metabolic engineering of escherichia coli for direct ... · 1,4-butanediol production in e. coli...

1,4-Butanediol Production in E. coli Yim, H. et al.

Supplementary Information

Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol Harry Yim1,*, Robert Haselbeck1,*, Wei Niu1,*, Catherine Pujol-Baxley1,*, Anthony Burgard1,*, Jeff Boldt1, Julia Khandurina1, John D. Trawick1, Robin E. Osterhout1, Rosary Stephen1, Jazell Estadilla1, Sy Teisan1, H. Brett Schreyer1, Stefan Andrae1, Tae Hoon Yang1, Sang Yup Lee2, Mark J. Burk1, and Stephen Van Dien1,** 1 Genomatica, Inc., 10520 Wateridge Circle, San Diego, CA 92121 2 Department of Chemical and Biomolecular Engineering (BK21 program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea


Nature Chemical Biology: doi:10.1038/nchembio.580



Supplementary Materials and Methods Genetic Manipulations Chromosomal replacement of genes using the sacB gene. The primary method used for the insertion or deletion of genes in the chromosome of E. coli was based on the utilization of the sacB gene from Bacillus subtilis 1. The vector used is pRE118 (ATCC87693) deleted of the oriT and IS sequences. The resulting vector (3.6 kb in size and carrying the kanamycin resistance gene) was sequenced and called pRE118-V2. All clonings of fragments were done into the restriction sites KpnI and PstI of pRE118-V2, unless notified. All PCR amplification used genomic DNA from E. coli MG1655 (ATCC47076) as DNA template. The first integration event in the chromosome was selected on LB agar plates containing Kanamycin (25 or 50 mg/L). Correct insertions were verified by PCR using 2 primers: one located outside the region of insertion and one in the kanamycin gene (5’-aggcagttccataggatggc-3’). Clones with the correct insertion were selected for resolution. They were sub-cultured twice in plain liquid LB at the desired temperature and serial dilutions were plated on LB-no salt-sucrose 10% plates. Clones that grew on sucrose containing plates were screened for the loss of the kanamycin resistance gene on LB-low salt agar medium and the deletion/insertion of the fragment of interest was verified by PCR and sequencing of the encompassing region. Construction of deletions of ldhA, adhE and pflB. Native genes ldhA and adhE was deleted from the chromosome by Red-mediated recombination (http://www.ncbi.nlm.nih.gov/pubmed/10829079). For ldhA, the primers S-ldhA-CAT 5’ (5’GCCTACACTAAGCATAGTTGTTGATGAATTTTTCAATATCGCCATAGCTTatgggaattagccatggtcc-3’) and AS-ldhA-CAT (5’-ATTGGGGATTATCTGAATCAGCTCCCCTGGAATGCAGGGGAGCGGCAAGAgtgtaggctggagctgcttc-3’) were used to amplify chloramphenicol resistance flanked by FRT sites from pKD3 (http://www.ncbi.nlm.nih.gov/pubmed/10829079). For adhE, the primers S-AdhE-CAT (5’-ATTCGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGAGCATTatgggaattagccatggtcc-3’) and AS-AdhE-CAT (5’-GCCCAGAAGGGGCCGTTTATGTTGCCAGACAGCGCTACTGCTTAAGCGGAgtgtaggctggagctgcttc-3’) were used and for pflB, the primers S-pflB-Kan (5’-tgtcgaagtacgcagtaaataaaaaatccacttaagaaggtaggtgttacgtgtaggctggagctgcttc-3’) and AS-pflB-Kan (5’-ATAGATTGAGTGAAGGTACGAGTAATAACGTCCTGCTGCTGTTCTcatatgaatatcctccttag-3’). The primers were used to amplify a chloramphenicol or kanamycin resistance gene from pKD3 or pKD4, respectively and were flanked by FRT sites and homologies to ldhA, adhE and pflB. E. coli K-12 MG1655 carrying the Red helper plasmid pKD46 was grown in 100 ml LB medium with ampicillin and 1 mM L-arabinose at 30°C to an OD600 of 0.3, and electroporation-competent cells were prepared as described elsewhere2. A measure of 50 l of competent cells was mixed with 400 ng of the PCR fragment in an ice-cold 0.1 cm cuvette (Bio-Rad Inc., Hercules, CA, USA). Cells were electroporated at 1.8 kV with 25 mF and 200 , immediately followed by the addition of 1 ml of SOC medium (2% Bacto Tryptone (Difco), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) with 1 mM L-arabinose. After incubation for 2 h at 37°C, one-tenth portion was spread onto agar plate to select chloramphenicolR or kanamycinR transformants at 37°C. The individual deletions were then transduced into new host strains using P1vir and selecting for the appropriate antibiotic 3. The chloramphenicol or kanamycin resistant colonies were then transformed with the temperature sensitive plasmid pCP20, and plated on ampicillin (100 ug/ml) plates at 30°C. pCP20 contains the carries the yeast Flp recombinase


