Chemicals and enzymes
Glycolaldehyde dimer, d-xylono-1,4-lactone, dl-lactaldehyde, d-xylonic acid lithium salt were purchased from Sigma-Aldrich UK. Zinc chloride was purchased from Merck, Germany. Mammalian hydroxyacid oxidase 1 (or glycolate oxidase, HAO1) enzyme was obtained from MyBioSource USA and LDH (lactate dehydrogenase) from rabbit muscle was purchased from Sigma-Aldrich. 10-Acetyl-3,7-dihydroxyphenoxazine (or Amplex Red) was purchased from Sigma-Aldrich. All other substrates were purchased from Sigma-Aldrich in high purity grade. The E. coli BL21(DE3) strain was used for the pBAT4 (Peränen et al. 1996) based cytoplasmic expression vectors. LB growth media was prepared according to Sambrook and Russel (2001). Glucose release medium EnPresso B was obtained from Bio-Silta Ltd. Ampicillin resistance (100 μg ml−1) was used for the selection of all plasmids. d-xylono-1,4-lactone was analysed by 1H-NMR spectroscopy to verify it has not spontaneously opened to d-xylonic acid.
Cloning of the pathway genes
The d-xylonolactonase encoding gene from C. crescentus (Cc xylC, CC_0820, Accession: NP_419637, GI: 16125073, NCBI), the aldehyde dehydrogenase encoding gene from E. coli (Ec aldA, b1415, Accession: P25553.2, GI: 113602, NCBI), and E. coli 2-dehydro-3-deoxy-d-pentonate aldolase encoding gene (Ec yagE, b0268, Accession: P75682.2, GI: 357529065, NCBI) were purchased as synthetic genes, and codon optimized for E. coli (GenScript, China) in the pBAT4 vector (Peränen et al. 1996). The genes encoding Azospirillum brasiliense α-ketoglutarate semialdehyde dehydrogenase (Ab araE, Accession: Q1JUP4.1, GI: 40339944, NCBI) E. coli 1,3-propanediol oxidoreductase (Ec yqhD, Accession: ADK47404.1, GI: 301015215) and E. coli 1,2-propanediol oxidoreductase (Ec fucO, Accession: P0A9S1.2, GI: 357528800, NCBI) were obtained as synthetic genes codon optimized for E. coli (GenArt, Thermo Fisher, Germany) and inserted into the linearized (NcoI and XhoI digestion) pBAT4 vector (Peränen et al. 1996). The synthetized insert contained 50 bp overlapping regions in both 5′ and 3′ ends to the vector to allow cloning using the Gibson assembly method (Gibson et al. 2009). The plasmids were transformed into E. coli BL21(DE3) strain for protein production. The Cc xylC gene was ordered as two different plasmid constructs with an eight amino acid long Strep-tag II (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) either at the N-terminus or at the C-terminus. A Site-directed Mutagenesis Kit (New England Biolabs, USA) was later used to remove the C-terminal tag from the Cc xylC gene, since it did not work in the purification procedure. The Ec yagE gene was tagged with a Strep-tag II at the N-terminus. A six amino acid long His-tag was added to the N-terminus for the Ec aldA, Ab araE and Ec yqhD genes, or to the C-terminus for the Ec fucO gene. All DNA constructs were verified by sequencing to confirm that no changes in the nucleotide sequence had occurred. Sequencing was done by Source Bioscience Sequencing, UK. The Acinetobacter baylyii ADP1 α-ketoglutarate semialdehyde dehydrogenase gene (GI: 81613403, NCBI) with an N-terminal His6-tag cloned into pET22 was a kind gift from Dr. Alain Perret, France.
Five genes encoding dehydratases from the enolase superfamily with putative activity on d-xylonate, were obtained as synthetic genes, codon optimized for E. coli and inserted into the pBAT4 vector (GenScript, China). The resulting plasmids contained the genes for the putative d-xylonate dehydratases from Haloarcula marismortui (Hm XylD, GI: 55230170, NCBI), and Haloferax volcanii (Hv XylD, GI: 292493977, NCBI), the putative d-gluconate dehydratase SEN1436 from Salmonella enterica (Se GluDHT, GI: 667467043, NCBI) the mandelate racemase/muconate lactonizing enzyme-like protein from Rubrobacter xylanophilus (Rx MR/MLE, GI: 123368307, NCBI), and the d-galactonate dehydratase family member RspA from Pantoea ananatis (Pa GalDHT, UniProt: D4GJ14). The dehydratases from H. marismortui, H. volcanii, and S. enterica were tagged with an N-terminal Strep-tag II, and a His6-tag was added to the C-terminal or N-terminal for the enzymes from R. xylanophilus and P. ananatis, respectively.
