Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ron Milo ( ron.milo@weizmann.ac.il ). The pFDH plasmid generated during this study was deposited to Addgene (#131706). The pCBB plasmid as well as the autotrophic E. coli clones generated during this study are available from the Lead Contact upon completing a Materials Transfer Agreement.

We generated an engineered ancestor strain for chemostat evolution based on the Escherichia coli BW25113 strain (). We used P1 transduction () to transfer knockout alleles from the KEIO strain collection () to our engineered strain, and to knock out the genes phosphofructokinase (pfkA and pfkB) and 6-phosphate-1-dehydrogenase (zwf). Following the transduction of each knockout allele, the Kmselection marker was removed by using the FLP recombinase encoded by the pCP20 temperature-sensitive plasmid (). Loss of the selection marker and the temperature-sensitive plasmid were validated by replica-plating the screened colonies and PCR analysis of the relevant loci. The engineered ΔpfkA ΔpfkB Δzwf strain was then transformed with the pCBB plasmid () (GenBank: KX077536 ) and with a pFDH plasmid (Addgene plasmid #131706) with a constitutive promoter controlling the expression of the fdh gene. Following whole-genome sequencing, we noted that the ancestral strain possessed the following three mutations - fusA T125I, lrhA Δ9 bp (85-93/939), and integration of a mobile insertion sequence (IS) element into the promoter region of the xylE gene (−21, position 4,232,204). These mutations were acquired during early handling of the strain prior to chemostat inoculation.

Method Details

Generation of recombinant plasmids Popov and Lamzin, 1994 Popov V.O.

Lamzin V.S. NAD(+)-dependent formate dehydrogenase. LtetO-1 promoter with a constitutive one driving medium transcription levels (clone #10 from Braatsch et al. [2008 Braatsch S.

Helmark S.

Kranz H.

Koebmann B.

Jensen P.R. Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine-tuning. Zelcbuch et al. [2013 Zelcbuch L.

Antonovsky N.

Bar-Even A.

Levin-Karp A.

Barenholz U.

Dayagi M.

Liebermeister W.

Flamholz A.

Noor E.

Amram S.

et al. Spanning high-dimensional expression space using ribosome-binding site combinatorics. R selection marker on the plasmid with the aadA gene, which confers resistance to streptomycin. Details regarding the pCBB plasmid are reported in Antonovsky et al. (2016) Antonovsky N.

Gleizer S.

Noor E.

Zohar Y.

Herz E.

Barenholz U.

Zelcbuch L.

Amram S.

Wides A.

Tepper N.

et al. Sugar Synthesis from CO2 in Escherichia coli. To create the pFDH plasmid, an E. coli codon optimized DNA sequence based on the amino acid sequence of formate dehydrogenase from the methytholotrophic bacterium Pseudomonas sp. 101 (, UniProt: P33160 ) was synthesized and cloned with an N-terminal his-tag into a pZE21-MCS plasmid (Expressys, Germany). We replaced the Ppromoter with a constitutive one driving medium transcription levels (clone #10 from]) and a strong ribosome binding site (rbs B of]). We replaced the Kmselection marker on the plasmid with the aadA gene, which confers resistance to streptomycin. Details regarding the pCBB plasmid are reported in

Preparation and utilization of growth media Plasmid cloning and genomic modifications were carried out on a Luria Bertani medium with the relevant antibiotics (kanamycin (50 μg/ml), chloramphenicol (30 μg/ml, dissolved directly in the autoclaved M9 media and then filtered through a 0.22 μm PVDF filter) and/or streptomycin (100 μg/ml)). Engineered and evolved strains were grown on M9 minimal media supplemented with trace elements and the relevant carbon source(s). In the 13C-labeling experiments and for accurate estimation of growth parameters of the evolved cells on formate as the only organic compound, we used HPLC-grade water (Sigma Aldrich) and omitted EDTA from the trace elements. The trace elements components and their concentrations in the M9 media are: 50 mg/L EDTA (omitted during 13C labeling experiments and growth measurements), 31 μM FeCl 3 , 6.2 μM ZnCl 2 , 0.76 μM CuCl 2 ·2H 2 O, 0.42 μM CoCl 2 -6H 2 O, 1.62 μM H 3 BO 3 , 81 nM MnCl 2 ·4H 2 O.