http://www.ncbi.nlm.nih.gov/pubmed/10829079

http://www.ncbi.nlm.nih.gov/pubmed/10829079



gene, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Colonies were grown in LB medium without antibiotics at 42°C overnight and streak plated on prewarmed LB Agar plates and grown at 42°C. Individual clones were tested for sensitivity to ampicillin and chloramphenicol or kanamycin to isolate clones which had lost the pCP20 plasmid and had the chloramphenicol or kanamycin resistance gene lost via the Flp recombinase. Positive clones with deletions in adhE, ldhA and pflB contain a single FRT “scar” and were verified by PCR using primers which flanked their respective genes. ECKh-138: Replacement of the native E. coli lpdA with the K. pneumonia lpdA. The lpdA gene of K. pneumoniae was amplified by PCR using genomic DNA (ATCC700721D) as template and the following primers KP-lpdA-Bam (5’-acacgcggatccaacgtcccgg-3’) and KP-lpdA-Nhe (5’-agcggctccgctagccgcttatg-3’). The resulting fragment was cloned into the vector pCR-BluntII-TOPO (Invitrogen), leading to plasmid pCR-KP-lpdA. The E. coli fragments flanking the lpdA gene were amplified by PCR using the combination of primers: EC-aceF-Pst (5’-aagccgttgctgcagctcttgagc-3’) + Ec-aceF-Bam2 (5’-atctccggcggtcggatccgtcg-3’) and EC-yacH-Nhe (5’-aaagcggctagccacgccgc-3’) + EC-yacH-Kpn (5’-attacacgaggtacccaacg-3’). A BamHI-XbaI fragment containing the lpdA gene of K. pneumonia was isolated from plasmid pCR-KP-lpdA and was then ligated to the above E. coli fragments digested with PstI +BamHI and NheI-KpnI respectively, and the pRE118-V2 plasmid digested with KpnI and PstI. The resulting plasmid (called pRE118-M2.1 lpdA yac) was subjected to Site Directed Mutagenesis (SDM) using the combination of primers KP-lpdA-GluLys-F (5’-atcgcctacactaaaccagaagtgg-3’) and KP-lpdA-GluLys-R (5’-ccacttctggtttagtgtaggcgat-3’) for the mutation of the residue Glu 354 to Lys residue. PCR was performed with the Polymerase Pfu Turbo (Stratagene, Agilent Technologies, Santa Clara, CA, USA). The sequence of the entire fragment as well as the presence of only the desired mutations was verified. The construct was introduced into electro

competent cells of E. coli adhE::Frt- ldhA::Frt by transformation. Selection of the recombinant clones were done as described in the above section. Sequence of the insertion region as well as the presence of mutation was verified . One clone (named 4-4-a1) with mutation D354K was selected. This clone was

then transduced with P1 lysate of E. coli pflB::Frt 3 leading to strain ECKh-138. Sequence of the lpdA gene was verified in strain ECKh-138. ECKh-172 (Dmdh): mdh deletion. Deletion of mdh from the chromosome of ECKh-138 was performed using Red-mediated recombination4. The primers S-mdh-Kan 5’ (5’-TATTGTGCATACAGATGAATTTTTATGCAAACAGTCAGCCCTGAAGAAGGgtgtaggctggagctgcttc-3’) and AS-mdh-Kan (5’-ATTGGGGATTATCTGAATCAGCTCCCCTGGAATGCAGGGGAGCGGCAAGAcatatgaatatcctccttag-3’) were used to amplify kanamycin resistance from pKD4 and were flanked by FRT sites and ends with homologies to mdh. Integration of the PCR product and resolution of the FRT-Kan-FRT cassette was performed as above. ECKh-401: arcA deletion in ECKh-172. The upstream and downstream regions of the arcA gene of E. coli MG1655 were amplified by PCR using primers ArcA-up-EcoRI (5’- ataataatagaattcgtttgctacctaaattgccaactaaatcgaaacagg -3’) with ArcA-up-KpnI (5’-tattattatggtaccaatatcatgcagcaaacggtgcaacattgccg -3’) and ArcA-down-EcoRI (5’-tgatctggaagaattcatcggctttaccaccgtcaaaaaaaacggcg -3’) with ArcA-down-PstI (5’-ataaaaccctgcagcggaaacgaagttttatccatttttggttacctg -3’) respectively. These fragments were subsequently digested with the restriction enzymes EcoRI and KpnI (upstream fragment) and EcoRI and PstI (downstream). They were then ligated into the pRE118-V2 plasmid digested with PstI and KpnI, leading

to plasmid pRE118- arcA. The sequence of plasmid pRE118- arcA was verified. pRE118- arcA was introduced into electro-competent cells of E. coli strain ECKh-172. After integration and resolution on




