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

Highlights

• Cell-free enzymatic production of glucaric acid (GlucA) from glucose-1-phosphate.

• 20.2% molar yield of GlucA with a titre of 8.1 mM.

• Cofactor regeneration mitigates the supply of expensive NAD⁺.

• Pathway enzymes were immobilised onto zeolite and reused multiple times.

• Uronate dehydrogenase generates the GlucA lactone and not the open acid.

Abstract

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 myo-inositol. 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-1-phosphate with a titre of 14.1 ± 0.9 mM (3.0 ± 0.2 g l⁻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 g l⁻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/l for free enzymes and 0.170 ± 0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.

Introduction

Glucaric acid (GlucA) is a glucose-derived emergent platform chemical with the potential for applications such as a biodegradable and biocompatible chelating agent for the degradation of organic contaminants (Subramanian and Madras, 2016), removal of heavy metals in soil (Fischer and Bipp, 2002), calcium sequestration for a phosphate-free detergent component (Abbadi et al., 1999), as an adhesive (Morton and Kiely, 2000), an anti-corrosive (Koefod, 2010), anti-plasticiser and plastic-strengthener (Lu and Ford, 2018). It is a starting material for biodegradable polymers/poly(amide)s, for example, hydroxylated nylon (Chiellini et al., 1997; Kiely et al., 1994) and serves as a renewable precursor for petroleum-derived adipic acid in nylon 6.6 (Boussie et al., 2016). In addition, GlucA may be employed as a cancer-preventive agent (Hanausek et al., 2003) or as preventive agent for diarrhoea that often arises from treatments with anticancer drugs such as CPT-11 (Fittkau et al., 2004).

Increasing environmental concerns and resource shortages have demanded the investment of ‘greener’ synthetic routes for biochemicals and biocommodities. Amongst these, GlucA has become an attractive target for the biobased industry. Accordingly, the microbial production of biobased GlucA has been attempted repeatedly since fermentation-based manufacturing is considered to be cost-effective and environmentally friendly (Keasling et al., 2012; Pellis et al., 2018). Using in vivo metabolic engineering, the maximum GlucA titre and molar yields obtained from glucose as substrate was 2.5 g l⁻¹ and 25%, respectively (Moon et al., 2010) (Table 1). The reasons for these low titres and yields are i) high GlucA concentrations lower the cellular pH which is toxic to most microbes, ii) metabolite competition, i.e. in vivo metabolic reactions compete with the introduced pathway, iii) insufficient control over optimal enzyme ratios and iv) limitation of product export via cell membrane (Gupta et al., 2016; Moon et al., 2009, 2010; Averesch et al., 2018). A range of solutions have been proposed to overcome some of these limitations, such as using the more acid-tolerant yeast such as Saccharomyces cerevisiae (Chen et al., 2018; Gupta et al., 2016), the use of synthetic scaffolds and fusion proteins to control enzyme ratios and enzyme proximity (Liu et al., 2016; Moon et al., 2010), enzyme engineering (Shiue and Prather, 2014; Zheng et al., 2018), metabolic engineering (Qu et al., 2018; Raman et al., 2014; Reizman et al., 2015) or supplementation of the GlucA pre-precursor myo-inositol (Chen et al., 2018; Gupta et al., 2016; Liu et al., 2016; Shiue and Prather, 2014) (Table 1). However, due to the high genetic and metabolic complexity of microorganisms, a major improvement of microbial GlucA production has been lacking until now.

An alternative to the cell-based approach is cell-free biocatalysis (or in vitro biocatalysis), which allows decoupling of the production pathway from the cellular growth and optimisation of enzyme pathways by freely mixing enzymes until ideal ratios and process conditions have been identified (Petroll et al., 2019a; Sheldon and Brady, 2018; Sperl and Sieber, 2018). Accordingly, cell-free biocatalysis offers greater engineering flexibility and system control than the in vivo approach and is not restrained by product export across the cell membrane. Note that during the revision process of this manuscript, another cell-free GlucA pathway has been published, which is operated at moderate temperatures and yields 35 mM GlucA from sucrose (75% molar yield) after 70 h incubation (Su et al., 2019). Nevertheless, high enzyme and cofactor costs are still limiting the use of cell-free biocatalysis in industrial processes.

Here, we demonstrate a comprehensive framework to overcome limitations attributed to cell-free biocatalysis by i) employing a combination of thermostable and non-thermostable enzymes to exploit the highest system stability possible, ii) incorporating a cofactor regeneration system and iii) immobilising the pathway onto an inexpensive silica-based matrix for recycling. We describe the use of synthetic biology for the construction of the first immobilised cell-free GlucA pathway with higher productivities than previously reported.