Engineering a Carboxyl Methyltransferase for the Formation of a Furan‐Based Bioplastic Precursor

Abstract FtpM from Aspergillus fumigatus was the first carboxyl methyltransferase reported to catalyse the dimethylation of dicarboxylic acids. Here the creation of mutant R166M that can catalyse the quantitative conversion of bio‐derived 2,5‐furandicarboxylic acid (FDCA) to its dimethyl ester (FDME), a bioplastics precursor, was reported. Wild type FtpM gave low conversion due to its reduced catalytic efficiency for the second methylation step. An AlphaFold 2 model revealed a highly electropositive active site, due to the presence of 4 arginine residues, postulated to favour the binding of the dicarboxylic acid over the intermediate monoester. The R166M mutation improved both binding and turnover of the monoester to permit near quantitative conversion to the target dimethyl ester product. The mutant also had improved activity for other diacids and a range of monoacids. R166M was incorporated into 2 multienzyme cascades for the synthesis of the bioplastics precursor FDME from bioderived 5‐hydroxymethylfurfural (HMF) as well as from poly(ethylene furanoate) (PEF) plastic, demonstrating the potential to recycle waste plastic.


G
Table S1 Pure protein yields of WT FtpM and FtpM R166X mutants from 1 L LB culture.

Enzyme Protein yield (mg)
WT FtpM >60 [1] FtpM Compounds 1 and 10 were purchased from Fluorochem.Compound 2 was purchased from Alfa Aesar.Compound 3 was synthesised in-house as described previously. [1]Compounds 4 and 9 were purchased from Apollo Scientific.Compound 8 was kindly provided by Dr Thomas Farmer, University of York.Compounds 11-24 were purchased from Sigma-Aldrich.

TGA analysis
Thermogravimetric analysis (TGA) was performed using a Mettler Toledo TGA/DSC1 STARe System® using a 25-800°C temperature range with a heating rate of 10°C min -1 , under an 80 mL min -1 N2 flow.

Figure S17
Thermogravimetric analysis of the synthesized PEF.

DSC analysis
Differential Scanning Calorimeter (DSC) thermograms were measured using a Mettler Toledo "DSC1 STARe System®" in the −40-240°C temperature range using a heating/cooling rate of 10°C min -1 , with a N2 flow of 20 mL min -1 .

Figure S18
Differential scanning calorimetry analysis of the synthesized PEF (1 st cooling and 2 nd heating cycle).

GPC Analysis
Gel permeation chromatography (GPC) was run on a different PEF sample which was prepared using the same method described above to give the following molecular weight data: Mn= 7400 g mol -1 , Mw= 18900 g mol -1 , Mw/Mn= 2.54.GPC was performed on a system equipped with a Waters 1515 Isocratic HPLC pump, a Waters 2414 refractive index detector (35°C), a Waters 2707 auto sampler, and a PSS PFG guard column followed by two PFG-linear-XL (7 μm, 8 × 300 mm) columns in series at 40°C.Hexafluoroisoproanol (HFIP) with potassium trifluoroacetate (3 g L −1 ) was used as the eluent at a flow rate of 0.8 mL min −1 .The molecular weights were calculated against poly(methyl methacrylate) standards.

FtpM R166X mutant conversions with FDCA (2 mM SAM)
Figure S19 Results of assaying FtpM R166X mutants with FDCA 2 using 1 mM substrate, 500 μM enzyme, 2 mM SAM and 4 μM SAH-nuc in 50 mM MES pH 6.0.Reactions were incubated at 25°C for 16 h.Products detected via RP-HPLC.Red: FDCA; blue: FMME; green: FDME. Figure S22 FDCA calibration curve.A range of standards consisting of different concentrations of FDCA were analysed via RP-HPLC with 1 mM caffeine as an internal standard.The average FDCA: caffeine peak area ratio was plotted against FDCA concentration to generate the curve.

Figure
Figure S2Michaelis-Menten curve used to determine kinetic parameters of the methylation of FDCA 2 catalysed by FtpM R166F.

Figure S3
Figure S3Michaelis-Menten curve used to determine kinetic parameters of the methylation of FMME 3 catalysed by FtpM R166F.

Figure S4
Figure S4Michaelis-Menten curve used to determine kinetic parameters of the methylation of FDCA 2 catalysed by FtpM R166I.

Figure S5
Figure S5 Michaelis-Menten curve used to determine kinetic parameters of the methylation of FMME 3 catalysed by FtpM R166I.

Figure S6
Figure S6Michaelis-Menten curve used to determine kinetic parameters of the methylation of FDCA 2 catalysed by FtpM R166M.

Figure S7
Figure S7 Michaelis-Menten curve used to determine kinetic parameters of the methylation of FMME 3 catalysed by FtpM R166M.

Figure S8
Figure S8Michaelis-Menten curve used to determine kinetic parameters of the methylation of FDCA 2 catalysed by FtpM R166Q.

Figure S9
Figure S9Michaelis-Menten curve used to determine kinetic parameters of the methylation of FMME 3 catalysed by FtpM R166Q.

Figure S10
Figure S10Michaelis-Menten curve used to determine kinetic parameters of the methylation of FDCA 2 catalysed by FtpM R166H.

Figure S11
Figure S11 Michaelis-Menten curve used to determine kinetic parameters of the methylation of FMME catalysed by FtpM R166H.

Figure S12
Figure S12Michaelis-Menten curve used to determine kinetic parameters of the methylation of FDCA catalysed by FtpM R166K.

Figure S13
Figure S13Michaelis-Menten curve used to determine kinetic parameters of the methylation of FMME catalysed by FtpM R166K.

1 H
, 13 C and HSQC NMR spectroscopy characterizations were performed using a JEOL ECZ400R/S3 and a CDCl3/TFAd mixture as the solvent.

Figure S14 1
Figure S14 1 H-NMR analysis of the synthesized PEF.

Figure
Figure S15 13 C-NMR analysis of the synthesized PEF.

Figure S16
Figure S16 HSQC analysis of the synthesized PEF.

Table S2
RP-HPLC retention times.RP-HPLC conditions described in manuscript.