A novel framework for the cell-free enzymatic production of glucaric acid
Graphical abstract
Cell-free biocatalysis based on an immobilised synthetic enzyme pathway to produce glucaric acid in an economic and environmental-friendly process.
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.
Section snippets
Materials and methods
Plasmid construction, enzyme assays and standard methods are included in the Supplementary Information S1. All enzymes, their source and kinetic characteristics are listed in Table S1. All primers used in this study are listed in Table S2, and all plasmids, enzyme constructions used in this study, position of the linker fusion are presented in Table S3.
Pathway design and rational enzyme selection
The pathway was designed with G1P as starting material in the proof of concept experiments (Fig. 1A). This substrate can be derived by enzymatic phosphorolysis from various renewable resources such as cellulose, starch and sucrose without consumption of the expensive coenzyme adenosine 5′-triphosphate (ATP) (Fujisawa et al., 2017; Wu et al., 2017; You et al., 2017; Zhong et al., 2017). Thus, G1P serves as an entry point for ATP-free GlucA production from different renewable resources. The
Conclusions
This work demonstrates a proof of concept for the cell-free production of GlucA from either free or immobilised enzymes. To overcome limitations of in vivo and cell-free biocatalysis, a novel framework has been developed that includes i) the use of thermostable enzymes where possible, ii) enzyme immobilisation and recycling (for five out of the six enzymes) and iii) cofactor regeneration. These experiments constitute the first demonstration of an immobilised multi-enzyme pathway for the in vitro
Declaration of competing interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgements
KP was supported by an international Macquarie University Research Excellence Scholarship (iMQRES). AC is supported by a Cancer Institute New South Wales Early Career Fellowship (Project Number: ECF171114). We thank Atul Bhatnagara and Matthew J. McKay from the Australian Proteome Analysis Facility (Macquarie University, Sydney, Australia) for their support in performing LC-MS experiments and Nicole Cordina from the Macquarie University NMR facility from the Department of Molecular Sciences for
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