Coding Synthetic Chemistry Strategies for Furan Valorization into Bacterial Designer Cells

Abstract Following a synthetic chemistry blueprint for the valorization of lignocellulosic platform chemicals, this study showcases a so far unprecedented approach to implement non‐natural enzyme modules in vivo. For the design of a novel functional whole cell tool, two purely abiotic transformations, a styrene monooxygenase‐catalyzed Achmatowicz rearrangement and an alcohol dehydrogenase‐mediated borrowing hydrogen redox isomerization, were incorporated into a recombinant bacterial host. Introducing this type of chemistry otherwise unknown in biosynthesis, the cellular factories were enabled to produce complex lactone building blocks in good yield from bio‐based furan substrates. This whole cell system streamlined the synthetic cascade, eliminated isolation and purification steps, and provided a high degree of stereoselectivity that has so far been elusive in the chemical methodology.


Construction of co-expression plasmids
The DNA fragment encoding Styrene monooxygenase was amplified from vector pET-28a-StyAB. The DNA fragment encoding alcohol dehydrogenase READH or LKADH was amplified from vector pET-15b-ReADH and pET-15b-LkADH, separately. The DNA fragments encoding formate dehydrogenase was amplified from pET-28a-FDH. The PCR products were purified, double digested with the corresponding restriction enzymes, and ligated into the corresponding cassette of pRFDuet-1 T or pET-15b (Table S1).
The ligation mixture was transformed into E. coli Top10 competent cells. Single colonies were selected on LB agar plate supplemented with kanamycin (50μg /ml) or ampicillin (50 μg/ml). The expression plasmid was identified by sequencing the ORF regions of the target DNA.

Co-expression of SMO and ADHs
Recombinant E. coli BL21(DE3)ΔnemA harboring a single or two vectors (as listed in Table 2) were cultivated overnight at 37 °C in Luria-Bertani (LB) medium containing kanamycin (50μg/ml) and ampicillin (50μ g/ml), or only kanamycin (50μg/ml). Then 1 ml of overnight culture was inoculated into 100 ml of LB containing kanamycin (50μg/ml) and ampicillin (50μg/ml), or only kanamycin (50μg/ml), and then incubated at 37 °C for 2 h. The expression was induced by the addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and the incubation was continued for 15 h at 20 °C with rotary shaking at 200 rpm. The cells were harvested by centrifugation at 5000 rpm, and washed twice with potassium phosphate buffer (0.1 M, pH 7.0).

Achmatowicz rearrangement in vivo
The reaction was performed at 30 °C in 1 ml of potassium phosphate buffer (0.1 M, pH 7.0) containing fresh recombinant E. coli cells (0.1 g), the substrate 1a (1 mg) with shaking at 200 rpm for 7 h. The reaction was terminated by extraction with ethyl acetate (1:1, v/v). The samples were analyzed with chiral GC to determine the yield.

Redox isomerisation in vivo
The reaction was performed at 30 °C in 1 ml of potassium phosphate buffer (0.1 M, pH 7.0) containing fresh recombinant E. coli cells (0.1 g), the substrate pyranone (1 mg) with shaking at 200 rpm for 4 h. The reaction was terminated by extraction with ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The samples were analyzed with chiral GC to determine the yield and chiral HPLC to determine the enantiomeric excess.

Artificial pathway in vivo
The reaction was performed at 30 °C in 1 ml of potassium phosphate buffer (0.1 M, pH 7.0) containing fresh recombinant E. coli cells (0.1 g), the substrate (1 mg) and isopropanol (2ul) with shaking at 200 rpm for 20 h. The reaction was terminated by extraction with ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The samples were analyzed with chiral GC to determine the yield and chiral HPLC to determine the enantiomeric excess. The products were purified by silica gel column chromatography, eluted with cyclohexane/ethyl acetate (1: 1), and identified by NMR analysis. Reactions of 20 ml scale or multiple reactions were performed to accumulate enough products for the analysis when it is necessary.
The reaction was quenched with ice water (10 mL), extracted with diethyl ether (3 x 15 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, n-pentane: diethyl ether = 10:1) giving as colorless oil.