We would like to acknowledge Peter Kelly for help with the construction and expression of the pBbE*C-ACX constructs. We would also like to acknowledge Victor Chubukov for helpful discussions. Finally, this work was conducted under funding from Defense Advanced Research Projects Agency (Microbial Chemical Agent Neutralization, MicroClean: DARPA agreement # HR001134783 under DOE contract DE-AC02-05CH11231) and the Joint BioEnergy Institute (http://www.jbei.org), which is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.

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Figure 1. Introduction of new carbon utilization pathway for sarin degradation. Carbon sources, frequently glucose (blue circle), are taken up and metabolized to the central metabolite acetyl-CoA (red circle). (A) Hydrolysis of sarin by a phosphotriesterase (PTE) yields isopropyl methylphosphonate (IMPA). Further hydrolysis by a phosphodiesterase (PDE) releases isopropanol (IPA) and methylphosphonate. Endogenous proteins (PHN complex) in E. coli can release the phosphate for cell use. (B) Proposed pathway for isopropanol catabolism. Isopropanol is oxidized by an alcohol dehydrogenase (ADH) to form acetone, which is carboxylated by the ACX complex to yield acetoacetate. Acetoacetate is ligated to CoA using an acetoacetyl-CoA synthetase (AACS), and an acetoacetyl-CoA thiolase (AACT) releases acetylCoA to be used for cell growth.


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Figure 2. Biochemical lysate analysis of recombinant proteins. (A) SDS-PAGE of total cell protein upon ACX expression (500 µM IPTG) from the pBbE7c vector. Lane 1, ACX; 2, ACXn; 3, ACXnT; 4, ACXnT-dT7-v3. Blue arrows indicate the 3 subunits. (B) Activity rate comparison of the ADHs in the oxidation of isopropanol and following the production of reduced NAD(P)H at 340 nm. TB, TBadh; CB, CBadh; PF, PFadh; GS, GSadh2.


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Figure 3. Growth of ACX strains on isopropanol. (A) Growth of ADH-containing strains (JBEI-14133, JBEI-14127) and RFP-containing strain (JBEI-14132) on glucose (glc) in the presence and absence of isopropanol (IPA). (B) HPLC analysis of isopropanol at days 4 (solid) and 6 (hashed) showing isopropanol consumption in cultures of strains engineered with the isopropanol consumption pathway. (C) Growth of CBadh strain (JBEI-14129) reveals the requirement for metal supplementation, but not thiamine, in the medium. (D) HPLC analysis of growth medium from four day old cultures shows isopropanol utilization is correlated with metal supplementation and increased growth in DH1(DE3). PF, PFadh; CB, CBadh; RFP, red fluorescent protein; CA, carbonic anhydrase; IPA, 0.75% isopropanol; MM, minimal media; T, thiamine; MMS, minimal media with supplements; glc, 0.2% glucose.


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Figure 4. Expansion of IPA catabolism pathway to probe pathway bottlenecks. Growth of engineered E. coli DH1(DE3) on isopropanol (IPA). (A) Expression of ACX complex at 20 µM IPTG with increasing concentrations of anhydrotetracycline (aTet) inducing pBbS2a-CB-CA-RNEC (JBEI-14124) vs. the control pBbS2a-CB-CA (JBEI-14123). (B) HPLC analysis of isopropanol in the growth medium due to the presence of the remaining pathway enzymes. +, with RNaacs-ECaact (RN-EC); -, control. (C) Isopropanol-only growth comparison. Expression of ACX at 10 µM IPTG using pBbS5a-CB-CA-RN-EC (JBEI-14130) vs. the control pBbS5a-CBCA (JBEI-14129). (D) HPLC analysis of isopropanol from four day old cultures grown in the presence of only isopropanol. CB, CBadh; CA, carbonic anhydrase; RFP, red fluorescent protein; IPA, 0.75% isopropanol; glc, 0.2% glucose.


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Figure 2. Biochemical lysate analysis of recombinant proteins. (A) SDS-PAGE of total cell protein upon ACX expression (500 µM IPTG) from the pBbE7c vector. Lane 1, ACX; 2, ACXn; 3, ACXnT; 4, ACXnT-dT7-v3. Blue arrows indicate the 3 subunits. (B) Activity rate comparison of the ADHs in the oxidation of isopropanol and following the production of reduced NAD(P)H at 340 nm. TB, TBadh; CB, CBadh; PF, PFadh; GS, GSadh2. 143x177mm (300 x 300 DPI)


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Figure 3. Growth of ACX strains on isopropanol. (A) Growth of ADH-containing strains (JBEI-14133, JBEI14127) and RFP-containing strain (JBEI-14132) on glucose (glc) in the presence and absence of isopropanol (IPA). (B) HPLC analysis of isopropanol at days 4 (solid) and 6 (hashed) showing isopropanol consumption in cultures of strains engineered with the isopropanol consumption pathway. (C) Growth of CBadh strain (JBEI14129) reveals the requirement for metal supplementation, but not thiamine, in the medium. (D) HPLC analysis of growth medium from four day old cultures shows isopropanol utilization is correlated with metal supplementation and increased growth in DH1(DE3). PF, PFadh; CB, CBadh; RFP, red fluorescent protein; CA, carbonic anhydrase; IPA, 0.75% isopropanol; MM, minimal media; T, thiamine; MMS, minimal media with supplements; glc, 0.2% glucose. 234x274mm (300 x 300 DPI)



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Figure 4. Expansion of IPA catabolism pathway to probe pathway bottlenecks. Growth of engineered E. coli DH1(DE3) on isopropanol (IPA). (A) Expression of ACX complex at 20 µM IPTG with increasing concentrations of anhydrotetracycline (aTet) inducing pBbS2a-CB-CA-RN-EC (JBEI-14124) vs. the control pBbS2a-CB-CA (JBEI-14123). (B) HPLC analysis of isopropanol in the growth medium due to the presence of the remaining pathway enzymes. +, with RNaacs-ECaact (RN-EC); -, control. (C) Isopropanol-only growth comparison. Expression of ACX at 10 µM IPTG using pBbS5a-CB-CA-RN-EC (JBEI-14130) vs. the control pBbS5a-CB-CA (JBEI-14129). (D) HPLC analysis of isopropanol from four day old cultures grown in the presence of only isopropanol. CB, CBadh; CA, carbonic anhydrase; RFP, red fluorescent protein; IPA, 0.75% isopropanol; glc, 0.2% glucose. 323x218mm (300 x 300 DPI)


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87x35mm (300 x 300 DPI)


Engineering Bacteria to Catabolize the Carbonaceous Component

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