Construction of a cell-free enzymatic pathway for the production of lactic acid from spent coffee grounds

Microorganisms have been investigated extensively for their use in bio-based production of compounds for the chemical, pharmaceutical and food industry. New tools used in synthetic biology enable a fundamental rewiring of metabolic pathways within microbes. The engineering of cellular metabolisms aims to maximise production efficiencies for the sustainable, yet economically competitive manufacturing of chemicals from renewable biomass. Despite the extensive development in genetic tools, in vivo approaches face challenges such as interference with the host metabolism, restricted flexibility in metabolic design and limited controllability of pathway flux. Alternatively, cell-free biocatalysis has emerged as a promising technology, which is based on an almost unrestricted assembly of enzymes into synthetic and highly modular production pathways. The technology not only offers flexibility to select and combine virtually any enzyme, but also allows for rapid prototyping and testing of de novo assembled pathways. To date, cell-free biocatalysis has been mostly used for the production of chemicals from refined substrates such as glucose and other carbohydrates. Since the economic viability of biotechnological production processes depends heavily on substrate cost, the use of highly abundant and low-cost biomass such as waste products improves the cost-efficiency and the environmental benefit of cell-free biocatalysis. In this work a novel cell-free enzymatic pathway was constructed for the conversion of carbohydrates obtained from spent coffee grounds into lactic acid. Lactic acid is a versatile chemical with a wide range of different applications in medical, textile and food industries and has received most of its attention for the use in form of the biodegradable polymer polylactic acid. Spent coffee grounds are a highly abundant and industrially underutilised waste compound rich in carbohydrates, especially mannose. The catabolism of mannose in most microorganisms relies on the Embden-Meyerhof-Parnas pathway, which is high in enzyme and cofactor cost and therefore suboptimal for a synthetic cell-free approach. The new cell-free pathway described here is based on a non-phosphorylative, putative mannose metabolism from the thermophilic archaeon Thermoplasma acidophilum. Initial identification and characterisation of a thermostable mannonate dehydratase from this organism allowed the construction of a four-enzyme pathway for the conversion of mannose into lactic acid. All the enzymes in the pathway were derived from thermophilic organisms and were recombinantly-expressed, purified and assembled into one-pot reactions. Several reaction conditions (e.g. substrate, cofactor and enzyme concentrations) were studied and optimised towards a conversion yield of up to 71.5 %. In order to demonstrate the feasibility of renewable substrates for cell-free biocatalysis, mannose was obtained by dilute acid hydrolysis from spent coffee grounds and converted into lactic acid using the novel pathway. The conversion from spent coffee grounds into lactic acid was further analysed via high-performance liquid chromatography and showed similar yields as those obtained from pure mannose. Complete identification of reaction intermediates and analysis of pathway flux was possible in reactions containing 13C-labelled substrate via nuclear magnetic resonance spectroscopy. Identification of unspecific side reactions explained the loss of carbon and the conversion below 100% of theoretical yield. Thiswork demonstrates the power of cell-free pathway engineering by construction of an alternative and so far undescribed carbohydrate pathway to convert waste biomass into valuable bio-based chemicals in a sustainable way.