Expression and purification of the pathway enzymes
The N-terminal Strep-tagged Cc xylB gene with a TEV site encoding the Caulobacter crescentus d-xylose dehydrogenase (Cc XylB) was expressed in S. cerevisiae under the PGK promoter, and Cc XylB was purified from the yeast cell extract in a single step using Strep-Tactin Sepharose. The buffer of the purified enzyme was changed to 50 mM phosphate buffer pH, 7 using PD-10 columns (GE Healthcare).
For purification of C. crescentus d-xylonolactonase (Cc XylC), E. coli BL21(DE3) cells containing the Cc xylC gene in the pBAT4-XylC plasmid were grown in Luria Broth medium (Bertani 1951) supplemented with 100 μg ml−1 ampicillin, at + 37 °C, 225 rpm to an OD600 0.6–0.8. After the addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to induce expression of the gene of interest, the culture was further grown at + 30 °C, 225 rpm overnight. The cells were harvested by centrifugation at 4000×g for 20 min at 4 °C and suspended in ice-cold lysis buffer (50 mM Tris–Cl buffer, pH 7.5, containing 1 mM DTT, 1× protease inhibitor cocktail (Complete EDTA-free, Roche), DNAse I, RNase A, and lysozyme (Sigma-Aldrich)), and lysed by sonication. The cell-free sample was diluted ten times with 50 mM Tric-Cl, pH 7.5 and loaded onto a 20 ml DEAE FF 16/10 ion-exchange column (GE Healthcare) equilibrated with 50 mM Tris–Cl buffer, pH 7.5. After washing with 25 column volumes (CV) of equilibration buffer, the sample was eluted with a stepwise NaCl gradient (0–120 mM NaCl for 15 CV followed by 120–200 mM NaCl for 5 CV). The fractions were analyzed for Cc XylC content by SDS-PAGE analysis (10% Criterion SF gel, BioRad), and the fractions containing Cc XylC were pooled, concentrated and the buffer was changed to 50 mM Tris–Cl, pH 7.5 by Vivaspin 20 centrifugal concentrator (MWCO 10,000 Da, Sartorius AG, Goettingen, Germany).
The expression and purification of C. crescentus d-xylonate dehydratase (Cc XylD) was performed as described previously (Andberg et al. 2016). Briefly, the N-terminal Strep-tagged Cc xylD gene was expressed in E. coli BL21(DE3) cells at + 30 °C overnight. The enzyme was purified from the bacterial cell extract using a Strep-Tactin affinity column.
The E. coli 2-dehydro-3-deoxy-d-pentonate aldolase encoding gene (Ec yagE) was expressed in E. coli BL21(DE3) cells at + 30 °C overnight using enzymatic glucose release medium EnPresso B (Bio Silta Ltd). The cells were harvested by centrifugation 1800×g for 15 min at + 4 °C and suspended in ice-cold lysis buffer (100 mM Tris–Cl buffer and 150 mM NaCl, pH 8, supplemented with protease inhibitors, lysozyme, DNase I, and RNase A, and lysed by sonication. The sample was centrifuged at 24,000×g for 45 min at + 4 °C where after the clear cell extract was loaded on a 5 ml Strep Tactin column equilibrated with 100 mM Tris–Cl buffer and 150 mM NaCl, pH 8. The Ec YagE enzyme was eluted with 2.5 mM desthiobiotin and the fractions were analysed by SDS-PAGE. The fractions containing Ec YagE were pooled and concentrated on Vivaspin 20 (10000 MWCO) and the buffer was changed to 50 mM Tris–Cl, pH 8 using a PD-10 column (GE Healthcare Life Sciences).
The E. coli aldehyde dehydrogenase (Ec aldA) gene was expressed in E. coli BL21(DE3) in the pBAT4-AldA_N-6×His plasmid in LB medium supplemented with ampicillin at + 30 °C, 150 rpm overnight. Cells were harvested by centrifugation at 4000×g for 15 min at 4 °C and suspended in ice-cold lysis buffer (50 mM Tris–HCl pH 8, 300 mM NaCl buffer) and disrupted with two passes through a French press at 10,000 psi. The resulting cell lysate was incubated for 30 min in the presence of protease inhibitors, DNase I, RNase A and lysozyme after which the insoluble fraction was separated by centrifugation at 37,000×g for 20 min at 4 °C. For the purification of E. coli aldehyde dehydrogenase (Ec AldA), 20 ml of the cell-free extract was loaded onto a 5 ml HiTrap Chelating HP column (GE Healthcare) charged with NiSO4 and equilibrated with 50 mM Tris–HCl, pH 8, 300 mM NaCl. After the column was washed with 50 ml equilibration buffer, the bound fraction was eluted with a gradient (10 column volumes) from 0 to 500 mM imidazole. One millilitre fractions were collected during elution, and protein purity in the fractions was analysed with SDS-PAGE. The fraction containing Ec AldA were pooled and the buffer was changed to 50 mM Tris–HCl buffer, pH 7.5, 100 mM NaCl by gel filtration using an EconoPac 10 DG desalting column (Bio-Rad).