Growth tests of autotrophic E. coli strain The growth experiments were conducted in a DASBox mini fermentation system (Eppendorf, Germany). The starting volume of each bioreactor was 150 mL M9 media supplemented with 30 mM or 35 mM sodium formate (Sigma Aldrich) as the carbon source, and trace elements (without the addition of EDTA and vitamin B1). Bacterial cells were seeded from a 15 mL starter at an OD 600 of 0.12-0.14 (resulting in a 1:10 dilution by volume). Growth temperature was set to 37°C, and the chemostat was aerated at a rate of 6 L/hr with 90% air supplemented with 10% CO 2 . Values from the various probes were logged at 5 min intervals and used for analysis as described below. Once a day, 2 mL samples were removed from the bioreactor and used for media analysis (after filtration through a 0.22 micron PVDF Millex-GV syringe filter unit (Merck Millipore)) and for offline OD measurements (see below). Once the culture reached the stationary phase, ≈15 mL of the media were resuspended in fresh M9 media, as above, to a total of 150 ml, and the growth test was repeated. Optical density measurements were performed online, using the integrated DASGIP® OD4 module and sensors. The values were converted into OD 600 by taking samples from the growth medium at various optical densities and measuring the OD 600 of each sample offline with a spectrophotometer (Ultrospec 10 Cell density meter, Amersham Biosciences) and a standard 10 mm polystyrene cuvette (Sarstedt, Germany). We fitted a linear relation between the DASGIP® OD4 measurements and the OD 600 measurements. After diluting the cells, the DASGIP® OD4 module was calibrated to give a value of 0 at the beginning of the second growth test. In this case, we fitted a linear relation between OD 600 measurements of samples from the culture and the readings of the DASGIP® OD4 sensor, using the same slope as the one employed for the linear fit from the first growth test. Growth rates were determined by transforming OD 600 measurements into logarithmic scale with a base of 2 and then calculating the growth rate over a sliding window interval of 150 sample points, in each window fitting a linear relation between log 2 (OD 600 ) and time (in hours). The slope of each fit represents the estimated growth rate (in doublings per hour). We then calculated the average of the highest growth rate in the four experiments (two growth cycles for each of the two formate concentrations - 30 mM and 35 mM) to give our best estimate of the maximal growth rate. The doubling time was calculated as the inverse of the growth rate, expressed in units of hours per doubling. To estimate the uncertainty of our calculated growth rates due to the calibration error, we sub-sampled from the data to get 100 different linear relations (slopes and intercepts) between the DASGIP® OD4 measurements and the OD 600 measurements. For each sampled set of parameters, we calculated the growth rate based on the same procedure described above. We used the mean and standard deviation of these 100 growth rates as our best estimators of the growth rate and its standard deviation in each growth test. We propagated the calibration error in each experiment assuming the calibration error is correlated across experiments.

Yield calculation for autotrophic growth Y = B ( t ) − B ( t 0 ) S ( t ) − S ( t 0 ) (Equation 1)

where B is the biomass weight in units of gram cell dry weight (gCDW) and S is the amount of formate in units of moles. The biomass weight was inferred from the measured optical densities of the samples at 600 nm (OD 600 ) via the conversion factor from OD 600 to gCDW, which ranges between 0.3 gCDW × L-1 per OD 600 for E. coli cells ( Glazyrina et al., 2010 Glazyrina J.

Materne E.M.

Dreher T.

Storm D.

Junne S.

Adams T.

Greller G.

Neubauer P. High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor. -1 per OD 600 ( Folsom and Carlson, 2015 Folsom J.P.

Carlson R.P. Physiological, biomass elemental composition and proteomic analyses of Escherichia coli ammonium-limited chemostat growth, and comparison with iron- and glucose-limited chemostat growth. -1 per OD 600 for the conversion. The yield was calculated based on the values of samples taken during the exponential phase of the growth according to the following equation:where B is the biomass weight in units of gram cell dry weight (gCDW) and S is the amount of formate in units of moles. The biomass weight was inferred from the measured optical densities of the samples at 600 nm (OD) via the conversion factor from ODto gCDW, which ranges between 0.3 gCDW × Lper ODfor E. coli cells () to 0.5 gCDW × Lper OD). We used the mean value of 0.4 ± 0.1 gCDW × Lper ODfor the conversion.

