Carbohydrate-binding module O-mannosylation alters binding selectivity to cellulose and lignin

Improved understanding of the effect of protein glycosylation is expected to provide the foundation for the design of protein glycoengineering strategies. In this study, we examine the impact of O-glycosylation on the binding selectivity of a model Family 1 carbohydrate-binding module (CBM), which has been shown to be one of the primary sub-domains responsible for non-productive lignin binding in multi-modular cellulases. Specifically, we examine the relationship between glycan structure and the binding specificity of the CBM to cellulose and lignin substrates. We find that the glycosylation pattern of the CBM exhibits a strong influence on the binding affinity and the selectivity between both cellulose and lignin. In addition, the large set of binding data collected allows us to examine the relationship between binding affinity and the correlation in motion between pairs of glycosylation sites. Our results suggest that glycoforms displaying highly correlated motion in their glycosylation sites tend to bind cellulose with high affinity and lignin with low affinity. Taken together, this work helps lay the groundwork for future exploitation of glycoengineering as a tool to improve the performance of industrial enzymes.

Improved understanding of the effect of protein glycosylation is expected to provide the foundation for the design of protein glycoengineering strategies.

The cell walls of terrestrial plants primarily comprise the polysaccharides cellulose, hemicellulose, and pectin, as well as the heterogeneous aromatic polymer, lignin. In nature, carbohydrates derived from plant polysaccharides provide a massive carbon and energy source for biomass-degrading fungi, bacteria, and archaea, which together are the primary organisms that recycle plant matter and are a critical component of the global carbon cycle. Across the various environments in which these microbes break down lignocellulose, a few known enzymatic and chemical systems have evolved to deconstruct polysaccharides to soluble sugars.^1–6 These natural systems are, in several cases, being evaluated for industrial use to produce sugars for further conversion into renewable biofuels and chemicals.From an industrial perspective, overcoming biomass recalcitrance to cost-effectively produce soluble intermediates, including sugars for further upgrading remains the main challenge in biomass conversion. Lignin, the evolution of which in planta provided a significant advantage for terrestrial plants to mitigate microbial attack, is now widely recognized as a primary cause of biomass recalcitrance.⁷ Chemical and/or biological processing scenarios of lignocellulose have been evaluated⁸ and several approaches have been scaled to industrial biorefineries to date. Many biomass conversion technologies overcome recalcitrance by partially or wholly removing lignin from biomass using thermochemical pretreatment or fractionation. This approach enables easier polysaccharide access for carbohydrate-active enzymes and/or microbes. There are however, several biomass deconstruction approaches that employ enzymes or microbes with whole, unpretreated biomass.^9,10 In most realistic biomass conversion scenarios wherein enzymes or microbes are used to depolymerize polysaccharides, native or residual lignin remains.^11,12 It is important to note that lignin can bind and sequester carbohydrate-active enzymes, which in turn can affect conversion performance.¹³Therefore, efforts aimed at improving cellulose binding selectivity relative to lignin have emerged as major thrusts in cellulase studies.^14–25 Multiple reports in the past a few years have made exciting new contributions to our collective understanding of how fungal glycoside hydrolases, which are among the most well-characterized cellulolytic enzymes given their importance to cellulosic biofuels production, bind to lignin from various pretreatments.^15,17 Taken together, these studies have demonstrated that the Family 1 carbohydrate-binding modules (CBMs) often found in fungal cellulases are the most relevant sub-domains for non-productive binding to lignin,^15,17,20,26 likely due to the hydrophobic face of these CBMs that is known to be also responsible for cellulose binding (Fig. 1).²⁷Open in a separate windowFig. 1Model of glycosylated CBM binding the surface of a cellulose crystal. Glycans are shown in green with oxygen atoms in red, tyrosines known to be critical to binding shown in purple, and disulfide bonds Cys8–Cys25 and Cys19–Cys35 in yellow.Furthermore, several studies have been published recently using protein engineering of Family 1 CBMs to improve CBM binding selectivity to cellulose with respect to lignin. Of particular note, Strobel et al. screened a large library of point mutations in both the Family 1 CBM and the linker connecting the catalytic domain (CD) and CBM.^21,22 These studies demonstrated that several mutations in the CBM and one in the linker led to improved cellulose binding selectivity compared to lignin. The emerging picture is that the CBM-cellulose interaction, which occurs mainly as a result of stacking between the flat, hydrophobic CBM face (which is decorated with aromatic residues) and the hydrophobic crystal face of cellulose I, is also likely the main driving force in the CBM-lignin interaction given the strong potential for aromatic–aromatic and hydrophobic interactions.Alongside amino acid changes, modification of O-glycosylation has recently emerged as a potential tool in engineering fungal CBMs, which Harrison et al. demonstrated to be O-glycosylated.^28–31 In particular, we have revealed that the O-mannosylation of a Family 1 CBM of Trichoderma reesei cellobiohydrolase I (TrCel7A) can lead to significant enhancements in the binding affinity towards bacterial microcrystalline cellulose (BMCC).^30,32,33 This observation, together with the fact that glycans have the potential to form both hydrophilic and hydrophobic interactions with other molecules, led us to hypothesize that glycosylation may have a unique role in the binding selectivity of Family 1 CBMs to cellulose relative to lignin and as such, glycoengineering may be exploited to improve the industrial performance of these enzymes. To test this hypothesis, in the present study, we systematically probed the effects of glycosylation on CBM binding affinity for a variety of lignocellulose-derived cellulose and lignin substrates and investigated routes to computationally predict the binding properties of different glycosylated CBMs.     