The N-terminally His-tagged Acinetobacter baylii ADP1 α-ketoglutarate semialdehyde dehydrogenase (Ab α-KGSA DH) was expressed and purified as described in (Aghaie et al. 2008). Briefly, the E. coli BL21/DE3 cells were grown in Terrific Broth medium containing 0.5 M sorbitol, 5 mM betaine, and 100 µg ml−1 carbenicillin at 37 °C until reaching an A600 of 2. After addition of 1 mM IPTG the culture was further grown at + 20 °C, 225 rpm overnight. The enzyme was purified from the cell-free extract in a single step using Ni–NTA chromatography and the enzyme was stored in 50 mM Tris–Cl buffer, pH 8, 50 mM NaCl, 10% glycerol and 1 mM DTT. The protein concentration of Ab α-KGSA DH was determined with the Bio Rad DC kit, using BSA as standard.
The genes corresponding to Azospirillum brasilense α-ketoglutarate semialdehyde dehydrogenase (Ab araE), E. coli 1,2-propanediol oxidoreductase (Ec fucO), and E. coli 1,3-propanediol oxidoreductase (Ec yqhD) were expressed in E. coli BL21(DE3) in the corresponding pBAT4-AraE_HisN, pBAT4-FucO_HisC, or pBAT4-YqhD_HisN plasmids. The cultivations were done in LB medium supplemented with ampicillin (225 rmp, overnight) at + 28 °C, + 22 °C, or at + 22 °C for Ab AraE, Ec FucO or Ec YqhD, respectively. The cells were harvested by centrifugation suspended in ice-cold lysis buffer (40 mM sodium phosphate buffer and 100 mM NaCl, pH 8 containing protease inhibitors, DNAse I, RNase A, and lysozyme, and disrupted by sonication. The samples were centrifuged at 12,000×g for 10 min at + 4 °C, where after the clear cell extract was adjusted to the binding buffer (20 mM sodium phosphate, 20 mM imidazole and 500 mM NaCl, pH 7.4) and loaded on a 5 ml HiTrap Chelating Sepharose column charged with Ni2+. The enzymes were eluted with 20 mM sodium phosphate, 200 mM imidazole and 500 mM NaCl, pH 7.4) and the fractions were analysed by SDS-PAGE. The fractions containing the enzymes of interest were pooled, concentrated, and the buffer was changed to 50 mM Tris–Cl, pH 7.5 using PD-10 columns for Ab AraE, or to 40 mM sodium phosphate buffer pH 7.4 using Econo-Pac column for Ec FucO and Ec YqhD.
Enzyme activity measurements
The lactonase activity of Cc XylC was measured using d-xylono-1,4-lactone made fresh daily as the substrate. A circular dichroism (CD) -based assay for lactonase was performed in 10 mM Tris–HCl buffer, pH 6–8, using 1 mM lactone, with varying amounts of divalent metal ions and an aliquot of enzyme. CD spectra were recorded on a Chirascan CD spectrometer (AppliedPhotophysics, UK) equipped with a Peltier thermally controlled cuvette holder. Spectra were recorded using two scans, a bandwidth of 1 nm and a wavelength step of 0.5 nm, and the values were corrected for buffer contribution. The cuvette used for all measurements was a 1 mm and the temperature was set to 25 °C in all measurements.
The activity of Cc XylC was also followed by 1H-NMR. The reactions were carried out in 600 µl of 50 mM Na-phosphate buffer, pH 6.8 containing 10% of D2O (Aldrich) using 2 mM xylonolactone with 100 µM metal ions or EDTA. After recording a zero spectrum, 10 µg of Cc XylC was added and the reactions were followed at 22 °C by recording a series of 2 min 1H-NMR spectra. All NMR spectra were recorded on a 600 MHz Bruker Avance III NMR spectrometer equipped with QCI (H1/C13/N15/P31) cryoprobe and SampleJet sample changer. A modified version of SampleJet firmware allowed monitoring reactions in parallel while using the preheating block as an incubator. The water signal was suppressed by the so called 1D NOESY presaturation using Bruker’s pulse program noesygppr1d.
