Raffinose, a trisaccharide abundantly found in soybeans, is a potential alternative carbon source for biorefineries. Nevertheless, residual intermediate di- or monosaccharides and low catabolic efficiency limit raffinose use through conventional microbial hosts. This study presents a Vibrio-based platform to convert raffinose efficiently. Vibrio sp. dhg was selected as the starting strain for the Adaptive Laboratory Evolution (ALE) strategy to leverage its significantly higher metabolic efficiency. We conducted ALE on a solid minimal medium supplemented with raffinose to prevent the enrichment of undesired phenotypes due to the shared effect of extracellular raffinose hydrolysis among multiple strains. As a result, we generated the VRA10 strain that efficiently utilizes raffinose without leaving behind degraded di- or monosaccharides, achieving a notable growth rate (0.40 h−1) and raffinose consumption rate (1.2 g/gdcw/h). Whole genome sequencing and reverse engineering identified that a missense mutation in the melB gene (encoding a melibiose/raffinose:sodium symporter) and the deletion of the two galR genes (encoding transcriptional repressors for galactose catabolism) facilitated rapid raffinose utilization. The further engineered strain produced 6.2 g/L of citramalate from 20 g/L of raffinose. This study will pave the way for the efficient utilization of diverse raffinose-rich byproducts and the expansion of alternative carbon streams in biorefinery applications.

中文翻译：

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生成基于 Vibrio 的平台，通过在固体培养基上的 Adaptive Laboratory Evolution 高效转化棉子糖


棉子糖是一种大量存在于大豆中的三糖，是生物精炼厂的潜在替代碳源。然而，残留的中间体二糖或单糖和低分解代谢效率限制了棉子糖通过常规微生物宿主的使用。本研究提出了一个基于 Vibrio 的平台，可以有效地转化棉子糖。Vibrio sp. dhg 被选为适应性实验室进化 （ALE） 策略的起始菌株，以利用其显着更高的代谢效率。我们在补充有棉子糖的固体最小培养基上进行了 ALE，以防止由于细胞外棉子糖水解在多个菌株之间的共同效应而富集不需要的表型。结果，我们产生了 VRA10 菌株，该菌株可有效利用棉子糖，而不会留下降解的二糖或单糖，实现了显着的生长速率 （0.40 h-1） 和棉子糖消耗率 （1.2 g/gdcw/h）。全基因组测序和逆向工程确定 melB 基因（编码二糖/棉子糖：钠同向转运蛋白）中的错义突变和两个 galR 基因（编码半乳糖分解代谢的转录抑制因子）的缺失促进了棉子糖的快速利用。进一步工程菌株从 6.2 g/L 棉子糖中产生 20 g/L 柠檬毛酸盐。这项研究将为生物炼制应用中各种富含棉子糖的副产物和替代碳流的扩展铺平道路。