Formaldehyde (FALD) is a crucial feedstock in the production of various everyday products because of its strong reactivity and versatility as a C1 building block, making it highly valued in numerous industries [1], [2]. It could be produced from CO [3], CO2 [4], HCOOH [5], [6] and CH4 [7] through biological and chemical means. Under atmospheric conditions, FALD exists as a gas, and its aqueous solution proves to be unstable [8]. Furthermore, converting FALD into high value-added chemicals remains a persistent challenge.

In recent years, biocatalysis has gained prominence as a green and sustainable biotechnology, playing a crucial role in the transition towards a more environmentally responsible global society. More and more high value-added chemicals are generated from simple one-carbon compounds, which has been emerging as the spotlight of biocatalysis [8]. A computationally designed enzyme, formolase (FLS), was emerged in 2015. In the report, a thiamine diphosphate-dependent benzaldehyde lyase was redesigned and synthesized through protein engineering techniques [9]. The reaction catalyzed by FLS selectively produces 1,3-dihydroxyacetone (DHA) from FALD. Then, FALD is commonly employed as a prebiotic compound in the synthesis of sugar-like substances [10], such as L-erythrulose [11] and ethylene glycol [12]. A synthetic acetyl-CoA (SACA) pathway was designed and constructed by repurposing glycolaldehyde synthase and acetyl-phosphate synthase, and a carbon yield of approximately 50% was achieved [13]. The pathway opens up possibilities for the future production of acetyl-CoA-derived chemicals using C1 resources.

Acetoin, also recognized as 3-hydroxybutanone, is primarily utilized in the food industry as a flavor-enhancing food additive, specifically for butter and cheese products [14], [15]. It is widely applied in medicine, agriculture and the chemical industry. Furthermore, it has been listed as one of the 30 priority platform compounds for development by the US Department of Energy [16]. Currently, most commercial acetoin is produced through chemical synthesis. However, the chemical method is environmentally harmful with low yield and poor safety in the food and cosmetic industries. Methods of industrial-scale acetoin production were developed through biotechnology, such as microbial fermentation and whole-cell biocatalysis [17]. Nevertheless, these methods are still unsatisfactory for economical manufacturing processes. Furthermore, in vitro biosynthesis has been gradually emerging as a potential approach for acetoin production [18]. It would be better to synthesize acetoin from a C1 source that comes from a variety of sources and is available at low prices, which could potentially lead to a reduction in acetoin manufacturing costs compared to other substrates such as 2,3-butanediol and pyruvate. Additionally, this process would help alleviate resource shortages through the utilization of renewable resources.

In this work, a cell-free multi-enzyme catalytic pathway for producing acetoin from FALD was designed and constructed which was composed of three scales including FALD utilization, glycolysis and acetoin synthesis. DHA was generated from FALD and was then converted into glycerone phosphate, catalyzed by dihydroxyacetone kinase (DAK). Following this, glycerone phosphate entered the glycolytic pathway, where it was converted into pyruvate. Ultimately, pyruvate underwent a two-step decarboxylation process, resulting in the formation of acetoin. By selecting enzymes in the pathway and combining substrates and necessary coenzymes to construct an in vitro multi-enzyme catalytic system, FALD was successfully transformed into acetoin.