Department of Animal Sciences, Ohio State Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, Ohio, USA.
Bioenergy and Water Treatment Management Program, Agricultural Technical Institute, The Ohio State University, Wooster, Ohio, USA.
Appl Environ Microbiol. 2020 Apr 17;86(9). doi: 10.1128/AEM.00196-20.
The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in , was successfully disrupted. The levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges. Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic species is perhaps one of the most vigorously pursued, tools for engineering species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of were developed. Overall, although our study shows that levan is not the only EPS produced by , it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.
在 2,3-丁二醇 (2,3-BD) 发酵过程中，多糖 (EPSs) 的形成会增加培养基的粘度，这给 2,3-BD 的下游加工带来了相当大的技术和经济挑战。为了消除 2,3-BD 发酵过程中 EPS 的产生，我们使用同源重组使 中 EPS 生物合成途径失活。成功敲除了编码纤维二糖蔗糖酶的基因，该酶是 中 EPS 生物合成的主要酶。 纤维二糖蔗糖酶缺失突变体在蔗糖和葡萄糖上分别产生 2.5±0.1 和 1.2±0.2 g/L EPS，而野生型在相同底物上分别产生 21.7±2.5 和 3.1±0.0 g/L EPS。与野生型相比，纤维二糖蔗糖酶缺失突变体在蔗糖和葡萄糖上形成 EPS 的水平分别降低了 8.7 倍和 2.6 倍，而 2,3-BD 的产量没有明显降低。EPS 生物合成的失活导致生长显著增加。在葡萄糖和蔗糖上，纤维二糖蔗糖酶缺失突变体的细胞生物量（分别为 8.1±0.8 和 6.5±0.3 g/L）比在相同底物上生长的野生型（分别为 6.0±0.1 和 4.6±0.3 g/L）增加了 1.4 倍。对纤维二糖蔗糖酶缺失突变体遗传稳定性的评估表明，它在五十多代中保持遗传稳定，无论是否添加抗生素，生长或 2,3-BD 形成都没有观察到下降。因此，纤维二糖蔗糖酶缺失突变体有可能作为一种工业生物催化剂，用于经济高效的大规模 2,3-BD 发酵过程，而不会产生与 EPS 相关的挑战。鉴于目前对下一代燃料和化学品生产的关注和研究投资浪潮，其中也许最受追捧的是无毒 属生产的 2,3-丁二醇 (2,3-BD)，因此迫切需要用于工程化 属的工具。在 2,3-BD 发酵过程中产生的多糖 (EPS) 是下游加工过程中的一个问题。具体来说，EPS 会对 2,3-BD 与发酵液的分离产生负面影响，从而增加 2,3-BD 生产的总成本。本文的结果表明，在 中失活纤维二糖蔗糖酶基因可减少 EPS 的积累。此外，还开发了一种新的 EPS 测定方法和一种简单的原生质体方法，用于增强 的转化。总体而言，尽管我们的研究表明，甘露聚糖并不是 产生的唯一 EPS，但它代表了朝着开发经济高效的 2,3-BD 发酵过程迈出的重要一步，该过程在下游加工过程中不会出现与 EPS 相关的并发症。
