One pot synthesis of l-xylose from formaldehyde with an improved benzoylformate decarboxylase

Abstract

Formaldehyde (FALD), which can be derived from CO₂, is a promising C1 feedstock for enzymatic synthesis of valuable chemicals, like sugars, starch, acetyl-CoA, and lactic acid. Rare sugars, like L-xylose, have great potential for pharmaceutical applications, but their production requires precise control of stereocenters, a challenge that can be addressed by enzymatic catalysis. To date, no enzymatic route to produce L-xylose from only FALD has been reported. In this report, thermal resistance of a highly active and specific glycolaldehyde (GALD) producing benzoylformate decarboxylase variant from Polynucleobacter necessarius (PnBFD-M1) was improved via rational design, yielding PnBFD-M2 (PnBFD-M1-K188I/Q192I/A282P). PnBFD-M2 exhibits improved thermal resistance (T_m +11.2 °C; t_1/2 at 37 °C increased from 44 min to 2247 min) and moderately enhanced initial GALD production rate (from 302.9 μmol per (min g protein) to 350.3 μmol per (min g protein)). Analysis of the surface hydrophilicity and molecular dynamics simulations indicate that the improved thermal resistance is based on hydrophobic substitutions that stabilize the contact of two helices and increased rigidity. The small increase of activity seems to be caused by the reduced flexibility of loops located near the active site. The engineered PnBFD-M2 was successfully coupled with a fructose-6-phosphate aldolase variant (FSA-A129T/A165G) to achieve the first one-pot enzymatic synthesis of L-xylose from FALD as sole feedstock, reaching a titer of 2.36 g L⁻¹ and a yield of 78% (outperforming PnBFD-M1: 1.76 g L⁻¹ and 59%). PnBFD-M2 is one of the most efficient GALD from FALD producing enzymes known to date: almost no side products are formed (<1% 1,3-dihydroxyacetone is produced) and PnBFD-M2 has higher thermal resistance and higher activity compared to GALS-F397Y/C398M (t_1/2 at 37 °C is <120 min). The presented L-xylose pathway is an interesting biocatalytic route to produce L-xylose directly from FALD showing the potential of C1 valorization.