LB-no salt-sucrose plates as described above, the deletion of the arcA gene in the chromosome of the resulting strain ECKh-401 was verified by sequencing. ECKh-422: mutation of the citrate synthase. The gltA gene of E. coli MG1655 was amplified by PCR using primers gltA-up (5’- ggaagagaggctggtacccagaagccacagcagga-3’) and gltA-PstI (5’-gtaatcactgcgtaagcgccatgccccggcgttaattc -3’). The amplified fragment was cloned into pRE118-V2 after digestion with KpnI and PstI. The resulting plasmid was called pRE118-gltA. This plasmid was then subjected to SDM using primers R163L-f (5’- attgccgcgttcctcctgctgtcga-3’) and R163L-r (5’-cgacagcaggaggaacgcggcaat -3’) to change the residue R163 to a Lys residue. The sequence of the entire fragment was verified by sequencing. An rpsL-neo cassette was then introduced into the gltA locus of ECKh-401 using the Counter-selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany), leading to a strain that is streptomycin sensitive and neomycin resistant. The mutated gltA gene was then introduced into electro-competent cells of ECKh-401::rpsL-neo, selecting for streptomycin resistance and neomycin sensitivity, leading to strain ECKh-422. The region encompassing the mutated gltA gene of strain ECKh-422 was verified by sequencing. Insertion of non-PTS sucrose operon into host chromosome. To generate a PCR product containing the non-PTS sucrose genes flanked by regions of homology to the rrnC region, two oligos were used to PCR amplify the csc genes from Mach1™ (Invitrogen). This strain is a descendent of W strain which is an E. coli strain known to be able to catabolize sucrose 5. The sequence was derived from E. coli W strain KO11 (accession AY314757)6 and includes genes encoding a sucrose permease (cscB), D-fructokinase (cscK), sucrose hydrolase (cscA), and a LacI-related sucrose-specific repressor (cscR). The first 53 amino acids of cscR was effectively removed by the placement of the AS primer. The sequences of the oligos were: rrnC 23S del S –CSC 5’-TGT GAG TGA AAG TCA CCT GCC TTA ATA TCT CAA AAC TCA TCT TCG GGT GAC GAA ATA TGG CGT GAC TCG ATA C-3' and rrnC 23S del AS –CSC 5’-TCT GTA TCA GGC TGA AAA TCT TCT CTC ATC CGC CAA AAC AGC TTC GGC GTT AAG ATG CGC GCT CAA GGA C-3’. Underlined regions indicate homology to the csc operon and bold sequence refers to sequence homology upstream and downstream of the rrnC region. After purification, the PCR product was electroporated into MG1655 electrocompetent cells which had been transformed with pRedET (tet) and prepared according to manufacturer’s instructions (http://www.genebridges.com/gb/pdf/K001%20Q%20E%20BAC%20Modification%20Kit-version2.6-2007-screen.pdf). The PCR product was designed so that it integrated into genome into the rrnC region of the chromosome. It effectively deleted 191 nt upstream of rrlC (23S rRNA), all of the rrlC rRNA gene and 3 nt downstream of rrlC and replaced it with the sucrose operon. Transformants were grown on M9 minimal salts medium with 0.4% sucrose and individual colonies tested for presence of the sucrose operon by diagnostic PCR. The entire rrnC::crcAKB region was transferred into the BDO host strain ECKh-422 by P1 transduction, resulting in ECKh-436. Replacement of the Stuffer Fragment in the pZ-based Expression Vectors. Vector backbones were obtained from Dr. Rolf Lutz of Expressys (http://www.expressys.de/). The vectors and strains are based on the pZ Expression System developed by Dr. Rolf Lutz and Prof. Hermann Bujard7. Vectors obtained were pZE13luc, pZA33luc, pZS*13luc and pZE22luc and contained the luciferase gene as a stuffer fragment. To replace the luciferase stuffer fragment with a lacZ-alpha fragment flanked by appropriate restriction enzyme sites, the luciferase stuffer fragment was first removed from each vector by digestion with EcoRI and XbaI. The lacZ-alpha fragment was PCR amplified from pUC19 with the following primers:


http://www.genebridges.com/gb/pdf/K001%20Q%20E%20BAC%20Modification%20Kit-version2.6-2007-screen.pdf

http://www.genebridges.com/gb/pdf/K001%20Q%20E%20BAC%20Modification%20Kit-version2.6-2007-screen.pdf

http://www.expressys.de/



lacZalpha-RI 5’GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGGCCGTCGTTTTAC3’ lacZalpha 3'BB 5’-GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAGA-3’. This generated a fragment with a 5’ end of EcoRI site, NheI site, a Ribosomal Binding Site, a SalI site and the start codon. On the 3’ end of the fragment contained the stop codon, XbaI, HindIII, and AvrII sites. The PCR product was digested with EcoRI and AvrII and ligated into the base vectors digested with EcoRI and XbaI (XbaI and AvrII have compatible ends and generate a non-site). Because NheI and XbaI restriction enzyme sites generate compatible ends that can be ligated together (but generate a site after ligation that is not digested by either enzyme), the genes cloned into the vectors could be “Biobricked” together (http://openwetware.org/wiki/Synthetic_Biology:BioBricks). Briefly, this method enables joining an unlimited number of genes into the vector using the same 2 restriction sites (as long as the sites do not appear internal to the genes), because the sites between the genes are destroyed after each addition. Initially, expression was low from these vectors, and they were subsequently modified using the Phusion® Site-Directed Mutagenesis Kit (NEB, Ipswich, MA, USA) to insert the spacer sequence AATTAA between the EcoRI and NheI sites. This eliminated a putative stem loop structure in the RNA that bound the RBS and start codon. All vectors have the pZ designation followed by letters and numbers indicating the origin of replication, antibiotic resistance marker and promoter/regulatory unit. The origin of replication is the second letter and is denoted by E for ColE1, A for p15A and S for pSC101 (as well as a lower copy number version of pSC101 designated S*) – based origins. The first number represents the antibiotic resistance marker (1 for Ampicillin, 2 for Kanamycin, 3 for Chloramphenicol). The final number defines the promoter that regulated the gene of interest (1 for PLtetO-1, 2 for PLlacO-1 and 3 for PA1lacO-1). For the work discussed here we employed three base vectors, pZS*13S, pZA33S and pZE13S, modified for the biobricks insertions as discussed above.

Analytical Methods

Two analytical platforms, LCMS/MS and GCMS, were employed for analysis of cell culture and fermentation samples. Good correlation between the two techniques (variation within 10-15%) provided method cross-validation and ensured data accuracy. LCMS/MS analysis was performed on API3200 triple quadrupole system (AB Sciex, Life Technologies, Carlsbad, CA), interfaced with Agilent 1200 HPLC, utilizing electrospray ionization and MRM based acquisition methods. BDO, 4HB, GBL and internal standard (1,5-pentanediol or isoleucine) were monitored in positive ionization mode. Negative ionization was used, when desired, for quantitation of acidic analytes, e.g. pyruvate, lactate, succinate and 4HB. Chromatographic separation was conducted on Zorbax Eclipse XDB C18 4.6x30mm, 1.8um particle size, maintaining column at 40⁰C, flow rate 0.6 mL/min. Injection volume was 3-5 uL. Eluents consisted of water with 0.1% formic acid and methanol with 0.1% formic acid, and fast 0.5 min 5-95% methanol gradient was used, resulting in 3 min long LCMS method. Filtered samples were diluted in water, containing internal standards, dilution factor varied from 200 to 10,000 depending on concentrations of metabolites of interest. Dilution in water of 100-fold of greater resulted in no ion suppression effects observed for media composition and conditions used in


