Biodegradation of high concentrations of formaldehyde using Escherichia coli expressing the formaldehyde dismutase gene of Methylobacterium sp. FD1

Figures & data

Table 1. Primers for PCR amplification of the formaldehyde dismutase gene of FD1.

Primer	Sequence (5′-3′)	Objective DNA fragment
FDM-mid-F1	AGCGAYCAGCAYATCTAYCA	Middle region
FDM-mid-R1	TCCCASTGGATSGCCATCAT	Middle region
FDM-TAIL-R1	CGCAATCGACCTCGTCGTTGACGTTC	Upstream region
FDM-TAIL-R2	ATCCGTGTGCCGCTCCTTGCAGTTG	Upstream region
FDM-TAIL-R3	ACCGAGCAAAGATCGCCGACCCTGA	Upstream region
AD2	WGGWANCWGAWANGCA	Upstream region
FDM-TAIL-F1	GGAGCACTCGGAGGCGGTGATCAATA	Downstream region
FDM-TAIL-F2	ATCTATACCGCAGCCGACCCGAAGG	Downstream region
FDM-TAIL-F3	CGAAGAAGGGTCAGCTCCCCATCGAC	Downstream region
AD1	NGTCGASWGANAWGAA	Downstream region
FD1-FDM-F	GAAGGAGATATACATATGGCCGCAAATGGAAACCGCGTC	Full length
FD1-FDM-R	GTGATGGTGGTGATGATGTTACGAGGCCTTTTTCACGCTGCC	Full length

The underlines indicated the vector sequence and the box indicated stop codon.

Figure 1. Hydrophobic interaction chromatography of FD1 formaldehyde dismutase. The solid line, relative formaldehyde dismutase activity; the dashed line, the concentration of ammonium sulfate. (A) and (B) were the fractions with relatively high formaldehyde dismutase activity. Experimental conditions are specified in Materials and methods.

Figure 2. SDS-PAGE of the fractions from hydrophobic interaction chromatography with formaldehyde dismutase activity. Lanes A and B, the representatives of A-fraction and B-fraction, respectively, from Figure 1. Lane M, protein markers. Arrows indicate the molecular weights of bands from the two fractions. Experimental conditions are specified in Materials and methods.

Table 2. NanoLC–MS/MS analysis of the gel bands from SDS-PAGE of the fractions from hydrophobic interaction chromatography with formaldehyde disumutase activity.

Gel band	Entry protein name [organism]	Protein code	Sequence coverage %	MASCOT score	No. matched peptides
A43	Aldehyde dismutase [Cupriavidus sp. SK-3]	gi 917041957	26	578	22
A41	Extracellular ligand-binding receptor [Methylobacterium oryzae CBMB20]	gi 689277923	39	334	8
	ABC transporter substrate-binding protein [bacteria]	gi 759635525	33	322	9
	Aldehyde dismutase [Cupriavidus sp. SK-3]	gi 917041957	26	523	16
B60	Methanol dehydrogenase (large subunit) [bacteria]	gi 759669109	36	972	25
B43	Phosphoglycerate kinase [Methylobacterium]	gi 518935653	21	236	7
	Isocitrate dehydrogenase [bacteria]	gi 759662868	45	839	21
	Methanol dehydrogenase (large subunit) [bacteria]	gi 759669109	4	62	2
	Formaldehyde dehydrogenase, glutathione-independent [Paraburkholderia caribensis]	gi 1016073698	5	120	4
B36	Type I glyceraldehyde-3-phosphate dehydrogenase [bacteria]	gi 501274167	33	602	13

Experimental conditions are specified in Materials and methods.

Figure 3. Degradation of formaldehyde and production of formic acid and methanol by resting cells of E. coli transformed with the FD1 formaldehyde dismutase gene and decrease in viable cells of the recombinant E. coli. The reaction mixture contained 1 OD-unit/mL of the cells. The solid lines, the recombinant E. coli with pRham-FDM; the dashed line, the recombinant E. coli with pRham (no insert); Closed circles, formaldehyde; closed triangles, formic acid; closed squares, methanol; open circles, viable cells of the recombinant E. coli in the buffer with 0.33 M formaldehyde; open triangles viable cells of the recombinant E. coli in the buffer without formaldehyde. Experimental conditions are specified in Materials and methods. All experiments were performed in triplicates, and the mean ± standard deviation is shown.

Figure 4. SDS-PAGE of protein extracts and precipitate fractions of E. coli transformed with the formaldehyde dismutase gene of FD1. For all samples, 10 μg of total protein were loaded. Lanes 1–4, the recombinant E. coli with pRham-FDM; lanes 5 and 6, the recombinant E. coli with pRham (no insert); lanes 1, 3, and 5, cell-free protein extracts; lanes 2, 4, and 6, precipitate fractions; lanes 1 and 2, no rhamnose induction; lanes 3, 4, 5, and 6, rhamnose induction. A triangle indicates a specific band. Experimental conditions are specified in Materials and methods.

Figure 5 Amino acid alignment of the formaldehyde dismutase of FD1 with (putative) enzymes related to formaldehyde dismutase. The sequences used for the alignment are as follows: MspFD1-FDM, formaldehyde dismutase of Methylobacterium sp. FD1 (accession No. LS269930) in class Alphaproteobacteria; CspSK3-ADM, putative aldehyde dismutase of Cupriavidus sp. SK-3 (WP_051648669) in class Betaproteobacteria; Tbo-ADM, putative aldehyde dismutase of T. bouteillei (WP_038092476) in class Cyanobacteria; MspGXS13-ADM, putative aldehyde dismutase of Methylobacterium sp. GXS13 (WP_058191240) in class Alphaproteobacteria; Pbo-FDH, putative glutathione-independent formaldehyde dehydrogenase of P. borealis (WP_076349806) in class Planctomycetia; Rsp-ADM, putative aldehyde dismutase of Rhodococcus spp. (WP_003943355) in class Actinobacteria; SspTu6071-ADM, putative aldehyde dismutase of Streptomyces sp. Tu6071 (WP_043257889) in class Actinobacteria; Fsp-ADM, putative aldehyde dismutase of Flavobacteriaceae (WP_027374817) in class Flavobacteriia; PpuF61-FDM, formaldehyde dismutase of P. putida F61 (L25862) of Gammaproteobacteria; PpuC83-FDH, glutathione-independent formaldehyde dehydrogenase of P. putida C83 (D21201) of Gammaproteobacteria. Identical residues in all of the (putative) enzymes are indicated by black highlight. Asterisks, the NAD(H)-binding residues; open circles, catalytic zinc-binding residues; closed circles, structural zinc-binding residues of the glutathione-independent formaldehyde dehydrogenase of P. putida C83 [2].

Figure 6. Degradation of high concentrations of formaldehyde by resting and lyophilized cells of E. coli transformed with the formaldehyde dismutase gene of FD1. (A) Resting cells. The reaction mixture contained 1 OD-unit/mL (circles) and 2 OD-units/mL (triangles) of the cells. (B) Lyophilized cells. The reaction mixture contained 1 OD-unit/mL (circles), 2 OD-units/mL (triangles), 5 OD-units/mL (squares), and 10 OD-units/mL (diamonds) of the cells. Experimental conditions are specified in Materials and methods. All experiments were performed in triplicates, and the mean ± standard deviation is shown.

Tanaka N, Kusakabe Y, Ito K, et al. Crystal structure of formaldehyde dehydrogenase from Pseudomonas putida: the structural origin of the tightly bound cofactor in nicotinoprotein dehydrogenases. J Mol Biol. 2002;324:519–533.

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