Ec AldA, Ab AraE and Ab α-KGSA DH dehydrogenases are NAD+- or NADP+-dependent enzymes whose activities were measured by following the increase in absorbance at 340 nm. Assays were performed at 22 °C, in 50 mM Tris–HCl buffer, pH 7, using 1–5 mM glycolaldehyde, 1 mM NAD+ (for Ec AldA and Ab AraE) or 1 mM NADP+ (for Ab α-KGSA DH), 0.5 mM DTT (for Ec AldA), 10 mM MgCl2 (for Ab AraE and Ab α-KGSA DH) and an aliquot of enzyme. The Ec FucO and Ec YqhD oxidoreductase activities were measured at 340 nm by following the oxidation of NAD(P)H at 22 °C in 50 mM Na-phosphate buffer, pH 7.5 using 10 mM glycolaldehyde and 1 mM NADH (for Ec FucO) or 1 mM NADPH (for Ec YqhD) as substrates.
The rate of glycolate, lactate or ethylene glycol formation in the last step of the different pathways was measured by following the NAD(P)H formation or consumption depending on the final dehydrogenase/oxidoreductase in the respective pathway. Assays were performed in microtitre plate at 22 °C in 50 mM Tris–HCl buffer pH 7, using 1 mM d-xylonolactone or d-xylonate, 2 mM NAD(P)+ or NAD(P)H, 10 mM Mg2+ and 0.5 mM DTT. The details of this method used to monitor the overall pathway are explained in more detail in the figure legends.
The production of glycolate starting from d-xylose utilising a 5-enzyme pathway was followed through an enzymatic assay using HAO1, which converts glycolate to glyoxylate and produced H2O2, which can be detected using HRP and Amplex red. The assay was carried out in a two-phase process, where the first phase contained 50 mM Tris–HCl pH 7.0, 1 mM d-xylose, 10 mM MgCl2, 2 mM NAD+, 2 µg Cc XylB, 8 µg Cc XylC, 4 µg Cc XylD, 4 µg Ec YagE and 4 µg Ec AldA (in a total volume of 200 µl). In this experiment, the concentration of the single enzyme in each 5 enzymatic step was varied keeping all others as in the basic reaction presented above. After this first 30 min reaction, the reaction was stopped by using 10.000 kDa cut-off Vivaspin centrifugal concentrator to separate the enzymes from the reaction product. The amount of glycolate produced in the first reaction was then measured after this separation step by using 50 µM Amplex Red, 0.4 µg HRP and 1 ug HAO1. This reaction was monitored at 560 nm for 1 h.
Protein analysis
The purity of the enzymes was checked on SDS-PAGE gels using 4–20% or 10%, Criterion™ TGX Stain-Free™ Protein Gels (Bio-Rad, USA). For Western blot analysis the antibody used for proteins containing the Strep-tag II was Strep-Tactin® conjugated to alkaline phosphatase (IBA, Germany), for proteins containing a His6 -tag an Anti-His tag Mouse Monoclonal (IgG2b) antibody was used (Trend Pharma & Tech Inc., Canada).
The protein concentrations for the purified enzymes were calculated from A280 using a theoretical extinction coefficient calculated by ProtParam (http://web.expasy.org/protparam/). The extinction coefficient for each enzyme is shown in parenthesis: Cc XylB (ε = 43,680 M−1 cm−1), Cc XylC (ε = 50,670 M−1 cm−1), Cc XylD (ε = 70,360 M−1 cm−1), Ec YagE (ε = 27,055 M−1 cm−1) Ec AldA (ε = 59,485 M−1 cm−1), Ab AraE (ε = 43,430 M−1 cm−1), Ec FucO (ε = 37,150 M−1 cm−1), and Ec YqhD (ε = 42,400 M−1 cm−1).
In order to determine the protein oligomeric state of Cc XylC, analytical gel filtration was conducted at 25 °C using a ACQUITY UPLC Protein BEH SEC 125 Å column (Waters Corporation) at a flowrate of 0.3 ml min−1. Cc XylC (0.6 μg protein) was injected on the column, pre-equilibrated in 100 mM sodium phosphate buffer pH 6.8. The absorbance of the eluate was monitored at 280 nm. Calibration of elution times was performed with the globular proteins ovalbumin (44.2 kDa, 0.3 ml min−1), ribonuclease A (13.7 kDa, 0.3 ml min−1), and uracil (112 Da, 0.3 ml min−1). The void volume of the column was determined with thyroglobulin (669 kDa, 0.1 ml min−1).
Nucleotide sequence accession numbers
The nucleotide sequences reported in this study have been deposited in NCBI GenBank database under the accession numbers MH836586–MH836597.