Formate uptake rate calculation y ( t ) = a − d 1 + ( t c ) b + d (Equation 2)

We then calculated the derivative of the fitted logistic function at each time point during the course of the growth to estimate the total formate consumption rate. The formate consumption rate was normalized to the amount of cellular biomass by using the OD 600 of the culture at the same time point, and converting it to dry cellular mass assuming a factor of 0.4 gCDW × L-1 per OD 600 . We report the mean uptake rate and its standard error across the four different growth tests (two growth cycles for each of the two formate concentrations - 30 mM and 35 mM). Throughout each of the four growth experiments, we measured the concentration of formate in the growth medium at different time points by using both HPLC and an enzymatic assay (see Analysis of media composition section). We fitted the measured formate concentration over the course of each growth experiment with a four parameter logistic function of the form:We then calculated the derivative of the fitted logistic function at each time point during the course of the growth to estimate the total formate consumption rate. The formate consumption rate was normalized to the amount of cellular biomass by using the ODof the culture at the same time point, and converting it to dry cellular mass assuming a factor of 0.4 gCDW × Lper OD. We report the mean uptake rate and its standard error across the four different growth tests (two growth cycles for each of the two formate concentrations - 30 mM and 35 mM).

Chemostat evolution experiment -1 (equivalent to a doubling time of ≈33 hours) at 37°C. The chemostat was fed media containing 4 g/L sodium formate and 0.5 g/L D-xylose as sole carbon sources. This amount of xylose in the feed makes xylose the limiting nutrient for cell growth in the chemostat. On days 47,166, 214, and 343 of the evolution experiment, the level of D-xylose in the feed media was reduced to 0.28, 0.13, 0.05, and 0 g/L, respectively. The concentration of formate was increased to 6 g/L on day 357, after the autotrophic growth phenotype was observed, and chloramphenicol (30 mg/L) and streptomycin (100 mg/L) were added to the feed media. Aeration of the chemostat was done through a DASGIP MX4/4 stand-alone gas-mixing module (Eppendorf, Germany) with a composition of 10% CO 2 and 90% air at a flow rate of 40 sL/hr. To monitor the chemostat, a weekly sampling protocol was performed. Samples were taken for media analysis and phenotyping (inoculation of the bacteria on minimal media containing formate and lacking D-xylose). We calculated the biomass dependency metric of each sample as the ratio between the xylose carbon concentration (g carbon/L) in the feed and the carbon concentration in the culture biomass. The biomass carbon concentration was calculated with a conversion factor of 0.2 g carbon per 1 OD 600 ( Glazyrina et al., 2010 Glazyrina J.

Materne E.M.

Dreher T.

Storm D.

Junne S.

Adams T.

Greller G.

Neubauer P. High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor. Folsom and Carlson, 2015 Folsom J.P.

Carlson R.P. Physiological, biomass elemental composition and proteomic analyses of Escherichia coli ammonium-limited chemostat growth, and comparison with iron- and glucose-limited chemostat growth. The evolution experiment was conducted in a Bioflo 110 chemostat (New Brunswick Scientific, USA) at a working volume of 0.7 L and a dilution rate of 0.02 h(equivalent to a doubling time of ≈33 hours) at 37°C. The chemostat was fed media containing 4 g/L sodium formate and 0.5 g/L D-xylose as sole carbon sources. This amount of xylose in the feed makes xylose the limiting nutrient for cell growth in the chemostat. On days 47,166, 214, and 343 of the evolution experiment, the level of D-xylose in the feed media was reduced to 0.28, 0.13, 0.05, and 0 g/L, respectively. The concentration of formate was increased to 6 g/L on day 357, after the autotrophic growth phenotype was observed, and chloramphenicol (30 mg/L) and streptomycin (100 mg/L) were added to the feed media. Aeration of the chemostat was done through a DASGIP MX4/4 stand-alone gas-mixing module (Eppendorf, Germany) with a composition of 10% COand 90% air at a flow rate of 40 sL/hr. To monitor the chemostat, a weekly sampling protocol was performed. Samples were taken for media analysis and phenotyping (inoculation of the bacteria on minimal media containing formate and lacking D-xylose). We calculated the biomass dependency metric of each sample as the ratio between the xylose carbon concentration (g carbon/L) in the feed and the carbon concentration in the culture biomass. The biomass carbon concentration was calculated with a conversion factor of 0.2 g carbon per 1 OD). The optical density of each extracted sample was measured using a spectrophotometer (Ultrospec 10 Cell density meter, Amersham Biosciences) and a standard 10 mm polystyrene cuvette (Sarstedt, Germany). For the following time points, only biomass concentration values are reported in the paper and not the metric of biomass dependency, which requires steady chemostat operation: Days 0-4, when the culture was grown in batch mode; days 134-137, when an especially low OD was measured for unknown reasons (possibly due to a malfunction) and the calculated biomass dependency values are thus extremely, and probably artificially, high (11 and 25 xylose carbon/biomass carbon); days 167-195. A technical malfunction on day 167 led most of the culture to be flushed out of the chemostat. The chemostat was switched to batch mode to enable recovery with fresh media. Until day 190, the OD remained low and thus a glycerol stock sample taken on day 166 was used as an extra inoculum. On day 195, the chemostat mode was restored.

