An enzyme cascade for competitive & sustainable production of glucaric acid
Dr Haibin Chen and Dr Thomas Daussmann of Enzymaster introduce a novel enzymatic process to produce a top value-added sugar acid
Figure 1- Enzyme cascade process to convert D-glucose to D-glucaric acid
Biobased chemicals are derived from renewable feedstocks( primarily C 5 and C 6 sugars), whereas petrochemicals and coal-based chemicals rely on nonrenewable resources. The current chemical industry is still largely built around processing non-renewables, for example by converting C 2
~ C 6 hydrocarbons into commodities, fine chemicals, polymers and pharmaceuticals, achieving high atomefficiency via advanced catalysts and continuous processes.
Despite biobased chemicals being hailed as the future of the industry, renewable feedstock processing remains limited. Existing petrochemical infrastructure cannot easily convert
C 5 and C 6 sugars into high-value chemicals, and current reliance on fermentation often leads to poor atomefficiency. Much glucose is diverted to cell mass and unmarketable non-target metabolites instead of target products.
Carboxylic acids, especially chiral species, are critical chemical building blocks. Since non-renewable hydrocarbons are poor starting materials for these compounds, most carboxylic acids( e. g. L-lactic acid, succinic acid, L-malic acid, L-tartaric acid, 2-ketoglutaric acid, citric acid, isocitric acid) are derived from renewable feedstocks via fermentation.
However, a fermentation process sets a high-cost base due to general poor atom-efficiency of converting feedstocks and non-straightforward downstream processing( DSP) for product isolation from the fermentation broth, which in several cases hinders their large-scale application as commodities.
Enzyme cascade to GA
Glucaric acid( GA), a building block for industry and human health, has been ranked a top value-added chemical from biomass since 2004.1 Although GA can theoretically be produced by oxidising the C 1 and C 6 carbons of glucose, commercial-scale production via chemical or traditional biological routes remains challenging.
Over the past two decades, nitric acid oxidation of glucose has been the most practical chemical method( supplying GA as a high-value dietary supplement), while cell factory routes( e. g. metabolically engineered E. coli or yeast) start with glycolysis pathway to generate myo-inositol, which is converted to D-glucuronic acid via myo-inositol oxygenase( MIOX) and then to GA via uronate dehydrogenase( UDH). 2
Unfortunately, the titre of GA from these biotransformations is too low for commercial viability. A direct cell-free enzymatic cascade process would circumvent the complex fermentation and DSP challenges. Here, we report a novel AI-guided enzyme engineering-
enabled cascade process for efficient glucose-to-GA conversion, comprising three enzymatic steps( Figure 1): 1. Oxidation of glucose to gluconic acid( a commercially mature step using glucose oxidase)
2. Oxidation of gluconic acid to L-guluronic acid via engineered galactose oxidase( GOase)
3. Oxidation of L-guluronic acid to GA via engineered UDH
All steps use molecular oxygen as the oxidant, with water as the sole by-product, ensuring environmental sustainability. A key challenge was that gluconic acid is not a native substrate for GOase, nor is L-guluronic acid for UDH, making it very challenging to discover starting variants from wild-type enzymes.
Enzyme engineering & process development
In fact, no wild-type GOase has been reported to exhibit oxidising activity towards C 6 carbon of glucose or gluconic acid. In the enzyme discovery phase, a novel GOase backbone was firstly designed via our generative protein AI model as a starting point for further engineering.
This de novo GOase backbone showed low activity in selectively oxidising C 6 carbon of gluconic acid. It was subjected to seven rounds of directed evolution to enhance its catalytic activity and
42 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981