http://openwetware.org/wiki/Synthetic_Biology:BioBricks



this study. Quantitation dynamic ranges varied from 0.05-50uM for BDO and 4HB to 0.5-200uM for GBL quantitation. Quantitation was performed using Analyst software. LCMS/MS approach was a preferred method for monitoring BDO, 4HB and GBL due to its simplicity and fast turnaround time enabling high throughput operation. In GCMS approach, BDO, 4HB, lactate and succinate in culture broth samples were derivatized by trimethylsilylation and quantitatively analyzed by GCMS, using a procedure adapted from the literature with some modifications 8. The developed method demonstrated sensitivity down to low μM level, linearity in 0.05-15mM range, as well as good reproducibility. 100μL filtered samples were dried down in a speedvac concentrator for approximately 1 hour at ambient temperature, followed by the addition of 20μL 10mM cyclohexanol solution, as an internal standard, in dimethylformamide. 100μL N,O-bis(trimethylsilyl)triflouro-acetimide (BSTFA) with 1% trimethylchlorosilane, was added, and the mixture was incubated at 70⁰C for 30 min. The derivatized samples were centrifuged for 5 min and directly injected into GCMS (Agilent 6890N/5973N). An Rtx-5SIL MS capillary column (Restek, 30m x 0.25mm ID x 0.25 μm film thickness) was used. The GC was operated in a split injection mode introducing 1μL of sample at 20:1 split ratio. The injection port temperature was 250⁰C. Helium was used as a carrier gas, and the flow rate was maintained at 1.0 mL/min. A temperature gradient program was optimized to ensure good resolution of the analytes of interest and minimum matrix interference. The oven was initially held at 80⁰C for 1.5min, then ramped to 140⁰C at 10⁰C/min and 3min hold, followed by fast ramping to 300⁰C at 100⁰C/min and final hold for 5min. The MS interface transfer line was maintained at 280⁰C. The data were acquired using ‘lowmass’ MS tune settings and 30-400 m/z mass-range scan. The total analysis time was 17 min including 4min solvent delay. Standard calibration curves were constructed using analyte solutions in the corresponding cell culture or fermentation medium to match sample matrix as close as possible. GBL and ethanol were assayed by GCMS as well, following liquid-liquid extraction from fermentation broth into chloroform (GBL) or benzyl alcohol (ethanol) using an HP-InnoWax capillary column (30m, 0.25mm ID, 0.25um film thickness). For higher ethanol content, above 10mM, a simple dilution in methanol was utilized prior to GC sample introduction. GCMS data were processed using ChemStation software (Agilent). Organic acids, acetate, pyruvate, lactate and succinate, were measured by HPLC (Agilent 1100) equipped with diode array detector, using an ion exclusion HPX-087H column (Bio-Rad) with 5mM sulfuric acid as the mobile phase at 0.6mL/min flow rate, 45⁰C column temperature, and UV absorption at 210nm 9. All chemicals and reagents were from Sigma-Aldrich (St. Louis, MO, USA), with the exception of BDO which was purchased from Mallinckrodt Baker (Phillipsburg, NJ). Residual glucose in the fermentation samples was measured using an Analox analyzer (Analox Instruments, London, UK). Enzyme Assays Cells were harvested by centrifugation at 4,500 rpm (Beckman-Coulter, Allegera X-15R; Fullerton, CA, USA) for 10 min. The pellets were resuspended in 0.3 mL BugBuster (Novagen/EMD; San Diego, CA, USA) reagent with benzonase and lysozyme, and lysis proceeded for 15 minutes at room temperature with gentle shaking. Cell-free lysate was obtained by centrifugation at 14,000 rpm (Eppendorf centrifuge 5402; Hamburg, Germany) for 30 min at 4oC. Protein concentrations of cellular extracts were determined as described by Bradford 10.




Succinyl-CoA synthetase (SucCD): The enzyme activity was determined by following the formation of succinyl-CoA from succinate and CoA in the presence of ATP. The experiment followed a procedure described by Cha 11. Succinate semialdehyde dehydrogenase (CoA-dependent) (SucD): The enzyme activity was determined by following the NAD(P)H formation at 340 nm in the presence of succinate semialdehyde and CoA following a published procedure 12. 4-hydroxybutyrate dehydrogenase (4Hbd): The enzyme activity was determined by monitoring the oxidation of NADH at 340 nm in the presence of succinate semialdehyde, following a published procedure13. 4-hydroxybutyrate CoA transferase (Cat2): The enzyme activity was determined by monitoring the 4HB-CoA/butyryl-CoA formation from acetyl-CoA and 4HB/butyrate using HPLC method, following a modified procedure from Scherf 14.

