Novy Vera, Nielsen Fredrik, Cullen Daniel, Sabat Grzegorz, Houtman Carl J, Hunt Christopher G
US Department of Agriculture, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI, 53726, USA.
Department of Biology and Bioengineering, Division of Industrial Biotechnology, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden.
Biotechnol Biofuels. 2021 Apr 26;14(1):105. doi: 10.1186/s13068-021-01955-5.
On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes' hydrolytic strength towards the same substrates.
Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation.
Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.
利用里氏木霉进行原位酶生产，通过利用真菌基因调控机制，使酶分泌能够持续适应用于培养的底物，从而提高木质纤维素糖化的产量并降低总成本。为了利用这一点，必须了解底物特性与真菌反应之间的相互关系。然而，真菌形态学或基因表达研究往往缺乏对底物结构和化学性质的表征。在此，里氏木霉QM6a在三种软木底物上进行培养：北方漂白软木硫酸盐浆（NBSK）以及分别经过稀酸催化蒸汽预处理（LP - STEX）或温和碱性氧化（LP - ALKOX）处理的黑松。通过对相似起始材料进行不同预处理，我们为真菌提供了经过系统修饰的底物。这使得能够阐明底物诱导的真菌反应变化，并测试所分泌酶对相同底物的水解强度。
酶活性随时间的变化过程与半纤维素含量和纤维素可及性相关。具体而言，通过蛋白质组分析观察到，在复杂底物（LP - STEX；LP - ALKOX）上产生的蛋白质中，侧链裂解半纤维素酶的量增加。共聚焦激光扫描显微镜图像显示，真菌微观形态对纤维素可及性和初始培养粘度的变化有反应。后者是由表面电荷和纤维尺寸引起的，可能限制了传质，导致真菌处于应激状态下的形态。用浓缩的里氏木霉上清液补充基本的纤维素酶混合物可提高三种底物的糖化效率，其中纤维素、木聚糖和甘露聚糖的转化率分别提高了27%至45%和2800%。对于在LP - STEX和LP - ALKOX上产生的蛋白质对相同底物的糖化效果改善最为显著，在最佳情况下，效率达到了最先进的商业酶制剂的水平。
在LP - STEX和LP - ALKOX上培养里氏木霉产生了一种蛋白质混合物，该混合物将基本纤维素酶混合物的水解强度提高到了软木底物上最先进的性能水平。这表明可以利用真菌的适应机制在无需事先了解特定底物要求的情况下实现酶水解性能的增强。
