Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden.
Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA.
Appl Environ Microbiol. 2021 Apr 13;87(9). doi: 10.1128/AEM.03017-20.
The native ability of to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of , as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 ± 0.00 and 0.18 ± 0.00 g g, respectively, compared to 0.15 ± 0.01 g g for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, while wild type showed 42 ± 4 h on glucose and >80 h on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 h with a growth rate of 0.11 ± 0.01 h and resulted in immediate growth on glucose at 0.24 ± 0.01 h Additional introduction of a G-to-V mutation at position 148 in resulted in immediate growth on fructose at 0.32 ± 0.03 h These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings. is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing, and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of can be engineered to (i) reduce glucose accumulation, (ii) study cellodextrin transport systems , (iii) allow experiments at >120 g liter soluble substrate concentration, or (iv) reduce costs for labeling studies.
能够高效地溶解纤维素，这使它成为通过整合生物加工生产可持续生物燃料的一个有趣平台。除了其他改进措施之外，通过改善和可重复的己糖消耗，以及对其代谢的基础研究，将有益于工业实施 和 的发展。为了研究 在葡萄糖或果糖上的生长情况以及潜在的分子机制，在碳限制恒化器中进行了实验室进化实验，其中葡萄糖或果糖的浓度逐渐增加，而纤维二糖的浓度逐渐降低。与野生型在纤维二糖上的 0.15±0.01 g g 生物质产量相比， 在葡萄糖和果糖上的生长分别实现了 0.09±0.00 和 0.18±0.00 g g 的生物质产量。单菌落分离株在单糖上没有或只有很短的延迟期，而野生型在葡萄糖上的延迟期为 42±4 h，在果糖上的延迟期>80 h。由于 在葡萄糖、果糖和纤维二糖上都有良好的生长，因此选择果糖分离株进行基于基因组序列的反向代谢工程。缺失一个假定的转录调节因子（Clo1313_1831），该因子上调了果糖激酶的活性，将果糖上的延迟时间缩短至 12 h，生长速率为 0.11±0.01 h，并导致在葡萄糖上立即生长，生长速率为 0.24±0.01 h。在 中的第 148 位引入 G 到 V 的突变，导致在果糖上立即生长，生长速率为 0.32±0.03 h。这些见解可以指导用于基础研究运输和上糖酵解以及在工业环境中最大化产物产量的菌株工程。 是通过整合生物加工生产生物乙醇的可持续和具有成本效益的重要候选者。除了无与伦比的纤维素解构之外，工业应用和基础研究还将受益于提高葡萄糖和果糖的消耗。本研究表明，可以对 进行进化，以实现葡萄糖或果糖的可重复组成型生长。随后的基因组测序、基因编辑和生理特征鉴定确定了两个潜在的突变，这些突变在 （糖的运输或代谢的）调节中起作用。鉴于这些发现，在以前使用基于己糖的培养基进行的研究中，可能已经（并且无意识地）发生了这些突变，这可能具有广泛的调节后果。通过对这些基因的靶向修饰，可以对 工业和研究菌株进行工程改造，（i）减少葡萄糖积累，（ii）研究纤维二糖运输系统，（iii）允许在>120 g liter 可溶性底物浓度下进行实验，或（iv）降低标记研究的成本。