13C Isotopic labeling experiment A culture of evolved cells grown on naturally labeled sodium formate in an elevated CO 2 (10%, naturally labeled) incubator (New Brunswick S41i CO 2 incubator shaker, Eppendorf, Germany) were diluted 8-fold into fresh M9 media with either 30 mM 12C or 13C-formate sodium salt (Sigma Aldrich) to a total volume of 10 mL of culture. In the “open” labeling setup, growth was carried out in 125 mL glass shake flasks with breathable sealing sticker-films (AeraSeal, Excel Scientific, USA), which allow free exchange of gases between the headspace of the growth vessel and the gas mixture of the incubator. The flasks were placed inside an elevated CO 2 (10%) shaker-incubator (New Brunswick) with 37°C. After ≈3 doublings, the cells were again diluted 8-fold into fresh media of the same type. This procedure was repeated several times for at least 10 doublings within each of the conditions. Then, the cells were harvested for subsequent analysis of protein-bound amino acids and intracellular metabolites. In the “closed” labeling setup, growth was carried out in 250 mL glass shake flasks with a transparent extension, which allows the measurement of the optical density of the culture without opening it. After ≈3 doublings, the cells were diluted 8-fold into flasks covered with an air-tight rubber septa (SubaSeal, Sigma Aldrich). Then, the headspace of the flask was flushed with a gas mixture containing 10% 13CO 2 (Cambridge Isotope Laboratories, USA) + 90% air or 10% 12CO 2 + 90% air generated by a DASGIP MX4/4 stand-alone gas-mixing module (Eppendorf, Germany). The flasks were then placed in a 37°C shaker incubator. This procedure was repeated several times for at least 10 doublings for each of the conditions. Then, the cells were harvested for subsequent analysis of protein-bound amino acids and intracellular metabolites. The glass flasks used in the labeling experiments were pretreated by heating in a 460°C furnace for 5 hours to evaporate any excess carbon sources that could remain in the vessels from previous utilizations. Number of replicates (growth flasks) in each condition with the evolved isolated clone: (a) 13CO 2 + 13C-formate (n = 3). (b) 13CO 2 + 12C-formate (n = 5). (c) 12CO 2 + 13C-formate (n = 3). (d) 12CO 2 + 12C-formate (n = 1 for this trivial control). Number of replicates (growth flasks) in each condition with a sample taken from the chemostat after day 350: (a) 13CO 2 + 13C-formate (n = 3). (b) 13CO 2 + 12C-formate (n = 2). (c) 12CO 2 + 13C-formate (n = 3). The labeling of WT E. coli BW25113 cells using U13C 6 -glucose was performed with n = 1 of this well established control.

Sample preparation for LCMS analysis Antonovsky et al. (2016) Antonovsky N.

Gleizer S.

Noor E.

Zohar Y.

Herz E.

Barenholz U.

Zelcbuch L.

Amram S.

Wides A.

