Engineering probiotics to biosynthesize natural substances for microbiome-brain research

项目摘要 为了推进微生物群-大脑研究，这项合作提案的总体目标是提供服务， 通过工程益生菌（例如，E. coli Nissle 1917（EcN），长双歧杆菌， 植物乳杆菌）生物合成各种天然存在的化学物质作为益生元-益生菌 结合物，将作为重要的实验室试剂销售，以研究天然产物对 通过调节肠道微生物组来治疗神经系统疾病。作为概念验证，EcN产生高水平的 5-羟色氨酸（5-HTP;血清素的天然生物合成前体）将被喂食给小鼠以评估其 生物利用度、药代动力学、耐受性、微生物组调节和毒性。我们最近 开发了一种新的微生物方法，通过合理地工程化苯丙氨酸4- 将色氨酸羟化酶转化为色氨酸5-羟化酶，并在 模型微生物大肠杆菌（K-12衍生物）。第一阶段的拟议研究将侧重于（目标1）测试 在E.大肠杆菌菌株BW 25113可以是 在益生菌菌株EcN（5-HTP+EcN）和（Aim 2）中使用，测试5-HTP+EcN没有不利的 效果，并且所产生的5-HTP是生物可利用的。我们选择EcN作为宿主菌株是因为它的安全性 已经有很好的记录，它可以直接与肠道中的粘膜树突状细胞相互作用，并促进 生物合成的生物分子本身的吸光度。此外，它可以在肠道中停留数周， 实现生物合成化合物的控释和缓释。HGG Research LLC是一家 该公司将开发在益生菌菌株中合成各种天然化合物的方法。的研究 目前在格鲁吉亚大学的学术合作伙伴将评估生物利用度， 微生物组和毒性。通过在不同益生菌中表达各种植物化学物质的研究设计 在小鼠模型中使用无诱导物表达系统的菌株将是该STTR之后的下一阶段 项目益生菌在肠道中生物合成天然产物与口服喂养相比具有几个优点 这些化合物。我们相信建议的研究具有创新性和重大意义;以及 代表了健康研究、代谢工程和合成生物学的前沿， 生物化学和工程学

PROJECT SUMMARY To advance microbiome-brain studies, the general goals of this collaborative proposal are to provide services to research community by engineering probiotics (e.g., E. coli Nissle 1917 (EcN), Bifidobacteria longum, Lactobacillus plantarum) to biosynthesize various naturally occurring chemicals as prebiotic-probiotic conjugates, which will be marketed as important laboratory agents to study the effects of natural products on neurological diseases by modulating gut microbiome. As a proof of concept, EcN that produces high levels of 5-hydroxytryptophan (5-HTP; a natural biosynthetic precursor of serotonin) will be fed to mice to evaluate its bioavailability, pharmacokinetic, tolerability, modulation of microbiome and toxicity. We have recently developed a novel microbial approach for 5-HTP production by rationally engineering a phenylalanine 4- hydroxylase into a tryptophan 5-hydroxylase and reconstituting a novel 5-HTP biosynthetic mechanism in a model microorganism E.coli (K-12 derivative). The proposed research in phase I will focus on (Aim 1) testing whether the novel microbial approach for producing high level 5-HTP in the E. coli strain BW25113 can be utilized in the probiotic strain EcN (5-HTP+EcN) and (Aim 2) on testing that the 5-HTP+EcN has no adverse effects, and the produced 5-HTP is biologically available. We choose EcN as the host strain because its safety has been well documented, and it can directly interact with mucosal dendritic cells in the gut and promote the absorbance of biomolecules biosynthesized by itself. Furthermore, it can reside in the gut for weeks and realize controlled release and sustained delivery of biosynthesized compounds. HGG Research LLC is a company that will develop methods to synthesize various natural compounds in probiotic strains. Research by the academic partner, currently at the University of Georgia, will evaluate the bioavailability, modulation of microbiome and toxicity. The design of studies by expressing various phytochemicals in different probiotic strains using an inducer-free expression system in a mouse model would be the next stage following this STTR project. Biosynthesis of natural products by probiotics in gut has several advantages compared to oral feeding of these compounds. We believe that the proposed research is innovative and of great significance; and represents a frontier in health research, metabolic engineering and synthetic biology with implications in biology, chemistry, and engineering.