A novel framework for the cell-free enzymatic production of glucaric acid

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myoinositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-l-phosphate with a titre of 14.1 +/- 0.9 mM (3.0 +/- 0.2 gl(-1)) and a molar yield of 35.2 +/- 2.3% using non-immobilised enzymes, and a titre of 8.1 +/- 0.2 mM (1.70 +/- 0.04 gl(-1)) and a molar yield of 20.2 +/- 0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 +/- 0.02 g/h/1 for free enzymes and 0.170 +/- 0.004 g/h/1 for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.