Evaluating and clustering retrosynthesis pathways with learned strategy

With recent advances in the computer-aided synthesis planning (CASP) powered by data science and machine learning, modern CASP programs can rapidly identify thousands of potential pathways for a given target molecule. However, the lack of a holistic pathway evaluation mechanism makes it challenging to systematically prioritize strategic pathways except for using some simple heuristics. Herein, we introduce a data-driven approach to evaluate the relative strategic levels of retrosynthesis pathways using a dynamic tree-structured long short-term memory (tree-LSTM) model. We first curated a retrosynthesis pathway database, containing 238k patent-extracted pathways along with ∼55 M artificial pathways generated from an open-source CASP program, ASKCOS. The tree-LSTM model was trained to differentiate patent-extracted and artificial pathways with the same target molecule in order to learn the strategic relationship among single-step reactions within the patent-extracted pathways. The model achieved a top-1 ranking accuracy of 79.1% to recognize patent-extracted pathways. In addition, the trained tree-LSTM model learned to encode pathway-level information into a representative latent vector, which can facilitate clustering similar pathways to help illustrate strategically diverse pathways generated from CASP programs.

Tree-structured long short-term memory neural model learns to understand the retrosynthesis design strategies from patent-extracted retrosynthetic pathway data.     

Two new compounds derived from bufalin

The biotransformation of bufalin by cell suspension cultures of Platycodon grandiflorus was investigated and two new biotransformed products were obtained,which was 3-epi-telocinobufagin and 3-epi-bufalin-3-O-β-D-glucoside.     

Fluorinated nitroxides as ESR probes of solvation

Some newly synthesized fluorinated nitroxides, such as t-butyl perfluoroalkyl nitroxides Bu^tN(O) R_f (R_f=CF₃, 5; C₂F₅, 6; n-C₃F₇, 7) and s-butyl perfluoroacyl nitroxides Bu^sN(O) COR_f (R_f=CF₃, 9; n-C₃F₇, 10) have been employed as ESR probes of solvation in different common organic solvents. In aprotic solvents, the measured a_N values for each of the nitroxyl probes show a linear correlation with the cybotactic polar solvent parameters E_T (Dimroth) and Z (Kosowar), i.e. a_N=bE_T+c, and a_N=b′Z+c′. The physical significance for the slope (b or b′), the slope×E_T or slope×Z, the extrapolated intercept on a_N axis, c or c′, are linked, respectively, to the sensitivity of a specific nitroxide toward solvation, the magnitude of the overall solvation effect on the a_N value, and the intrinsic a_N value of each nitroxide in the ideal gaseous state. The intercept on the a_N axis may also serve as a new measure of electronegativity for perfluoroalkyl groups, CF₃, C₂F₅, n-C₃F₇, and perfluoroacyl groups, CF₃CO, n-C₃F₇CO. In protic solvents, i.e. alcohols and carboxylic acids, however, a_N values of all the probes, kept almost no change with the increase in E_T and Z. Furthermore, the plots of a_N versus non-cybotactic solvent constants, such as dipolar moment (μ) and dielectric constant (ε), all show random variations.     

