Formaldehyde is a known toxic compound, and functional formaldehyde detoxification is crucial for the survival of all living cells. Such detoxification systems are of particular importance for methylotrophic microorganisms that rely on formaldehyde as a central metabolite in their one-carbon metabolism. Understanding formaldehyde dissimilation pathways in non-methylotrophic industrial microorganisms is necessary for ongoing research aiming at engineering methylotrophy into their metabolism (synthetic methylotrophy). There is a variety of formaldehyde dissimilation pathways across microorganisms, often based on the activity of formaldehyde dehydrogenases. In this study, we investigated the role of the yycR gene of Bacillus subtilis putatively encoding a novel, uncharacterized zinc-type alcohol dehydrogenase-like protein. We showed that the B. subtilis Δ yycR mutant displayed a reduced formaldehyde tolerance level and confirmed the enzymatic activity of recombinantly produced and purified YycR as formaldehyde dehydrogenase in vitro . Biochemical analyses demonstrated that YycR activity is optimal at 40°C, with the highest measured activity at pH 9.5, formaldehyde is the preferred substrate, and the kinetic constants are K _m of 0.19 ± 0.05 mM and V _max of 2.24 ± 0.05 nmol min ⁻¹ . Altogether, we showed that YycR is a novel formaldehyde dehydrogenase with a role in formaldehyde detoxification in B. subtilis , providing valuable insights for future research on synthetic methylotrophy in this organism. IMPORTANCE: Formaldehyde is a key metabolite in methanol assimilation for many methylotrophic microorganisms, and at the same time, it is toxic to all living cells, which means its intracellular concentrations must be tightly controlled. An in-depth understanding of methanol detoxification systems in industrially relevant microorganisms is a prerequisite for the introduction of methanol utilization pathways into their metabolism (synthetic methylotrophy). Bacillus subtilis , an industrial workhorse conventionally used for the production of enzymes, is known to possess two formaldehyde detoxification pathways. Here, we identify a novel formaldehyde dehydrogenase in this bacterium as a path towards creating innovative prospect strategies for strain engineering towards synthetic methylotrophy.

The β-blocker (S)-esmolol, has been synthesized in 97% enantiomeric excess and 26% total yield in a four-step synthesis, with a transesterification step of the racemic chlorohydrin methyl 3-(4-(3-chloro-2-hydroxypropoxy)phenyl)propanoate, catalysed by lipase B from Candida antarctica from Syncozymes, Shanghai, China. The β-blocker (S)-penbutolol, has been synthesized in 99% enantiomeric excess and in 22% total yield. The transesterification step of the racemic chlorohydrin 1-chloro-3-(2-cyclopentylphenoxy)propan-2-ol was catalyzed by the same lipase as used for the esmolol building block. We have used different bases for the deprotonation step of the starting phenols, and vinyl butanoate as the acyl donor in the transesterification reactions. The reaction times for the kinetic resolution steps catalysed by the lipase varied from 23 to 48 h, and were run at 30–38 °C. Specific rotation values confirmed the absolute configuration of the enantiopure drugs, however, an earlier report of the specific rotation value of (S)-esmolol is not consistent with our measured specific rotation values, and we here claim that our data are correct. Compared to the previously reported syntheses of these two enantiopure drugs, we have replaced toluene or dichloromethane with acetonitrile, and replaced the flammable acetyl chloride with lithium chloride. We have also reduced the amount of epichlorohydrin and bases, and identified dimeric byproducts in order to obtain higher yields.

The β-blocker (S)-betaxolol has been synthesized in 99% enantiomeric excess (ee) from the commercially available precursor 4-(2-hydroxyethyl)phenol. The racemic chlorohydrin 1-chloro-3-(4-(2-(cyclopropylmethoxy)ethyl)phenoxy)propan-2-ol was esterified with vinyl acetate catalyzed by lipase B from Candida antarctica, which gave the R-chlorhydrin (R)-1-chloro-3-(4-(2-(cyclopropylmethoxy)ethyl)phenoxy)propan-2-ol in 99% ee with 38% yield. The enantiomeric excess of the R-chlorohydrin was retained in an amination reaction with isopropylamine in methanol to yield (S)-betaxolol in 99% ee and with 9% overall yield. We are under way to improve the yield.

Understanding the mechanisms underlying substrate recognition and selectivity is essential for developing new biotechnological applications of carbohydrate-binding modules (CBMs). Many members of CBM family 2 (CBM2) prefer binding to crystalline substrates and show diverse substrate specificities despite sharing similar folds. We show here that CBM2 specificity cannot be rationalized solely based on sequence and fold. Rather, specific binding of CBM2s to crystalline surfaces depends on a balance of topological complementarity, hydrophobic dehydration, and hydrogen-bonding interactions. We compare the binding modes of Sc CBM2 and Ba CBM2 on cellulose, chitin, and polyethylene terephthalate (PET) with respect to protein and substrate properties. Using MD simulations and NMR, we explore the CBMs’ surface topology, hydration, solvent accessibility, and electrostatics. The results indicate that the two CBMs may bind to crystalline substrates in opposite orientations, and that the interplay between hydrophobic and π-stacking interactions along the aromatic triad and hydrogen-bonding interactions on either side determines their orientation. We further show that higher water density at the aromatic triad correlates with higher substrate affinity. Lastly, we probe the interaction dynamics of Ba CBM2 with PET nanoparticles and the monomer bis(2-hydroxyethyl) terephthalate (BHET) using NMR relaxation and solvent paramagnetic relaxation enhancement (sPRE) experiments, revealing a mixture of transient, low-population states involved in PET binding. These insights clarify the interplay among factors that drive CBM2 substrate recognition and highlight the importance of evaluating surface hydration and protein dynamics in future CBM2 studies.

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind β-1,3- and β-1,6-glucans with high specificity. Cp CBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the protein’s binding sites plays distinct roles in ligand binding. One binding site, referred to as the β site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on Cp CBM92A. The other two binding sites, referred to as α and γ, preferentially bind longer segments of β-1,3- and β-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.