-ketoglutarate decarboxylase (SucA): The SucA enzyme activity was measured by following a

previously reported method 15, monitoring the reduction of ferricyanide at 430 nm in the presence of -ketoglutarate. Aldehyde and Alcohol dehydrogenase: Reduction of acyl-coA or aldehyde derivatives by AdhE2 enzyme was conducted in one milliliter volumes containing 50 mM potassium-MOPS pH = 6.5, 4 mM NADH, 0.1

mM acyl-CoA or 1-10 mM aldehyde, and 1-20 l active extract. Reactions were started by the addition of enzyme, and change in absorbance at 340 nM was monitored kinetically over a four minute period to determine rates of reactions. Reduction of acyl-CoA derivatives by C. beijerinckii Ald enzyme was conducted as previously described 16. Citrate Synthase: Citrate synthase activity was determined by following the formation of free coenzyme A (HS-CoA), which is released from the reaction of acetyl-CoA with oxaloacetate 17. The free thiol group of HS-CoA reacts with 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) to form 5-thio-2-nitrobenzoic acid (TNB). The assay mixture contained 100 mM Tris/HCl buffer (pH 7.5), 20 mM acetyl-CoA, 10 mM DTNB, and 20 mM oxaloacetate. The enzymatic reaction was initiated by the addition of oxaloacetate and the progress of the reaction (appearance of TNB) followed in a plate reader (Biotek Synergy 2, BioTek, Winooski, VT, USA) at a wavelength of 410 nM for 4 minutes. To determine inhibition by NADH, 2 mM NADH was added to the standard reaction and the reaction kinetic compared to the reaction without NADH. Clones that showed less inhibition than the citrate synthase wild type were further evaluated. A unit of specific activity is defined as the µmol of product converted per minute per mg protein.

13C-flux analysis Flux distribution of ECKh-422 containing the BDO pathway was determined using

13C-labeled glucose.

Four different conditions were used, each with biological replicates: 1) 79% unlabeled glucose, 21%

uniform-13

C glucose; 2) 9% unlabeled glucose, 91% uniform-13

C glucose; 3) 89% 1-13

C glucose, 11%

uniform-13

C glucose; 4) 40% 1-13

C glucose, 60% uniform-13

C glucose. Cultures were harvested at 24

hours, and proteinogenic amino acids analyzed for isotopomer composition by GC-MS 18

. Two unlabeled

cultures were grown in parallel, and samples taken every two hours to determine glucose uptake rates,

product secretion rates, and growth curve. The rate and isotopomer data was used to calculate intracellular

flux distribution and associated error bars as described previously 19

.




Supplementary Results Tables

Supplementary Table 1. Enzyme activities of SucD and 4HBd

strain specific activities (U/mg)

host pZE13S (high copy) pZA33S (medium copy) SucD 4HBd

1 MG1655 lacIQ sucD (Pg) 2.32

2 MG1655 lacIQ sucD (Ck) 0.05

3 MG1655 lacIQ 4hbd (Re) 0.25

4 MG1655 lacIQ 4hbd (Pg) 3.31

5 MG1655 lacIQ 4hbd (Ck) 2.57

a One unit (U ) of SucD activity equals 1 mol of NAD(P)H oxidized per min at 23 C. One unit (U) of 4HBd

actitivy equals 1 mol of NADH oxidized per min at 23 C. b Abbreviation, Pg, Porphyromonas gingivalis; Ck, Clostridium kluyveri, Re, Ralstonia eutropha.

Supplementary Table 2. Enzyme activities (U/mg) of 4HB-producing strains at 24 h

host plasmid (copy number) SucCD SucD 4HBd

1 MG1655 lacIQ pZS*13S-sucCD-sucD-4hbd (high) 2.94 1.45 12.07

2 MG1655 lacIQ pZA33S-sucCD-sucD-4hbd (medium) 7.07 4.09 32.06

3 MG1655 lacIQ pZE13S-sucCD-sucD-4hbd (low) 8.44 3.27 24.46

a One unit (U) of SucCD activity equals 1 mol of succinyl-CoA formed per min at 23 C. One unit (U ) of SucD

activity equals 1 mol of NAD(P)H oxidized per min at 23 C. One unit (U) of 4HBd actitivy equals 1 mol of

NADH oxidized per min at 23 C.




Supplementary Table 3. Aldehyde and alcohol dehydrogenase activities (U/mg) of AdhE-like genes

on 2-carbon and 4-carbon substrates.a

Aldehyde dehydrogenase Alcohol dehydrogenase

Gene source and ID number Butyryl-CoA Acetyl-CoA Butyraldehyde Acetaldehyde

C. acetobutylicum AdhE (002) 20 0.0076 0.0046 0.0264 0.0247

C. acetobutylicum AdhE2 (003) 21 0.0060 0.0072 0.0080 0.0075

E. coli AdhE (011) 0.0069 0.0095 0.0265 0.0093

C. acetobutylicum BdhB (013) 22 N.D.b N.D.b 0.0130 0.0142

AdhE from metagenomic library

(023) 23 0.0089 0.0137 0.0178 0.0235

C. beijerinckii ald (025) 24 0 0.0001 N.D.c N.D.c

C. acetobutylicum bdhA (026) 22 0 0.0005 0.0024 0.0008

a One unit of activity equals 1 mol of NADH oxidized per min at 23 C. Cells were harvested 4 hours after

induction with 0.25 mM IPTG and assayed for ALD and ADH activity b Not determined: enzyme is alcohol dehydrogenase only

c Not determined: enzyme is aldehyde dehydrogenase only

Supplementary Table 4. Composition of biomass hydrolysate used for BDO production

Compound Concentration, g/L

Glucose 75.51

Xylose 33.12

Glycerol 16.05

Acetic acid 5.55

5-hydroxymethylfurfural 0.28

Furfural 0.40

Glucolygomers 21.22

Xylolygomers 16.42

Acetyls (total) 0.83




Figures

Supplementary Figure 1. Shortlisted biochemical pathways leading from alpha-ketoglutarate and

succinyl-CoA to 1,4-butanediol that pass through a 4-hydroxybutyrate intermediate.

Supplementary Figure 2. Shortlisted biochemical pathways leading from alpha-ketoglutarate to

1,4-butanediol that pass through a 5-hydroxy-2-oxopentanoic acid intermediate.