Mechanism transition of self-pumped phase conjugation in KTa1 −xNbxO3:Fe crystals

Mechanism transitions of Self-Pumped Phase Conjugation (SPPC) with wavelength and doping concentration are observed in KTN:Fe (KTa_{1 –x} Nb _x O₃:Fe with _x = 0.48) crystals. The SPPC mechanism in KTN: Fe (0.4 wt. %) crystal transforms from Stimulated Photorefractive Backscattering and Four-Wave Mixing (SPB-FWM) to cat (or total internal reflection) as the wavelength increases from 514.5 nm to 620 nm. SPPC at 514.5 nm is formed with the cat mechanism in a 0.2 wt. % doped KTN:Fe crystal, while with the SPB-FWM mechanism in a 0.4 wt. % doped one. These mechanism transitions are discussed with respect to the dependence of the backscattering gain coefficient of the crystals on wavelength and doping concentration.     

将A-L理论推广到无限维李代数

为使由Alhassid与Levine所提出的动力学李代数方法（简称A－L理论）能适用于更多的散射体系，在h(∞)中引入了有效集合C（有限维）的概念．按照微扰理论的意义，C中的代数元所对应的群参量是较低次微扰的结果，而不属于C的代数元所对应的群参量则相当于较高次做扰所产生的修正结果．因此可以近似地利用C来代替h(∞)．这样，不仅简化了计算程序并且对于很多具有现实意义的散射过程的计算成为可能．     

共线散射体系A+BC的平-振能量传递──无限维李代数处理方法

利用无限维李代数方法处理了在BC分子能谱中含有二级与三级非简谐项的散射体系A＋BC的平-振能量传递问题，获得了散射过程的含有主要动力学参量的跃迁矩阵元和跃迁几率的解析表达式     

选择脱卤还原反应的研究

Ramasamy、Osuka等用NaHTe/EtOH研究了二卤代物的脱邻二溴、偕二溴反应和α-溴代羰基化合物的脱卤还原反应,我们选择结构不同的卤代物以NaHTe还原,发现在水介质和催化量碲代替计算量碲时也能顺利地进行这类反应,其反应条件温和,产率高,是有机官能团转化的好方法。     

Indirect Determination of Captopril with Sodium Nitrate and Thiocyanate by Floatation Separation of Copper

A new method for indirect determination of captopril (CPT) with NaNO₃ and NH₄SCN by floatation and separation of copper has been studied. In the weak acid, a small amount of Cu(II) can be reduced to Cu(I) by CPT, then Cu(I) reacted with the SCN, which can float on the surface of the liquid phase with NaNO₃. A good linear relationship is observed between the floatation yield (E%) of Cu(II) and the amount of captopril. The linear range is 2?32 mg/L. On the ground, captopril can be indirectly determined by determining E(%) of Cu(II). The method is simple, rapid, reliable and has good selectivity. The developed method can be applied to indirect determination of captopril with satisfactory results.     

Gear-like array of (H_2O)_(12) as building blocks in one-dimensional supramolecular assembly

The preparation and crystal structure of complex Co(Hsae)_2·2H_2O (1,H_2sae=N-salicylidene-2-iminoethanol) are reported.X- ray analysis revealed that every six Co(Hsae)_2 forms a cyclic chip and every 12 water forms a novel gear-like cluster.Acting as building blocks,the gear-like water cluster and complex chip are connected in A-B fashion and extend into one-dimensional supramolecular chain.Hydrogen bond is the primary bridging force in the formation of supramolecular framework.