Tailoring CO2 fixation through glucose and acetone as co-substrates for mevalonate biosynthesis with high carbon atom economy

Mevalonate (MVA) is a pivotal platform intermediate for the biosynthesis of terpenoids and other high-value isoprenoids with broad applications in pharmaceuticals, chemicals, and materials. However, MVA biosynthesis through the glycolytic pathway requires three molecules of acetyl-CoA (Ac-CoA), accompanied by the release of three molecules of CO 2 , resulting in limited carbon atom efficiency with a theoretical maximum of only 66.6%. Here, we developed a previously unreported glucose–acetone co-substrate strategy in Escherichia coli to improve the theoretical carbon atom yield of MVA biosynthesis by coupling acetone assimilation with CO 2 incorporation. We first selected and identified a highly active acetoacetate-CoA synthetase, and subsequently established a glucose and acetone co-substrate pathway in vivo. Further improvement in pathway efficiency was achieved through integrated metabolic engineering strategies, including suppression of competing acetoacetate-consuming pathways, reinforcement of CO 2 hydration via carbonic anhydrase overexpression and cell-surface display, and modulation of intracellular ATP supply to meet the energetic demands of acetone assimilation. 13 C isotopic tracing provided direct evidence that CO 2 was incorporated into the MVA biosynthetic pathway. Ultimately, fed-batch fermentation in a 5 L bioreactor achieved an MVA titer of 2130 mg/L. Overall, this work establishes a novel co-substrate framework for MVA biosynthesis that integrates acetone assimilation with CO 2 fixation, providing an interesting idea for developing a low-carbon and sustainable biomanufacturing strategy.