1.2.1-

Succinyl-CoA

1.1.1.-

1.1.1.- 2.7.2.- 2.3.1.- 1.2.1.- 1.1.1.-

Succinic semialdehyde 4-hydroxybutyrate 4-hydroxybutyryl-phosphate 4-hydroxybutyryl-CoA 4-hydroxybutanal 1,4-butanediol

1.1.1.-

1.2.1.-

Alpha-ketoglutarate

Glutamate 4-aminobutyrate

4.1.1.-

1.4.1.-2.6.1.-

1.4.1.-2.6.1.-

4.1.1.-

2.8.3.-6.2.1.-

Alpha-ketoglutarateAlpha-ketoglutaryl-phosphate

2.7.2.-

2,5-dioxopentanoic acid

1.1.1.-

5-hydroxy-2-oxopentanoic acid

1.1.1.-

4-Hydroxybutanal 1,4-butanediol

4.1.1.-1.2.1.-

Alpha-ketoglutaryl-CoA

1.1.1.-

1.2.1.-

FIGURE 2

1.2.1.-

4-hydroxybutyryl-CoA

2.3.1.-

2.8.3.-6.2.1.-




Supplementary Figure 3. Shortlisted biochemical pathways leading from glutamate to 1,4-

butanediol that pass through a 5-hydroxy-2-oxopentanoic acid intermediate.

Supplementary Figure 4. Shortlisted biochemical pathways leading from acetyl-CoA to 1,4-

butanediol that pass through a 4-hydroxybutyryl-CoA intermediate.

Supplementary Figure 5. Biosynthesis of 4HB from glucose via succinate route by E. coli strains

grown anaerobically for 24 h. Host strain, MG1655lacIQ; construct 1, plasmid pZS*13S-sucCD-sucD-

4hbd; construct 2, plasmid pZA33S-sucCD-sucD-4hbd; construct 3, plasmid pZE13S-sucCD-sucD-4hbd;

construct 4, no plasmid. Blue bars, extracellular 4HB concentration; black bars, extracellular succinate

concentration; circles, culture OD.

Glutamate2-amino-5-hydroxypentanoic acidGlutamyl-CoA 5-hydroxy-2-oxopentanoic acidGlutamate-5-semialdehyde

1.1.1.-

Glutamate-5-phosphate

1.1.1.-


4.1.1.-

FIGURE 2

1.2.1.-


2.7.2.-

2.8.3.-6.2.1.- 1.2.1.-

1.2.1.-

1.1.1.-1.4.1.-2.6.1.-

2.3.1.-

Acetoacetyl-CoA3-hydroxybutyryl-CoA

Vinylacetyl-CoACrotonoyl-CoA

1.1.1.- 4.2.1.- 5.3.3.-


1.2.1.- 1.1.1.-


1.1.1.-

4.2.1.-

0.00

0.60

1.20

1.80

2.40

3.00

0.0

3.0

6.0

9.0

12.0

15.0

1 2 3 4

OD

60

0 n

m

con

cen

trat

ion

(m

M)

4HB succinate OD600




Supplementary Figure 6. Biosynthesis of 4HB from glucose via -ketoglutarate route by E. coli

strains grown anaerobically for 24 h. Host strain, MG1655lacIQ; construct 1, plasmid pZS*13S-sucA-

4hbd; construct 2, plasmid pZA33S-sucA-4hbd; construct 3, plasmid pZE13S-sucA-4hbd; construct 4,

pZA33S-4hbd. Blue bars, extracellular 4HB concentration; black bars, extracellular succinate

concentration; circles, culture OD. Construct with pZS*13S empty vector made no detectable 4HB (data

not shown).

0.00

0.60

1.20

1.80

2.40

3.00

0.0

3.0

6.0

9.0

12.0

15.0

1 2 3 4

OD

60

0 n

m

con

cen

trat

ion

(m

M)