Tepper N.

et al. Sugar Synthesis from CO2 in Escherichia coli. 600 turbidity of ≈0.1-0.15 were pelleted by centrifugation for 5 minutes at 8,000 g. The pellet was suspended in 1 mL of 6N HCl and incubated for 24 hours at 110°C. The acid was subsequently evaporated with a nitrogen stream, resulting in a dry hydrolysate. Dry hydrolysates were resuspended in 0.6 mL of MilliQ water, centrifuged for 5 minutes at 14,000 g. The supernatant was then injected into the LCMS. Hydrolyzed amino acids were separated using ultra performance liquid chromatography (UPLC, Acquity - Waters, USA) on a C-8 column (Zorbax Eclipse XBD - Agilent, USA) at a flow rate of 0.6 mL/min and eluted off the column using a hydrophobicity gradient. Buffers used: A) H 2 O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid with the following gradient: 100% of A (0-3 min), 100% A to 100% B (3-9 min), 100% B (9-13 min), 100% B to 100% A (13-14 min), 100% A (14-20 min). The UPLC was coupled online to a triple quadrupole mass spectrometer (TQS - Waters, USA). Data was acquired using MassLynx v4.1 (Waters, USA). Amino acids and metabolites used for analysis were selected according to the following criteria: We chose amino acids that have peaks at a distinct retention time and m/z values for all isotopologues and also showed correct 13C labeling fractions in control samples that contained protein hydrolyzates of WT cells grown with known ratios of 13C 6 -glucose to 12C-glucose. After harvesting the biomass, culture samples were prepared and analyzed as described in. Briefly, for protein-bound amino acids, ≈3 mL of culture at ODturbidity of ≈0.1-0.15 were pelleted by centrifugation for 5 minutes at 8,000 g. The pellet was suspended in 1 mL of 6N HCl and incubated for 24 hours at 110°C. The acid was subsequently evaporated with a nitrogen stream, resulting in a dry hydrolysate. Dry hydrolysates were resuspended in 0.6 mL of MilliQ water, centrifuged for 5 minutes at 14,000 g. The supernatant was then injected into the LCMS. Hydrolyzed amino acids were separated using ultra performance liquid chromatography (UPLC, Acquity - Waters, USA) on a C-8 column (Zorbax Eclipse XBD - Agilent, USA) at a flow rate of 0.6 mL/min and eluted off the column using a hydrophobicity gradient. Buffers used: A) HO + 0.1% formic acid and B) acetonitrile + 0.1% formic acid with the following gradient: 100% of A (0-3 min), 100% A to 100% B (3-9 min), 100% B (9-13 min), 100% B to 100% A (13-14 min), 100% A (14-20 min). The UPLC was coupled online to a triple quadrupole mass spectrometer (TQS - Waters, USA). Data was acquired using MassLynx v4.1 (Waters, USA). Amino acids and metabolites used for analysis were selected according to the following criteria: We chose amino acids that have peaks at a distinct retention time and m/z values for all isotopologues and also showed correctC labeling fractions in control samples that contained protein hydrolyzates of WT cells grown with known ratios of-glucose toC-glucose. 600 turbidity of ≈0.1-0.15 were pelleted by centrifugation for 5 minutes at 5,000 g. The pellet was suspended in 4 mL of a cold (−20°C) acetonitrile:methanol:water (40:40:20) extraction solution and incubated overnight at this temperature. The next day, the extracts were centrifuged (5 minutes at 16,000 g), and the supernatant was transferred into fresh tubes. Organic solvents were subsequently evaporated using a speedvac vacuum concentrator. The aqueous phase was evaporated by freeze drying. Dry extracts were stored at −80°C until the mass spectrometry analysis. Prior to injection into the mass spectrometer, the dry extracts were suspended in 200 μL of a 1:1 methanol:water solution, centrifuged (5 minutes at 16,000 g) and then the supernatant was transferred to a vial for injection. Metabolites were separated using liquid chromatography. A ZIC-pHILIC column (4.6 mm × 150 mm, guard column 4.6 mm × 10 mm; Merck) was used for liquid chromatography separation via a gradient elution with a solution of 20 mM ammonium carbonate, with 0.1% ammonium hydroxide, and acetonitrile at 0.1 mL/min. Detection of metabolites was performed using a Thermo Scientific Exactive high-resolution mass spectrometer with electrospray ionization, examining metabolites in a polarity switching mode over the mass range of 75–1,000 m/z. The identities of the compounds were verified by matching masses and retention times to a library of authenticated standards. Data analysis was performed using the Maven software suite ( Clasquin et al., 2012 Clasquin M.F.

