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Engineering a short, aldolase-based pathway for (R)-1,3-butanediol production in Escherichia coli.

Microbial processes can produce a wide range of compounds; however, producing complex and long chain hydrocarbons remains a challenge. Aldol condensation offers a direct route to synthesize these challenging chemistries and can be catalyzed by microbes using aldolases. Deoxyribose-5-phosphate aldolase (DERA) condenses aldehydes and/or ketones to β-hydroxyaldehydes, which can be further converted to value-added chemicals such as a precursor to cholesterol-lowering drugs. Here, we implement a short, aldolase-based pathway in Escherichia coli to produce (R)-1,3-BDO from glucose, an essential component of pharmaceutical products and cosmetics. First, we expressed a three step heterologous pathway from pyruvate to produce 0.3 g/L of (R)-1,3-BDO with a yield of 11.2 mg/g of glucose in wild-type E. coli K12 MG1655. We used a systems metabolic engineering approach to improve (R)-1,3-BDO titer and yield by: 1) identifying and reducing major by-products: ethanol, acetoin, and 2,3-butanediol; 2) increasing pathway flux through DERA to reduce accumulation of toxic acetaldehyde. We then implemented a two-stage fermentation process to improve (R)-1,3-BDO titer by 8-fold to 2.4 g/L and yield by 5-fold to 56 mg/g of glucose (11% of maximum theoretical yield) in strain BD24, by controlling pH to 7 and higher dissolved oxygen level. Furthermore, this study highlights the potential of the aldolase chemistry to synthesize diverse products directly from renewable resources in microbes.

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