4HB succinate OD600




ATGAAAGTGACCAATCAAAAAGAACTGAAACAAAAGCTGAATGAATTGCGTGAAGCGCAA

AAGAAGTTTGCCACCTATACTCAAGAGCAAGTTGATAAAATTTTTAAACAATGTGCCATTGC

CGCAGCGAAAGAACGTATCAACCTGGCGAAACTGGCAGTAGAAGAAACAGGTATTGGTCTG

GTGGAAGATAAAATTATCAAAAATCATTTTGCCGCAGAATATATTTACAATAAATATAAAAA

TGAAAAAACCTGTGGCATTATCGACCATGACGATAGCCTGGGCATTACCAAAGTTGCTGAAC

CAATTGGTATTGTTGCGGCCATTGTTCCGACCACTAATCCAACTTCCACCGCAATTTTCAAAT

CACTGATTTCGCTGAAAACACGCAACGCCATTTTCTTTTCACCACATCCACGTGCAAAAAAA

TCGACCATTGCTGCAGCAAAACTGATTCTCGATGCCGCGGTTAAAGCAGGCGCACCGAAAA

ATATTATCGGCTGGATTGATGAGCCGTCAATTGAACTTAGCCAAGATCTGATGAGTGAAGCT

GATATCATTCTGGCAACCGGCGGTCCTTCAATGGTTAAAGCGGCCTATTCCAGCGGTAAACC

TGCAATTGGTGTTGGCGCAGGAAATACGCCAGCAATTATCGATGAGAGCGCAGATATCGAT

ATGGCAGTAAGCTCCATTATTCTCAGCAAAACCTATGACAATGGCGTGATTTGCGCCTCTGA

ACAATCGATTCTGGTGATGAATAGCATTTACGAAAAAGTTAAAGAGGAATTTGTGAAACGC

GGCAGCTATATTCTCAATCAAAATGAAATCGCTAAAATAAAAGAAACCATGTTTAAAAATG

GCGCTATTAATGCTGACATCGTTGGTAAATCTGCTTATATCATTGCTAAAATGGCAGGTATTG

AAGTTCCGCAAACCACAAAAATACTGATTGGCGAAGTACAAAGCGTTGAAAAAAGCGAGCT

GTTCAGCCATGAAAAACTGTCACCGGTACTGGCAATGTATAAAGTTAAAGATTTTGATGAAG

CGCTGAAAAAAGCACAACGTCTGATTGAACTGGGTGGTAGTGGTCACACCTCATCGCTGTAT

ATCGATAGCCAAAACAATAAAGATAAAGTTAAAGAATTTGGTCTGGCAATGAAAACCTCAC

GCACATTTATTAACATGCCTTCTAGCCAGGGCGCAAGCGGCGATCTCTACAATTTTGCGATT

GCACCATCATTTACTCTGGGCTGCGGCACCTGGGGCGGCAACAGCGTATCGCAAAATGTTGA

GCCGAAACATCTGCTGAATATTAAAAGCGTTGCTGAACGCCGTGAAAATATGCTGTGGTTTA

AAGTGCCACAAAAAATTTATTTTAAATATGGCTGTCTGCGTTTTGCACTGAAAGAATTAAAA

GATATGAATAAGAAACGCGCCTTTATCGTCACCGATAAAGATCTGTTTAAACTGGGTTATGT

TAATAAAATCACCAAAGTACTGGATGAGATCGATATTAAATACAGCATTTTTACCGATATTA

AAAGCGATCCAACTATTGATAGCGTAAAAAAAGGTGCGAAAGAAATGCTTAACTTTGAACC

TGATACTATTATCTCTATTGGTGGTGGTTCGCCAATGGATGCCGCAAAGGTGATGCACTTGCT

GTATGAATATCCGGAAGCAGAAATTGAAAATCTGGCTATTAACTTTATGGATATTCGTAAGC

GCATTTGCAATTTCCCGAAACTGGGTACAAAGGCGATTAGCGTGGCTATTCCGACAACTGCT

GGTACCGGTAGCGAGGCAACACCTTTTGCCGTTATTACTAATGATGAAACAGGAATGAAATA

CCCGTTAACCTCTTATGAACTGACCCCAAACATGGCAATTATCGATACTGAATTAATGCTCA

ATATGCCGCGTAAATTAACCGCAGCAACTGGTATTGATGCACTGGTTCATGCTATTGAAGCC

TATGTTTCGGTGATGGCGACGGATTATACCGATGAACTGGCGTTACGCGCAATTAAAATGAT

TTTTAAATATTTGCCGCGCGCCTATAAAAATGGCACTAACGACATTGAAGCACGTGAAAAAA

TGGCGCATGCCTCTAATATTGCGGGGATGGCATTTGCCAATGCTTTCCTCGGTGTATGCCATT

CAATGGCGCATAAACTGGGGGCAATGCATCACGTTCCACATGGCATTGCCTGTGCGGTATTA

ATTGAAGAAGTTATTAAATATAACGCCACCGACTGTCCAACAAAGCAAACCGCATTCCCGCA

ATATAAAAGCCCGAATGCTAAGCGTAAATATGCTGAAATTGCAGAGTATCTGAATCTGAAA

GGTACTAGCGATACCGAAAAGGTGACCGCGTTAATTGAAGCGATTAGCAAGCTGAAGATTG

ATCTCAGTATTCCACAAAATATCTCTGCCGCTGGTATCAATAAAAAAGATTTTTATAACACG

CTGGATAAAATGAGCGAGCTGGCTTTTGATGACCAATGTACAACCGCTAATCCGCGCTATCC

ACTGATCAGTGAACTGAAGGATATCTATATCAAAAGTTTTTAA

Supplementary Figure 7. Nucleotide sequence of gene 002C.




Supplementary Figure 8. Production envelope of OptKnock strain design for BDO production.

Strain has deletions in adhE, ldhA, pflB, and mdh, and contains the BDO pathway reactions shown in

Figure 1. Basis is 20 mmol/hr-gDCW glucose uptake and a non-growth associated maintenance of 7.6

mmol ATP/hr-gDCW is assumed. Plot shows maximum attainable BDO yield as a function of the

maximum growth yield.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.00 0.03 0.06 0.09 0.12 0.15

BD

O Y

ield

(g

/g)

Biomass Yield (g/g)

Wild Type

OptKnock Mutant




Supplementary Figure 9. Production of BDO from glucose in 1L fed-batch fermentation using

first-generation engineered host. Strain is ECKh-138 containing the plasmids pZS*13S-sucCD-sucD-

4hbd/sucA and pZE23S-002C-34. Filled squares, BDO; filled circles, acetate; filled triangles, ethanol;

open squares, pyruvate; open circles, 4HB; open triangles, GBL. All concentrations are mM in

fermentation broth.

