INBIOTEC (Instituto de Biotecnología de León), Avda. Real 1 - Parque Científico de León, 24006, León, Spain.
Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, N-7491, Trondheim, Norway.
Appl Microbiol Biotechnol. 2023 Feb;107(2-3):691-717. doi: 10.1007/s00253-022-12335-w. Epub 2023 Jan 3.
Plant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54-1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50-55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer. KEY POINTS: • Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity. • Overexpression of the ORFs produced a novel P. rubens strain with improved activity. • The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown.
植物生物质是生物炼制的有前途的底物，也是具有多种工业应用的生物活性化合物、平台化学品和前体的来源。这些应用取决于其顽固结构的水解。然而，植物细胞壁的有效生物降解需要几种协同作用的酶类，并且需要新型酶才能实现有利可图的工业水解工艺。在本工作中，对青霉属菌株的酰基酯酶（FAE）活性进行了筛选，发现了一种有前途的候选菌株（青霉属 rubens Wisconsin 54-1255；先前为青霉属 chrysogenum），其中鉴定出两个 FAE-ORF，并随后进行了过表达。对酶提取物进行了分析，证实了相应基因产物（PrFaeA 和 PrFaeB）中存在 FAE 活性。用 PrFaeB 富集的酶提取物来确定 FAE 活性的最佳条件（pH5.0 和 50-55°C），并通过 MALDI-TOF/TOF 质谱进行蛋白质组分析。通过确定其他木质纤维素酶活性、非靶向代谢物分析以及在搅拌釜反应器中放大 FAE 生产完成了研究。本工作中描述的研究结果表明，青霉属 rubens 是一种很有前途的木质纤维素酶生产菌。要点：• 首次证实两个青霉属 rubens ORF 具有酰基酯酶活性。• ORF 的过表达产生了一种新型的青霉 rubens 菌株，其活性得到了提高。• 首次对青霉属木质纤维素酶提取物进行了深入的蛋白质组学研究。
