Genetic parts and hosts can be sourced from nature to realize new functions for synthetic biology or to improve performance in a particular application environment. Here, we proceed from the discovery and characterization of new parts to stable expression in new hosts with a particular focus on achieving sustained chitinase activity. Chitinase is a key enzyme for various industrial applications that require the breakdown of chitin, the second most abundant biopolymer on the earth. Diverse microbes exhibit chitinase activity, but for applications, the environmental conditions for optimal enzyme activity and microbe fitness must align with the application context. Achieving sustained chitinase activity under broad conditions in heterologous hosts has also proven difficult due to toxic side effects. Toward addressing these challenges, we first screen ocean water samples to identify microbes with chitinase activity. Next, we perform whole genome sequencing and analysis and select a chitinase gene for heterologous expression. Then, we optimize transformation methods for target hosts and introduce chitinase. Finally, to achieve robust function, we optimize ribosome binding sites and discover a beneficial promoter that upregulates chitinase expression in the presence of colloidal chitin in a sense-and-respond fashion. We demonstrate chitinase activity for >21 days in standard (Escherichia coli) and nonstandard (Roseobacter denitrificans) hosts. Besides enhancing chitinase applications, our pipeline is extendable to other functions, identifies natural microbes that can be used directly in non-GMO contexts, generates new parts for synthetic biology, and achieves weeks of stable activity in heterologous hosts.

中文翻译：

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从天然微生物筛选到外源宿主中持续的几丁质酶活性

遗传部分和宿主可以取自自然界，以实现合成生物学的新功能或提高特定应用环境中的性能。在这里，我们从新部件的发现和表征到在新宿主中的稳定表达，特别关注实现持续的几丁质酶活性。几丁质酶是需要分解几丁质（地球上第二丰富的生物聚合物）的各种工业应用的关键酶。多种微生物表现出几丁质酶活性，但对于应用而言，最佳酶活性和微生物适应性的环境条件必须与应用环境相一致。由于毒副作用，在异源宿主的广泛条件下实现持续的几丁质酶活性也被证明是困难的。为了应对这些挑战，我们首先筛选海水样本以识别具有几丁质酶活性的微生物。接下来，我们进行全基因组测序和分析，并选择几丁质酶基因进行异源表达。然后，我们针对目标宿主优化转化方法并引入几丁质酶。最后，为了实现强大的功能，我们优化了核糖体结合位点，并发现了一种有益的启动子，可以在胶体几丁质存在的情况下以感知和响应的方式上调几丁质酶的表达。我们在标准（大肠杆菌）和非标准（脱氮玫瑰杆菌）宿主中证明了几丁质酶活性>21天。除了增强几丁质酶应用之外，我们的管道还可扩展到其他功能，识别可直接用于非转基因环境的天然微生物，生成用于合成生物学的新部件，并在异源宿主中实现数周的稳定活性。