Levoglucosan (LG), an anhydrosugar produced from glucan pyrolysis, is abundant in nature and a byproduct of biomass burning and carbohydrate processing. Its annual production is estimated at 90 million metric tons, with LG comprising over 90%. Previous research identified *Bacillus smithii* S-2701M as an LG-utilizing thermophile, suggesting a metabolic pathway from LG to glucose initiated by LG dehydrogenase (LGDH). Two types of microbial LG metabolism are known: a eukaryotic pathway involving LG kinase and a bacterial pathway utilizing NAD-dependent LGDH. However, the bacterial pathway beyond LGDH remained unclear. This study aimed to elucidate the complete LG metabolic pathway in *B. smithii* S-2701M using recombinant proteins, focusing on the enzymes suspected to be involved in the conversion of LG to glucose. Understanding this pathway is crucial for developing effective bioprocesses for LG utilization and for contributing to sustainable biomass utilization strategies. The high abundance of LG makes its efficient conversion into usable forms a significant goal for various applications. Previous work had shown that *B. smithii* S-2701M possesses a thermophilic LGDH and exhibits glucose-forming activity. This study built upon these findings to detail the entire metabolic cascade.

Existing literature describes two main microbial metabolic pathways for levoglucosan (LG) utilization. Eukaryotic organisms, such as fungi and yeasts, employ an LG kinase to phosphorylate LG, cleaving the 1,6-linkage and producing glucose-6-phosphate, which is further metabolized through glycolysis. In contrast, bacterial metabolism involves NAD-dependent LG dehydrogenase (LGDH), initially characterized in *Arthrobacter* sp. 1-552, and later in *Pseudoarthrobacter phenanthrenivorans*. The crystal structure of LGDH has been determined, confirming its C3-specific oxidation of LG. However, the biochemical and genetic mechanisms following LG oxidation in bacteria remained largely unexplored. This study aimed to bridge this knowledge gap and detail the subsequent reactions to complete the bacterial LG to glucose pathway.

The researchers identified three genes (*lgdB1*, *lgdB2*, and *lgdC*) in the *B. smithii* S-2701M genome near the LGDH gene (*lgdA*). These four genes were expressed in *Escherichia coli* as GST-fusion proteins, purified, and their functions analyzed. Thin layer chromatography (TLC) analysis was used to track substrate conversion at each enzymatic step. The enzymatic reactions were conducted with various combinations of the four recombinant proteins (LgdA, LgdB1, LgdB2, and LgdC), using LG or 3-keto-LG as substrates, along with cofactors such as NAD+, Mn2+, and 2-mercaptoethanol (2-ME). The activity of LGDH (LgdA) was measured spectrophotometrically by monitoring the change in absorbance at 340 nm due to NADH. The structures of intermediate compounds were determined using high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The specific activity of LgdA was measured with and without additional Lgd proteins to assess potential synergistic effects or product inhibition. The influence of Mn2+ on the reactions was also investigated. The complete process involved gene amplification from the S-2701M genome using PCR and primers (listed in supplementary information), cloning into expression vectors (pGEX-6P-1), protein expression and purification through GST-affinity chromatography and protease cleavage, and detailed biochemical assays using TLC, spectroscopy, mass spectrometry, and NMR to characterize the enzymatic steps and reaction products. The enzymatic reactions were meticulously controlled to optimize conditions for conversion and analysis, including varying substrate concentrations, incubation times, and the presence or absence of cofactors.

The study identified a four-enzyme pathway for LG conversion to glucose: 1) LgdA (LGDH) catalyzes the C3-oxidation of LG to produce 3-keto-LG. 2) LgdB1 catalyzes the β-elimination of 3-keto-LG, producing 2-hydroxy-3-keto-D-glucal. 3) LgdB2 catalyzes the hydration of 2-hydroxy-3-keto-D-glucal to 3-keto-D-glucose (this compound was unstable and difficult to isolate directly). 4) LgdC (glucose-3-dehydrogenase) reduces 3-keto-D-glucose to glucose. TLC analysis confirmed the complete conversion of LG to glucose when all four enzymes were present. Individual enzymatic steps were verified using appropriate substrates and monitoring product formation via TLC. The addition of LgdB1 or LgdB2 increased LgdA's specific activity, suggesting that the conversion of 3-keto-LG alleviates product inhibition of LGDH. LgdB2 exhibited bifunctional activity, able to catalyze both β-elimination and hydration, albeit with lower affinity for β-elimination compared to LgdB1. Structural identification of the reaction products (intermediate and final) was achieved through mass spectrometry and NMR spectroscopy, confirming the proposed reaction pathway. The complete metabolic pathway involves oxidation (LgdA), elimination (LgdB1), hydration (LgdB2), and reduction (LgdC). The study showed that the Mn2+ ion was required for the hydration step catalyzed by LgdB2. The researchers found that the reaction of LgdA (LGDH) is the rate-limiting step in the pathway and the presence of LgdB1 and LgdB2 helped alleviate the product inhibition of LGDH. LgdB1 and LgdB2 were suggested to be novel enzymes with unique catalytic capabilities. The proposed pathway shares similarities with the mechanism of enzymes in glycoside hydrolase family 4 (GH4).

The identified four-enzyme pathway for LG conversion to glucose in *B. smithii* S-2701M represents a novel metabolic route, distinct from the previously known fungal LG kinase pathway. The sequential reactions of oxidation, β-elimination, hydration, and reduction, bear resemblance to the mechanism of GH4 enzymes, but with the key difference of involving four separate enzymes rather than a single polypeptide. The characterization of three novel enzymes (LgdB1, LgdB2, and LgdC) contributes significantly to our understanding of sugar metabolism. The identification of this pathway opens new avenues for efficient LG utilization. The enzyme-catalyzed conversion of LG to glucose has implications for biotechnological applications. The pathway could be integrated into recombinant systems for biofuel production or the synthesis of valuable compounds from renewable lignocellulosic biomass. The relatively simple and efficient conversion makes *B. smithii* S-2701M a potentially valuable host for bioproduction of useful compounds from LG. Further research could focus on optimizing this pathway for industrial applications and exploring its potential in various biotechnological processes.

This study completely elucidated the metabolic pathway for the conversion of levoglucosan (LG) to glucose in *Bacillus smithii* S-2701M, a pathway involving four enzymes, three of which are novel. The sequential reactions of oxidation, β-elimination, hydration, and reduction are similar to the mechanism of glycoside hydrolase family 4 enzymes but use four separate enzymes rather than a single polypeptide. This discovery provides valuable insights into microbial sugar metabolism and opens exciting avenues for biotechnological applications in biofuel production and sustainable biomass utilization. Future research could focus on optimizing the expression and activity of these enzymes for industrial-scale applications, exploring potential applications in various biotechnological processes, and using *B. smithii* S-2701M as a chassis for bioproduction from LG.

The study primarily used in vitro assays with purified recombinant proteins. The in vivo rates and regulation of this pathway within *B. smithii* S-2701M remain to be fully characterized. The instability of the 3-keto-D-glucose intermediate hampered its direct structural characterization; thus, the researchers used an indirect method to determine its structure. Furthermore, the research focused on a specific bacterial strain, and the pathway might vary in other LG-utilizing bacteria. The in vitro conditions may not completely mimic the natural cellular environment in *B. smithii* S-2701M, potentially affecting the exact rates and regulation of each enzymatic step.