Department of Biotechnology, K L E F University, Guntur 522 502, India.
Department of Physiology, Anatomy and Microbiology, College of Science, Health and Engineering, La Trobe University, Melbourne, Victoria 3086, Australia.
Int J Mol Sci. 2020 Feb 14;21(4):1299. doi: 10.3390/ijms21041299.
Cellulose is one of the most abundant and renewable biomass products used for the production of bioethanol. Cellulose can be efficiently hydrolyzed by Bacillus subtilis VS15, a strain isolate obtained from decomposing logs. A genome shuffling approach was implemented to improve the cellulase activity of Bacillus subtilis VS15. Mutant strains were created using ethyl methyl sulfonate (EMS), N-Methyl-N' nitro-N-nitrosoguanidine (NTG), and ultraviolet light (UV) followed by recursive protoplast fusion. After two rounds of shuffling, the mutants Gb2, Gc8, and Gd7 were produced that had an increase in cellulase activity of 128%, 148%, and 167%, respectively, in comparison to the wild type VS15. The genetic diversity of the shuffled strain Gd7 and wild type VS15 was compared at whole genome level. Genomic-level comparisons identified a set of eight genes, consisting of cellulase and regulatory genes, of interest for further analyses. Various genes were identified with insertions and deletions that may be involved in improved celluase production in Gd7.. Strain Gd7 maintained the capability of hydrolyzing wheatbran to glucose and converting glucose to ethanol by fermentation with Saccharomyces cerevisiae of the wild type VS17. This ability was further confirmed by the acidified potassium dichromate (KCrO) method.
纤维素是最丰富和可再生的生物质产品之一，用于生产生物乙醇。枯草芽孢杆菌 VS15 可以有效地水解纤维素，该菌株是从分解原木中分离得到的。采用基因组改组方法来提高枯草芽孢杆菌 VS15 的纤维素酶活性。使用乙基甲基磺酸酯（EMS）、N-甲基-N'-硝基-N-亚硝基胍（NTG）和紫外线（UV）处理后，进行递归原生质体融合，创建突变株。经过两轮改组，产生了突变株 Gb2、Gc8 和 Gd7，它们的纤维素酶活性分别比野生型 VS15 提高了 128%、148%和 167%。与野生型 VS15 相比，改组菌株 Gd7 的遗传多样性在全基因组水平上进行了比较。基因组水平比较确定了一组 8 个基因，包括纤维素酶和调节基因，这些基因可能与 Gd7 中提高纤维素酶产量有关。在 Gd7 中发现了各种具有插入和缺失的基因，这些基因可能参与了纤维素酶产量的提高。与野生型 VS17 的酿酒酵母一起，菌株 Gd7 仍然能够水解麦麸产生葡萄糖，并通过发酵将葡萄糖转化为乙醇。这一能力通过酸化重铬酸钾（KCrO）法进一步得到了证实。
