B. licheniformis is widely used in industrial production due to its efficient secretion system and broad metabolic capacity (Schallmey et al., 2004). In recent years, B. licheniformis has been recognized as a potential strain for industrial production of 2, 3-butanediol (2,3-BD) and is generally recognized as a safe (GRAS) by the U.S. Food and Drug Administration (FDA) (Xu et al., 2025a, Nakamura et al., 2018). As an important biobased four-carbon platform compound, 2,3-BD is widely used in food, chemical, aerospace fuel and other fields (Karayannis et al., 2025, Ji et al., 2011, Xu et al., 2025b). Its combustion value is comparable to methanol and ethanol, which makes it a fuel additive with great potential (Jo et al., 2024, Amraoui et al., 2022). Among its three isomers, the optically active D-(-)-2,3-BD and L-(-)-2,3-BD can be used as synthetic precursors of chiral compounds, while meso-2,3-BD can be used to produce renewable polyester and enantiomerically pure halogenated alcohols (Gao et al., 2013, Li et al., 2014). The market demand for 2,3-BD is rising at an annual growth rate of 4–7 %, and it is expected that by 2027, the global market value of 2,3-BD will reach 220 million US dollars (Xie et al., 2022). In addition to 2,3-BD, its synthetic intermediate acetoin also has a wide range of applications, such as natural additives in food and cosmetics, as well as vital intermediates in the synthesis of different chemicals. Therefore, enhancing the production capacity of acetoin and 2,3-BD is of great significance for reducing the dependence on limited petroleum resources (Lu et al., 2022, Narisetty et al., 2022, Wang et al., 2025), thereby promoting the development of green chemical industry.

In recent years, constructing engineered strains for high-titer production of 2,3-butanediol and acetoin glycol has become a hot research topic. It has been reported that LuLi et al. investigated the fermentation conditions of the Bacillus licheniformis mutant strain WX-02. They employed a three-stage stirring and batch feeding approach for fermentation, resulting in an accumulation of 2,3-BD reaching 110.04 g/L (Li et al., 2017). Additionally, an efficient gas-producing Escherichia coli strain was developed based on strategies to increase carbon flux and regulate NADH/NAD+ supply for producing 2,3-BD. After fermenting with sucrose as the carbon source for 24 h, strain IAM1183-LPBC achieved a 2,3-BD titer of 11.29 g/L (Lu et al., 2022). In batch-fed fermentation with co-expression of BDH and GAPDH in B. amyloliquefaciens, the concentration of 2,3-BD reached a maximum of 132.9 g/L at 45 h, with a productivity rate of 2.95 g/L·h (Bai et al., 2013).

Although B. licheniformis shows potential in the production of 2,3-BD and acetoin, there are still numerous challenges in its industrial application (He et al., 2025). First, in the microbial fermentation process, the efficiency of the metabolic pathway is low, resulting in an unsatisfactory yield and synthesis rate of 2,3-BD. In particular, the insufficient accumulation of acetoin restricts the further synthesis of 2,3-BD. Acetoin is converted by 2,3-BD dehydrogenase to produce 2,3-BD in a low oxygen environment (Celinska and Grajek, 2009) (Fig. 1), but the insufficient activity of its key enzymes and the accumulation of by-products during fermentation lead to a decrease in the synthesis rate of the target products (Maina et al., 2022, Pu et al., 2024, Shiloach et al., 2005). In B. licheniformis, the transcription of the acoABCL operon is regulated by the sigma 54-dependent transcriptional activator acoR (Thanh et al., 2010). In the presence of glucose, acoR expression is inhibited by metabolite control protein A (CcpA) (Ali et al., 2001). Since it has been reported that blocking acetoin catabolism facilitates 2,3-BD synthesis, we attempted to further increase the accumulation of 2,3-BD by regulating the production of acetoin. It was also found that the synthesis rate of 2,3-BD reached a maximum when acetoin and glucose were consumed rapidly, while the simultaneous consumption of pyruvate and glucose reduced the synthesis rate of 2,3-BD. This suggests that the reaction catalyzed by α-acetolactate synthase and/or α-acetolactate decarboxylase may be the limiting step. This bottleneck can be overcome by additionally optimizing the expression ratio of these enzymes in the 2,3-BD synthesis pathway (Boecker et al., 2021).

In this study, B. licheniformis MW03 with high production capacity of 2,3-BD and acetoin was screened and metabolically engineered by CRISPR-Cas9 gene editing technology to obtain a stable 2,3-BD high-yielding strain. First, whole-genome sequencing revealed mutations in key genes acoR (acetoin dehydrogenase regulatory factor) and budC (2,3-BD dehydrogenase). We then respectively knocked out these two key genes, and the acoR mutation increased acetoin production by 21.2 % (75.09 g/L) by inhibiting acetoin breakdown and may potentially regulate NADH/NAD+ balance, while the budC mutation blocked the acetoin reduction pathway and increased acetoin production by 49.2 % to 90.71 g/L. Moreover, the optical purity of D-(-)-2,3-BD was increased to 92.7 %. Gene complementation verified the functions of acoR and budC, after which synergistic optimization of carbon fluxes increased the production of 2,3-BD by 61.9 %. This study not only deepens our understanding of the metabolism of B. licheniformis, but also lays a foundation for the upgrading and industrial application of this organism.