Complex Biosystems Program, University of Nebraska, Lincoln, Nebraska, United States of America.
Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, Nebraska, United States of America.
PLoS One. 2021 May 3;16(5):e0245407. doi: 10.1371/journal.pone.0245407. eCollection 2021.
Within the field of bioproduction, non-model organisms offer promise as bio-platform candidates. Non-model organisms can possess natural abilities to consume complex feedstocks, produce industrially useful chemicals, and withstand extreme environments that can be ideal for product extraction. However, non-model organisms also come with unique challenges due to lack of characterization. As a consequence, developing synthetic biology tools, predicting growth behavior, and building computational models can be difficult. There have been many advancements that have improved work with non-model organisms to address broad limitations, however each organism can come with unique surprises. Here we share our work in the non-model bacterium Actinobacillus succinognes 130Z, which includes both advancements in synthetic biology toolkit development and pitfalls in unpredictable fermentation behaviors. To develop a synthetic biology "tool kit" for A. succinogenes, information gleaned from a growth study and antibiotic screening was used to characterize 22 promoters which demonstrated a 260-fold range of fluorescence protein expression. The strongest of the promoters was incorporated into an inducible system for tunable gene control in A. succinogenes using the promoter for the lac operon as a template. This system flaunted a 481-fold range of expression and no significant basal expression. These findings were accompanied by unexpected changes in fermentation products characterized by a loss of succinic acid and increase in lactic acid after approximately 10 months in the lab. During evaluation of the fermentation shifts, new tests of the synthetic biology tools in a succinic acid producing strain revealed a significant loss in their functionality. Contamination and mutation were ruled out as causes and further testing is needed to elucidate the driving factors. The significance of this work is to share a successful tool development strategy that could be employed in other non-model species, report on an unfortunate phenomenon that needs addressed for further development of A. succinogenes, and provide a cautionary tale for those undertaking non-model research. In sharing our findings, we seek to provide tools and necessary information for further development of A. succinogenes as a platform for bioproduction of succinic acid and to illustrate the importance of diligent and long-term observation when working with non-model bacteria.
在生物生产领域，非模式生物作为生物平台候选物具有很大的潜力。非模式生物具有天然的能力来消耗复杂的原料，生产工业上有用的化学品，并能在极端环境中生存，这些特性非常适合产品提取。然而，由于缺乏特征描述，非模式生物也带来了独特的挑战。因此，开发合成生物学工具、预测生长行为和构建计算模型可能会很困难。尽管已经有了许多改进，可以解决非模式生物的广泛限制问题，但每种生物都可能会带来独特的惊喜。在这里，我们分享了我们在非模式细菌 Actinobacillus succinognes 130Z 方面的工作，其中包括合成生物学工具包开发方面的进展和不可预测发酵行为方面的陷阱。为了开发用于 A. succinogenes 的合成生物学“工具包”，我们从生长研究和抗生素筛选中获取信息，用于表征 22 个启动子，这些启动子展示了荧光蛋白表达的 260 倍差异。最强的启动子被整合到一个诱导系统中，用于在 A. succinogenes 中使用 lac 操纵子的启动子作为模板进行基因的可调控制。该系统展示了 481 倍的表达范围，且没有明显的基础表达。这些发现伴随着发酵产物的意外变化，大约 10 个月后在实验室中发现琥珀酸的产量减少，乳酸的产量增加。在评估发酵变化时，在产琥珀酸的菌株中对合成生物学工具的新测试表明，其功能显著丧失。排除了污染和突变的原因，需要进一步的测试来阐明驱动因素。这项工作的意义在于分享一种可以应用于其他非模式物种的成功工具开发策略，报告 A. succinogenes 进一步发展所需要解决的不幸现象，并为从事非模式研究的人员提供一个警示故事。通过分享我们的研究结果，我们旨在为 A. succinogenes 作为琥珀酸生物生产平台的进一步发展提供工具和必要的信息，并说明在与非模式细菌合作时，勤奋和长期观察的重要性。
