Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
Institute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
Int J Mol Sci. 2024 Nov 7;25(22):11983. doi: 10.3390/ijms252211983.
24 (BL24) is an efficient, non-pathogenic producer of 2,3-butanediol (2,3-BD). However, during inulin fermentation, the strain produces large amounts of exopolysaccharides (EPS), which interfere with the process' performance. The present study aims to investigate the effect that inactivation of the gene, encoding levansucrase in BL24, has on 2,3-BD production efficiency. Knockout of the gene was accomplished via insertional inactivation. The -knockout variant formed 0.57 g/L EPS from sucrose and 0.7-0.8 g/L EPS from glucose and fructose, a 15- and 2.5-fold reduction relative to the wild type, respectively. Likewise, during batch fermentation with soluble inulin Frutafit CLR, the mutant BLΔ produced significantly less EPS than the wild type, allowing the maintenance of pH at values favoring 2,3-BD synthesis. At pH 6.50, BLΔ reached a record titer of 128.7 g/L 2,3-BD, with productivity of 1.65 g/L/h, and a yield of 85.8% of the theoretical maximum. The obtained concentration of 2,3-BD is two-fold higher compared to that of the wild type. Subsequent RT-qPCR assays confirmed a successful knockout. Three of the genes involved in inulin hydrolysis (, , and ) maintained their expression levels compared to the wild type, while that of increased. Although total EPS accumulation could not be completely eliminated via gene knockout alone, the overall reduction in EPS content has enabled the highest yield of 2,3-BD from inulin to date, a promising result for the industrial production from inulin-rich substrates.
24（BL24）是一种高效、非致病性的 2,3-丁二醇（2,3-BD）生产菌。然而，在菊糖发酵过程中，该菌株会产生大量的胞外多糖（EPS），从而干扰发酵过程的性能。本研究旨在探讨失活 BL24 中编码蔗聚糖蔗糖酶的 基因对 2,3-BD 生产效率的影响。通过插入失活法敲除了 基因。与野生型相比，-敲除变体分别从蔗糖和葡萄糖果糖中形成 0.57 g/L 和 0.7-0.8 g/L 的 EPS，减少了 15 倍和 2.5 倍。同样，在使用可溶性菊糖 Frutafit CLR 的分批发酵过程中，突变体 BLΔ产生的 EPS 明显少于野生型，从而使 pH 值维持在有利于 2,3-BD 合成的范围内。在 pH 6.50 时，BLΔ达到了 128.7 g/L 2,3-BD 的记录滴度，生产速率为 1.65 g/L/h，理论最大产率为 85.8%。与野生型相比，获得的 2,3-BD 浓度提高了一倍。随后的 RT-qPCR 检测证实了 基因的成功敲除。与野生型相比，三种参与菊糖水解的基因（ 、 和 ）的表达水平保持不变，而 的表达水平增加。尽管仅通过 基因敲除不能完全消除总 EPS 的积累，但 EPS 含量的总体减少使得从菊糖中获得迄今为止最高的 2,3-BD 产率，这是从富含菊糖的底物进行工业生产的有希望的结果。
