Abstract
The oxidative d-xylose pathway, i.e. Dahms pathway, can be utilised to produce from cheap biomass raw material useful chemical intermediates. In vitro metabolic pathways offer a fast way to study the rate-limiting steps and find the most suitable enzymes for each reaction. We have constructed here in vitro multi-enzyme cascades leading from d-xylose or d-xylonolactone to ethylene glycol, glycolic acid and lactic acid, and use simple spectrophotometric assays for the read-out of the efficiency of these pathways. Based on our earlier results, we focussed particularly on the less studied xylonolactone ring opening (hydrolysis) reaction. The bacterial Caulobacter crescentus lactonase (Cc XylC), was shown to be a metal-dependent enzyme clearly improving the formation of d-xylonic acid at pH range from 6 to 8. The following dehydration reaction by the ILVD/EDD family d-xylonate dehydratase is a rate-limiting step in the pathway, and an effort was made to screen for novel enolase family d-xylonate dehydratases, however, no suitable replacing enzymes were found for this reaction. Concerning the oxidation of glycolaldehyde to glycolic acid, several enzyme candidates were also tested. Both Escherichia coli aldehyde dehydrogenase (Ec AldA) and Azospirillum brasilense α-ketoglutarate semialdehyde dehydrogenase (Ab AraE) proved to be suitable enzymes for this reaction.
Highlights
The production of chemical building blocks and fuels from biomass is a promising option to replace the fossil raw material sources with renewable alternatives
For the in vitro metabolic pathway testing to convert d-xylose or d-xylonolactone to glycolate, l-lactate, or ethylene glycol, altogether 14 different enzymes were expressed in heterologous host E. coli or S. cerevisiae
The primary product of Cc XylB is xylonolactone, which is hydrolysed to linear d-xylonate form (Fig. 1). For this lactone ring opening reaction we were interested to study and characterise the lactonase Cc XylC, which is found in C. crescentus in the same operon as the Cc XylB dehydrogenase and crescentus d-xylonate dehydratase (Cc XylD) dehydratase enzymes (Fig. 1)
Summary
The production of chemical building blocks and fuels from biomass is a promising option to replace the fossil raw material sources with renewable alternatives. Biocatalysts, i.e. enzymes and microbes, offer a sustainable way to produce chemicals starting from lignocellulose-based sugars. Microbes have evolved to utilise both pentose and hexose sugars, and their multi-enzyme pathways can be applied for production of compounds found in natural metabolism. We have been interested to study utilisation of the pentose sugars d-xylose and l-arabinose, abundant especially in grasses, agricultural crops and hardwoods. In the Dahms pathway (Dahms 1974), the 2-keto-3-deoxy-xylonate is split by an aldolase to pyruvate and glycolaldehyde (see Fig. 1). These two pathways provide possibility for the biosynthesis of a variety of chemicals starting from pentose sugars. As shown by us and other groups, the Dahms pathway can be used, besides xylonic acid (Toivari et al 2012), for production of ethylene glycol (Liu et al 2013; Salusjärvi et al 2019), glycolic acid (Salusjärvi et al 2017, 2019), lactic acid (Penttilä et al 2014), and 1,4-butanediol (Liao and Yan 2011; Tai et al 2016)
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