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

0 10 20 30 40 50 60

Ex

tra

ce

llu

lar

co

nc

en

tra

tio

n (

mM

)

Time since induction (hours)

a




Supplementary Figure 10. Citrate synthase activity of wild-type (top) and R163L mutant (bottom)

GltA enzymes. Crude extracts of ECKh-401 and ECKh-422, containing the wild-type and R163L gltA,

respectively, were assayed as described in the supplementary materials and methods. Activity was

monitored by free CoA formation as indicated by the change in absorbance at 410 nm. Green triangles, no

NADH added; red squares, NADH added to assay mixture to a concentration of 2 mM.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200 250

abs

41

0 n

m

time (s)

gltA activity assay, WT

2 mM NADH

No NADH

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 50 100 150 200 250

abs

41

0 n

m

time (s)

gltA activity assay, R163L mut

2 mM NADH

No NADH




Supplementary Figure 11. Appearance of 13

C label in intracellular metabolites after pulse addition

of 13

C-glucose. ECKh-422 pZS*13S-sucCD-sucD-4hbd/sucA pZE23-002C-Cat2 was grown in

microaerobic fermentation using unlabeled glucose. After 24 hours, glucose feed was turned off and the

glucose concentration was allowed to drop to below 1 g/L. 1 g/L U-13

C-glucose was added to the tank,

and samples harvested at 6 minute intervals. Cells were rapidly quenched in cold methanol, and

intracellular metabolites measured by LC-MS as described previously 25

.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 10 20 30 40 50 60 70

13C

in

co

rpo

ratio

n

incubation time [min]

Alanine

1,4-butandiol

4-hydroxybutyrate

Glutamate




Supplementary Figure 12. Comparison of BDO and ethanol production in ECKh-138 containing

different aldehyde dehydrogenase genes. Strains were transformed with pZS*13S-sucCD-sucD-

4hbd/sucA and either pZE23-002C-Cat2 (left) or pZE23-025B-Cat2 (right), and cultured

microaerobically in M9 minimal medium supplemented with 20 g/L glucose and induced with 0.25 mM

IPTG. Cells were harvested 48 hours post-induction and analyzed for ethanol (blue bars) and BDO (red

bars).

0.00

5.00

10.00

15.00

20.00

25.00

30.00

pZE23-002C-Cat2 pZE23-025B-Cat2

mM

in c

ult

ure

bro

th Ethanol

BDO




ATGAATAAAGACACACTGATCCCTACAACTAAAGATTTAAAAGTAAAAACAAATGGTGAAA

ACATTAATTTAAAGAACTACAAAGATAATAGCAGTTGTTTCGGCGTATTCGAAAATGTTGAA

AATGCTATCAGCAGCGCTGTACACGCACAAAAGATATTATCGCTGCATTATACAAAAGAGC

AACGTGAAAAAATCATCACTGAGATACGTAAGGCCGCATTACAAAATAAAGAGGTGCTGGC

TACAATGATTCTGGAAGAAACACATATGGGACGTTATGAGGATAAAATATTAAAACATGAA

CTGGTAGCTAAATATACTCCTGGTACAGAAGATTTAACTACTACTGCCTGGAGCGGTGATAA

TGGTCTGACAGTTGTAGAAATGTCTCCATATGGTGTTATTGGTGCAATAACTCCTTCTACCAA

TCCAACTGAAACTGTAATTTGTAATAGCATTGGCATGATTGCTGCTGGAAATGCTGTAGTAT

TTAACGGACACCCATGCGCTAAAAAATGTGTTGCCTTTGCTGTTGAAATGATCAATAAGGCA

ATTATTAGCTGTGGCGGTCCGGAAAATCTGGTAACAACTATAAAAAATCCAACCATGGAGTC

TCTGGATGCCATTATTAAGCATCCTTCAATAAAACTGCTTTGCGGAACTGGCGGTCCAGGAA

TGGTAAAAACCCTGTTAAATTCTGGTAAGAAAGCTATTGGTGCTGGTGCTGGAAATCCACCA

GTTATTGTCGATGATACTGCTGATATTGAAAAGGCTGGTCGTAGCATCATTGAAGGCTGTTC

TTTTGATAATAATTTACCTTGTATTGCAGAAAAAGAAGTATTTGTTTTTGAGAATGTTGCAGA

TGATTTAATATCTAACATGCTGAAAAATAATGCTGTAATTATCAATGAAGATCAGGTATCAA

AATTAATCGATTTAGTATTACAAAAAAATAATGAAACTCAAGAATACTTTATCAACAAAAAA

TGGGTAGGTAAAGATGCAAAATTATTCCTCGATGAAATCGATGTTGAGTCTCCTTCAAATGT

TAAATGCATTATCTGCGAAGTGAATGCCAATCATCCATTTGTTATGACAGAACTGATGATGC

CAATATTGCCAATTGTGCGCGTTAAAGATATCGATGAAGCTATTAAATATGCAAAGATTGCA

GAACAAAATAGAAAACATAGTGCCTATATTTATAGCAAAAATATCGACAACCTGAATCGCTT

TGAACGTGAAATCGATACTACTATTTTTGTAAAGAATGCTAAATCTTTTGCTGGTGTTGGTTA

TGAAGCAGAAGGATTTACCACTTTCACTATTGCTGGATCTACTGGTGAGGGCATAACCTCTG

CACGTAATTTTACCCGCCAACGTCGCTGTGTACTGGCCGGCTAA

Supplementary Figure 13. Nucleotide sequence of gene 025B.

Supplementary Datasets

Supplementary datasets are included in the attached Microsoft Excel file.

Supplementary Datafile 1. List of reactions included in the genome-scale E. coli model used for

OptKnock analysis. Includes abbreviation, reaction stoichiometry, upper and lower bounds, and

associated genes.

Supplementary Datafile 2. Metabolite abbreviations and names, also indicating cytosolic or extracellular

location.

Supplementary Datafile 3. OptKnock results. Columns A-E are the gene deletions, column F is the

predicted maximum growth rate at a basis of 20 mmol/gDCW/hr glucose uptake, and column G is the

predicted BDO production (mmol/gDCW/hr) at this point.

Supplementary Datafile 4. Set of reaction operators used by the Biopathway Predictor algorithm and

corresponding reaction diagrams. If reversible, both directions are shown.




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