Department of Food Science and Human Nutrition, and Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Department of Bioenergy Science and Technology, and Department of Integrative Food, Bioscience and Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea.
J Microbiol Biotechnol. 2021 Jul 28;31(7):1035-1043. doi: 10.4014/jmb.2105.05018.
Although engineered fermenting cellobiose is useful for the production of biofuels from cellulosic biomass, cellodextrin accumulation is one of the main problems reducing ethanol yield and productivity in cellobiose fermentation with expressing cellodextrin transporter (CDT) and intracellular β-glucosidase (GH1-1). In this study, we investigated the reason for the cellodextrin accumulation and how to alleviate its formation during cellobiose fermentation using engineered fermenting cellobiose. From the series of cellobiose fermentation using expressing only GH1-1 under several culture conditions, it was discovered that small amounts of GH1-1 were secreted and cellodextrin was generated through trans-glycosylation activity of the secreted GH1-1. As GH1-1 does not have a secretion signal peptide, non-conventional protein secretion might facilitate the secretion of GH1-1. In cellobiose fermentations with expressing only GH1-1, knockout of gene involved in non-conventional protein secretion pathway significantly delayed cellodextrin formation by reducing the secretion of GH1-1 by more than 50%. However, in cellobiose fermentations with expressing both GH1-1 and CDT-1, knockout did not show a significant effect on cellodextrin formation, although secretion of GH1-1 was reduced by more than 40%. These results suggest that the development of new intracellular β-glucosidase, not influenced by non-conventional protein secretion, is required for better cellobiose fermentation performances of engineered fermenting cellobiose.
虽然工程化发酵纤维二糖可用于从纤维素生物质生产生物燃料，但纤维二糖发酵过程中，细胞外β-葡萄糖苷酶（GH1-1）和细胞外纤维二糖转运蛋白（CDT-1）的表达会导致细胞外纤维二糖的积累，从而降低乙醇产量和生产效率，这是主要问题之一。在这项研究中，我们研究了工程化发酵纤维二糖在纤维二糖发酵过程中积累纤维二糖的原因，以及如何减轻其形成。在几种培养条件下，仅表达 GH1-1 的一系列纤维二糖发酵实验中，发现少量 GH1-1 被分泌出来，并通过分泌的 GH1-1 的转糖基化活性生成纤维二糖。由于 GH1-1 没有分泌信号肽，因此非常规蛋白分泌可能有助于 GH1-1 的分泌。在仅表达 GH1-1 的纤维二糖发酵中，敲除非常规蛋白分泌途径中涉及的 基因，通过减少 GH1-1 的分泌，使 GH1-1 的分泌减少超过 50%，从而显著延迟纤维二糖的形成。然而，在同时表达 GH1-1 和 CDT-1 的纤维二糖发酵中，敲除 基因对纤维二糖的形成没有显著影响，尽管 GH1-1 的分泌减少了超过 40%。这些结果表明，需要开发不受非常规蛋白分泌影响的新型细胞内β-葡萄糖苷酶，以提高工程化发酵纤维二糖的纤维二糖发酵性能。