Melamud E.

Rabinowitz J.D. LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine. For intracellular metabolites, ≈8 mL of culture at ODturbidity of ≈0.1-0.15 were pelleted by centrifugation for 5 minutes at 5,000 g. The pellet was suspended in 4 mL of a cold (−20°C) acetonitrile:methanol:water (40:40:20) extraction solution and incubated overnight at this temperature. The next day, the extracts were centrifuged (5 minutes at 16,000 g), and the supernatant was transferred into fresh tubes. Organic solvents were subsequently evaporated using a speedvac vacuum concentrator. The aqueous phase was evaporated by freeze drying. Dry extracts were stored at −80°C until the mass spectrometry analysis. Prior to injection into the mass spectrometer, the dry extracts were suspended in 200 μL of a 1:1 methanol:water solution, centrifuged (5 minutes at 16,000 g) and then the supernatant was transferred to a vial for injection. Metabolites were separated using liquid chromatography. A ZIC-pHILIC column (4.6 mm × 150 mm, guard column 4.6 mm × 10 mm; Merck) was used for liquid chromatography separation via a gradient elution with a solution of 20 mM ammonium carbonate, with 0.1% ammonium hydroxide, and acetonitrile at 0.1 mL/min. Detection of metabolites was performed using a Thermo Scientific Exactive high-resolution mass spectrometer with electrospray ionization, examining metabolites in a polarity switching mode over the mass range of 75–1,000 m/z. The identities of the compounds were verified by matching masses and retention times to a library of authenticated standards. Data analysis was performed using the Maven software suite ().

Isotopic analysis composition of biomolecules 13C fraction of each metabolite was determined as the weighted average of the fractions of all the isotopologues of the metabolite, as depicted in the equation below: f 1 3 C = ∑ i = 0 n f i × i n (Equation 3)

where n is the number of carbons in the compound (e.g., for the amino acid serine, n = 3) and f i is the relative fraction of the i-th isotopologue that contains i 13C carbon atoms. The totalC fraction of each metabolite was determined as the weighted average of the fractions of all the isotopologues of the metabolite, as depicted in the equation below:where n is the number of carbons in the compound (e.g., for the amino acid serine, n = 3) and fis the relative fraction of the i-th isotopologue that contains iC carbon atoms.

Calculation of the effective 13C fraction 13C labeling of intracellular inorganic carbon (f 13CO2, effective ) by using the following equation (written for glutamate but can be equivalently used with proline instead): f 13 C O 2 ' e f f e c t i v e = ∑ i = 0 6 f a r g i − ∑ i = 0 5 f g l u i (Equation 4)

where f 13CO2, effective is the relative fraction of 13CO 2 out of the total pool of CO 2 (or more formally the inorganic C pool), and f arg_i and f glu_i are the fraction of the i-th isotopologue of arginine and glutamate respectively. We sum over all isotopologues (equal to the number of carbon atoms in the compound, 6 for arginine and 5 for proline or glutamate). We repeated the calculation using the measured isotopologue fractions of proline instead of those of glutamate. We used the average of those two calculations as a more robust estimator of the effective level of 13CO 2 and the associated uncertainty. We then used the computed labeled fraction of 13CO 2 to normalize the 13C-labeled fractions of all the measured metabolites using the following equation: f 1 3 C m e t j , c o r r e c t e d = f 1 3 C m e t j , m e a s u r e d f 1 3 C O 2 , e f f e c t i v e (equation 5)

