In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii
In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii
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DOI：
10.1021/acssynbio.0c00562
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发表时间：
2021-04-12
影响因子：
4.7
通讯作者：
Crook, Nathan
中科院分区：
文献类型：
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作者：
Durmusoglu, Deniz;Al'Abri, Ibrahim S.;Crook, Nathan
博拉氏酵母菌是一种益生菌酵母，在37℃下生长迅速，易于转化，并能在肠道中产生治疗性蛋白质。为了确定其产生由多基因途径编码的小分子的能力，我们测量了启动子、终止子、选择性标记和拷贝数控制元件所激活的蛋白质表达的数量和差异。接下来，我们在该菌株中展示了crispr介导的高效基因组编辑（>95%），使我们能够探测不同基因组位点的工程基因表达。我们利用这些策略来组装途径，使广泛的维生素前体（β -胡萝卜素）和药物（紫罗兰素）滴度。我们发现博氏沙门氏菌在无菌小鼠中稳定定植超过30天，并与共生微生物竞争生态位空间，在常规和抗生素治疗的小鼠中表现出短（1-2天）的肠道停留时间。使用这些工具，我们在无菌小鼠肠道中进行了14天的β -胡萝卜素合成（总共194 μ g），估计粪便中回收的额外β -胡萝卜素的总质量比初始益生菌剂量中存在的β -胡萝卜素高56倍。这项工作量化了哺乳动物肠道中的博氏沙门氏菌的异源小分子生产滴度，并提供了一套调节这些滴度的工具。
Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 degrees C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (beta-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled beta-carotene synthesis (194 mu g total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional beta-carotene recovered in feces was 56-fold higher than the beta-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.