State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, 266237, P. R. China.
Zibo Center Hospital, Zi Bo, 255036, P. R. China.
J Microbiol. 2020 Aug;58(8):687-695. doi: 10.1007/s12275-020-9630-5. Epub 2020 Jun 10.
The saprophytic fungus Trichoderma reesei has long been used as a model to study microbial degradation of lignocellulosic biomass. The major cellulolytic enzymes of T. reesei are the cellobiohydrolases CBH1 and CBH2, which constitute more than 70% of total proteins secreted by the fungus. However, their physiological functions and effects on enzymatic hydrolysis of cellulose substrates are not sufficiently elucidated. Here, the cellobiohydrolase-encoding genes cbh1 and cbh2 were deleted, individually or combinatively, by using an auxotrophic marker-recycling technique in T. reesei. When cultured on media with different soluble carbon sources, all three deletion strains (Δcbh1, Δcbh2, and Δcbh1Δcbh2) exhibited no dramatic variation in morphological phenotypes, but their growth rates increased apparently when cultured on soluble cellulase-inducing carbon sources. In addition, Δcbh1 showed dramatically reduced growth and Δcbh1Δcbh2 could hardly grew on microcrystalline cellulose (MCC), whereas all strains grew equally on sodium carboxymethyl cellulose (CMC-Na), suggesting that the influence of the CBHs on growth was carbon source-dependent. Moreover, five representative cellulose substrates were used to analyse the influence of the absence of CBHs on saccharification efficiency. CBH1 deficiency significantly affected the enzymatic hydrolysis rates of various cellulose substrates, where acid pre-treated corn stover (PCS) was influenced the least. CBH2 deficiency reduced the hydrolysis of MCC, PCS, and acid pre-treated and delignified corncob but improved the hydrolysis ability of filter paper. These results demonstrate the specific contributions of CBHs to the hydrolysis of different types of biomass, which could facilitate the development of tailor-made strains with highly efficient hydrolysis enzymes for certain biomass types in the biofuel industry.
腐生真菌里氏木霉长期以来一直被用作研究木质纤维素生物质微生物降解的模型。里氏木霉的主要纤维素酶是纤维二糖水解酶 CBH1 和 CBH2，它们构成了真菌分泌的总蛋白的 70%以上。然而，它们的生理功能及其对纤维素底物酶解的影响尚未得到充分阐明。在这里，通过使用里氏木霉的营养缺陷型标记回收技术，单独或组合地删除了编码细胞二糖水解酶的基因 cbh1 和 cbh2。当在含有不同可溶性碳源的培养基中培养时，所有三种缺失菌株（Δcbh1、Δcbh2 和 Δcbh1Δcbh2）在形态表型上没有明显变化，但在培养于可溶性纤维素诱导碳源时，其生长速度明显增加。此外，Δcbh1 的生长明显降低，Δcbh1Δcbh2 在微晶纤维素（MCC）上几乎无法生长，而所有菌株在羧甲基纤维素钠（CMC-Na）上的生长情况相同，这表明 CBHs 对生长的影响取决于碳源。此外，使用五种代表性的纤维素底物来分析缺失 CBHs 对糖化效率的影响。CBH1 缺失显著影响各种纤维素底物的酶解速率，其中预处理酸玉米秸秆（PCS）受影响最小。CBH2 缺失降低了 MCC、PCS 和预处理脱木质素玉米芯的水解，但提高了滤纸的水解能力。这些结果表明 CBHs 对不同类型生物质水解的特定贡献，这有助于为生物燃料工业中某些生物质类型开发具有高效水解酶的定制菌株。