where met j stands for the j-th measured metabolite (or protein-bound amino acid). We used the carbamoyl-phosphate moiety as a marker for the isotopic distribution of the intracellular inorganic carbon pool. Carbamoyl-phosphate is generated by carbamoyl phosphate synthetase from bicarbonate as the carbon substrate. Carbamoyl-phosphate is then condensed with ornithine in the L-arginine biosynthesis pathway. We looked at mass isotopologue distribution of L-arginine, which contains an extra carbon from carbamoyl-phosphate (the guanidinium group carbon), versus the mass isotopologue distribution of either L-proline or L-glutamate, which are similar to that of ornithine. We calculated the effectiveC labeling of intracellular inorganic carbon (f) by using the following equation (written for glutamate but can be equivalently used with proline instead):where fis the relative fraction ofCOout of the total pool of CO(or more formally the inorganic C pool), and fand fare the fraction of the i-th isotopologue of arginine and glutamate respectively. We sum over all isotopologues (equal to the number of carbon atoms in the compound, 6 for arginine and 5 for proline or glutamate). We repeated the calculation using the measured isotopologue fractions of proline instead of those of glutamate. We used the average of those two calculations as a more robust estimator of the effective level ofCOand the associated uncertainty. We then used the computed labeled fraction ofCOto normalize theC-labeled fractions of all the measured metabolites using the following equation:where metstands for the j-th measured metabolite (or protein-bound amino acid). Bennett et al., 2008 Bennett B.D.

Yuan J.

Kimball E.H.

Rabinowitz J.D. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. An analogous correction procedure using the labeled fractions aspartate and carbamoyl-aspartate was performed in a recent study () to account for incomplete labeling owing to incorporation of non-labeled inorganic carbon in the media.

Whole-genome sequencing Herz et al. (2017) Herz E.

Antonovsky N.

Bar-On Y.

Davidi D.

Gleizer S.

Prywes N.

Noda-Garcia L.

Frisch K.L.

Zohar Y.

Wernick D.G.

et al. The genetic basis for the adaptation of E. coli to sugar synthesis from CO2. Antonovsky et al. (2016) Antonovsky N.

Gleizer S.

Noor E.

Zohar Y.

Herz E.

Barenholz U.

Zelcbuch L.

Amram S.

Wides A.

Tepper N.

et al. Sugar Synthesis from CO2 in Escherichia coli. Herz et al. (2017) Herz E.

Antonovsky N.

Bar-On Y.

Davidi D.

Gleizer S.

Prywes N.

Noda-Garcia L.

Frisch K.L.

Zohar Y.

Wernick D.G.

et al. The genetic basis for the adaptation of E. coli to sugar synthesis from CO2. Barrick et al., 2014 Barrick J.E.

Colburn G.

Deatherage D.E.

Traverse C.C.

Strand M.D.

Borges J.J.

Knoester D.B.

Reba A.

Meyer A.G. Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq. DNA extraction (DNeasy blood & tissue kit, QIAGEN) and library preparation procedures were carried as previously described in. Tagging and fragmenting (‘tagmentation’) using the Nextera kit (Illumina kits FC-121–1031) was performed by mixing 1 μL containing 1.5 ng of genomic DNA, 1.25 μL of TD buffer, and 0.25 μL of TDE1. The mixture was mixed gently by pipetting and placed for incubation in a thermocycler for 8 min at 55°C. Next, “tagmented” gDNA underwent PCR-mediated adaptor addition and library amplification by mixing 11 μL of PCR master mix (KAPA KK2611/KK2612), 4.5 μL of 5 μM index1 (Nextera index kit FC-121-1011), 4.5 μL of 5 μM index2, and 2.5 μL of tagmented DNA in each well. The final total volume per well was 22.5 μl. The thermocycler was run with the following program: 1) 72°C for 3 min, 2) 98°C for 5 min, 3) 98°C for 10 s, 4) 63°C for 30 s, and 5) 72°C for 30 s. 6) Repeat steps (3)–(5) 13 times for a total of 13 cycles. 7) 72°C for 5 min. 8) Hold at 4°C. PCR cleanup and size selection were done in several steps: mixing 12 μL of magnetic beads SPRIselect reagent (Beckman Coulter B23317) with 15 μL of each PCR reaction. Incubation at room temperature for 5 min followed by 1 min on a magnetic stand. The clear solution was discarded, and the beads were mixed with 200 μL of freshly made 80% ethanol. An ethanol wash was performed twice, and the plate was then incubated at room temperature for 5 min to allow for the evaporation of residual ethanol. The sample was eluted with 30 μL of ultrapure water for 5 min at room temperature, and the beads were removed using the magnetic stand.The prepared libraries were sequenced by a Miseq machine (Illumina). Analysis of the sequencing data was performed as previously described inandusing the breseq software () with genomic and plasmid DNA sequences as references for alignments of sequencing reads. To exclude the possibility of contamination in the different experiments, we extracted the DNA from bacterial pellets taken at the end of the experiments, sequenced them as described and validated that following alignment of the sequencing reads to the reference genome and plasmid sequences; at least 95% of the reads were aligned.

