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首页> 外文期刊>Applied Microbiology >Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation
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Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation

机译：Paenibacillus polymyxa DSM 365中左旋胰酶基因的失活在2,3-丁二醇发酵过程中减少了外奥多糖生物合成

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The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa 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 P. polymyxa . The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa , was successfully disrupted. The P. polymyxa 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 P. polymyxa 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.IMPORTANCE 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 Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus 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 P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa , it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.
机译：通过Paenibacillus polymyxa的2,3-丁二醇（2,3-Bd）发酵在2,3-丁二醇（2,3-Bd）发酵期间的形成增加了培养基粘度，这反过来呈现出相当大的技术和经济挑战至2,3-BD下游加工。为了在2,3-BD发酵期间消除EPS生产，我们使用同源重组在P. polymyxa中禁用EPS生物合成途径。编码Levansucrase的基因，Pualdyxa在P. puminyyxa中负责EPS生物合成的主要酶。 P. polymyxa Levansucrase Null突变体分别产生2.5？±0.1和1.2？±0.2g /升EPS，而蔗糖和葡萄糖，而野生型21.7？±2.5​​和3.1？±0.0克/升EPS分别在相同的基材上。这些水平的EPS通过蔗糖和葡萄糖分别相对于野生型分别在蔗糖和葡萄糖上形成的EPS形成的8.7和2.6倍，并且在2,3-BD生产中没有显着降低。 EPS生物合成的失活导致增长的相当大。在葡萄糖和蔗糖上，与野生型相比，左旋核酶NULL突变体的细胞生物质（8.1〜±0.8和6.5？±0.3克/升）增加1.4倍（6.0？±0.1和4.6 ？±0.3克/升，分别在同一底物上生长。对左转ucrase Null突变体的遗传稳定性的评价显示，它保持遗传稳定超过50多种，生长或2,3-BD形成无可观察到的降低，有或没有抗生素补充。因此，P. polymyxa Levansucrase null突变体具有潜在的用作工业生物催化剂，用于具有与eps相关的挑战的成本效益的大型2,3-bd发酵过程。称为目前的注意力和研究投资下一代燃料和化学物质的生产，其中由非遗传的Paenibacillus物种产生的2,3-丁二醇（2,3-Bd）可能是最剧烈追求的，工程工程工具之一强烈追捧。在2,3-BD发酵期间的潜水糖（EPS）产生在下游加工过程中构成问题。具体地，EPS对发酵液产生2,3-BD分离产生负面影响，从而增加2,3-BD生产的总成本。本文呈现的结果表明，Pualdyxa中Levansucrase基因的灭活导致eps积累减少。另外，开发了一种用于EPS测定的新方法和使用原生质体的简单方案，用于增强P. polymyxa的改变。总体而言，虽然我们的研究表明，Levan不是P. polymyxa的唯一EPS，但它代表了在下游加工过程中开发缺乏eps相关并发症的成本效益的2,3-BD发酵的重要第一步。

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