Biosynthesis of D‐xylulose 5‐phosphate from D‐xylose and polyphosphate through a minimized two‐enzyme cascade

Abstract

Sugar phosphates cannot be produced easily by microbial fermentation because negatively-charged compounds cannot be secreted across intact cell membrane. D-xylulose 5-phosphate (Xu5P), a very expensive sugar phosphate, was synthesized from D-xylose and polyphosphate catalyzed by enzyme cascades in one pot. The synthetic enzymatic pathway comprised of xylose isomerase and xylulokinase was designed to produce Xu5P, along with a third enzyme, polyphosphate kinase, responsible for in site ATP regeneration. Due to the promiscuous activity of the ATP-based xylulokinase from a hyperthermophilic bacterium Thermotoga maritima on polyphosphate, the number of enzymes in the pathway was minimized to two without polyphosphate kinase. The reactions catalyzed by the two-enzyme and three-enzyme pathways were compared for Xu5P production, and the reaction conditions were optimized by examining effects of reaction temperature, enzyme ratio and substrate concentration. The optimized two-enzyme system produced 32 mM Xu5P from 50 mM xylose and polyphosphate after 36 h at 45°C. Biosynthesis of less costly Xu5P from D-xylose and polyphosphate could be highly feasible via this minimized two-enzyme pathway.

Simultaneous biosynthesis of (R)-acetoin and ethylene glycol from D-xylose through in vitro metabolic engineering

“…D -xylose has also been used as the building block for in vitro H 2 production with a yield of 96% ( Martín del Campo et al, 2013 ). D -xylulose 5-phosphate was also synthesized from D -xylose, achieving 64% conversion ( Kim and Zhang, 2016 ). Although optically pure ( R )-acetoin and EG was obtained from D -xylose by the cell-free biosystem, more intensive optimizations of the multi-enzymatic cascades in the ‘one-pot’ system were needed to improve product better.…”

“…In addition, enzyme-based biocatalysis, involving the necessary substrates and intermediates, has higher reaction selectivity under mild reaction conditions. So far, ‘one-pot’, in vitro biotransformations with balanced cofactors and ATP have been demonstrated for bio-hydrogen ( Martín del Campo et al, 2013 ), D -xylulose 5-phosphate ( Kim and Zhang, 2016 ), fructose 1,6-diphosphate ( Wang et al, 2017 ), and bio-food ( Qi et al, 2014 , You et al, 2013 ) production. Here, a coenzyme-balanced pathway for the in vitro , direct, simultaneous conversion of D -xylose to optically pure ( R )-acetoin and EG was constructed and optimized by assembling seven biocatalytic steps, without ATP requirements.…”

Stoichiometric Regeneration of ATP by A NAD(P)/CoA‐free and Phosphate‐balanced In Vitro Synthetic Enzymatic Biosystem

“…[8] To avoid the use of costly phosphate-containing energy compounds and the accumulation of inorganic phosphate, ATP regeneration can be achieved by in vitro synthetic biosystems (ivSBs) which consist of multiple enzymes in the form of either purified enzymes, crude cell extracts, resting cells, or lyophilized recombinant cells [9] for the realization of complex biological reactions in one vessel. [10] ivSB has a number of advantages over its in vivo counterpart (i. e. fermentation), including greater engineering flexibility, [11] easier process control and optimization, [1b,12] higher tolerance to cell-toxic compounds, [11,13] higher product yields, [1d,14] more convenient product separation, [15] and has been used for the phosphate-free regeneration of ATP in many cases. For example, Swartz and coworkers have designed ivSBs using cell extracts to utilize inexpensive phosphate-free compounds such as pyruvate and glucose for ATP regeneration.…”

Water Splitting for High‐Yield Hydrogen Production Energized by Biomass Xylooligosaccharides Catalyzed by an Enzyme Cocktail

“…The theory of enzyme promiscuity suggests that some enzymes may have side reactions that are often ignored even though enzymes are usually regarded as highly selective catalysts. For example, ATP‐dependent xylulokinase isolated from the hyperthermophilic bacterium Thermotoga maritima utilizes polyphosphate as a phosphate donor to generate xylulose 5‐phosphate . The use of promiscuous enzyme activities or the discovery of novel enzyme activities may help the construction of novel pathways to achieve desired reactions.…”