Analysis of media composition Media samples collected during the evolution experiment and batch-growth experiments were first filtered through a 0.22 micron PVDF Millex-GV syringe filter unit (Merck Millipore), and stored at −80°C. After thawing, the media samples they were analyzed with an Agilent 1200 high-performance liquid chromatography system (Agilent technologies, USA) equipped with a refractive index detector and an anion exchange Bio-Rad HPX-87H column (Bio-Rad, USA). The column was eluted with 5 mM sulfuric acid at a flow rate of 0.6 mL/min at 45°C. Samples with a formate concentration below the detection limit of the HPLC were analyzed by an enzymatic assay kit (Megazyme, Ireland). Media samples from the evolution experiments were each measured once. Media samples from the batch growth experiments were measured 3 times, with the mean ± SD is shown in Figure 2 C. The samples analyzed with the enzymatic kit were measured twice; the mean ± SD is reported.

Measurement of FDH activity in cell extracts Berríos-Rivera et al. (2002) Berríos-Rivera S.J.

Bennett G.N.

San K.-Y. Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase. 600 of 1, they were harvested by centrifugation (15 minutes; 4000 g; 25°C) and pellets were lysed with 0.5mL BugBuster ® ready mix (Merck Millipore) for 25 minutes at room temperature. Crude extracts were then centrifuged for 30 min at 4000 g, 4°C to remove the insoluble fraction. Total protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s multi-well plate protocol. The enzymatic activity assay was performed in the presence of 200mM PBS PH-7.0, 10mM B-mercaptoethanol, 100mM sodium formate and 2mM NAD+ (ACROS organics). 190 μL of assay mix were added to 96-well plate, three wells (repeats) for each sample. The assay mix was pre-incubated at 37°C inside the plate reader for 15 min. All protein lysates were diluted to 0.1 mg/mL. 10 μL of lysate from each sample were injected into three replicate wells. The increase in NADH concentration resulting from formate oxidation was monitored at 340 nm (Infinite 200 Pro (Tecan, Switzerland)) over time. We averaged the slopes between replicates and compared the slope averages between bacterial lysates that originally contained the mutated plasmid to those with unmutated plasmids and observed no significant difference between them. The experiment included lysate from wild-type E.coli as a negative control and pure formate dehydrogenase (C. boidinii, Sigma-Aldrich) protein with the same overall concentration as a positive control. The assay was adapted fromwith several modifications listed below. In brief, both the original pFDH and the mutated one (Δ8 bp position 918) were transformed into TSS competent E. coli BW25113 cells by heat shock and selected on LB agar plates with streptomycin. Five colonies of each type were picked, verified by PCR and grown overnight at 37°C in 2 mL of M9 minimal medium supplemented with 50 μg/mL Streptomycin, 0.4% glucose and 30mM formate. When cells reached an ODof 1, they were harvested by centrifugation (15 minutes; 4000 g; 25°C) and pellets were lysed with 0.5mL BugBuster ® ready mix (Merck Millipore) for 25 minutes at room temperature. Crude extracts were then centrifuged for 30 min at 4000 g, 4°C to remove the insoluble fraction. Total protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s multi-well plate protocol. The enzymatic activity assay was performed in the presence of 200mM PBS PH-7.0, 10mM B-mercaptoethanol, 100mM sodium formate and 2mM NAD(ACROS organics). 190 μL of assay mix were added to 96-well plate, three wells (repeats) for each sample. The assay mix was pre-incubated at 37°C inside the plate reader for 15 min. All protein lysates were diluted to 0.1 mg/mL. 10 μL of lysate from each sample were injected into three replicate wells. The increase in NADH concentration resulting from formate oxidation was monitored at 340 nm (Infinite 200 Pro (Tecan, Switzerland)) over time. We averaged the slopes between replicates and compared the slope averages between bacterial lysates that originally contained the mutated plasmid to those with unmutated plasmids and observed no significant difference between them. The experiment included lysate from wild-type E.coli as a negative control and pure formate dehydrogenase (C. boidinii, Sigma-Aldrich) protein with the same overall concentration as a positive control.