Engineering probiotic yeast for efficient sulforaphane delivery

WS00393217的项目摘要/摘要 主要调查者：查尔斯·登比，博士(BFS) 高效输送萝卜硫素的工程益生菌酵母 食用十字花科蔬菜，如花椰菜、球芽甘蓝和花椰菜 以降低罹患多种癌症的风险。最近的研究表明，这些健康 益处的部分原因是异硫氰酸酯的生化活性，这是一类在 这些蔬菜。萝卜硫醚(SFN)，一种由十字花科植物如 花椰菜和羽衣甘蓝，已显示出特别令人印象深刻的潜力，作为化学预防和癌症治疗 在体外和动物模型研究中。严格评估三氟化硫的健康益处一直是一项挑战 然而，人类。这些挑战源于SFN固有的不稳定性，以及 不同的食品制备方法和个人微生物组组成对SFN的生物利用度有影响。在这 我们计划设计一种益生菌酵母，它将从人类体内生物合成SFN。 胃肠道。这种益生菌酵母将提供稳定和直接的SFN来源，很容易 扩散到血液中，不依赖于特定的微生物组组成、食物制备 方法，或药物治疗方案。鉴于大量研究详细描述了SFN治疗的潜力 在前列腺癌细胞培养和动物模型中，我们将开发这种益生菌用于潜在的前列腺癌 癌症治疗。我们将使用的益生菌作为我们工程工作的宿主是安全的，并且 基因上易驯化的益生菌酵母，布拉氏链霉菌。经过工程改造的布氏链球菌菌株将提供一种方法 直接评估SFN在前列腺癌治疗中的健康益处，同时也提供了一种机制 有效地为癌症治疗和预防目的提供SFN。第一阶段的实验将开发一种 该菌株分泌一种高活性的黑芥子酶，该酶能够在 SFN的生物合成。为了实现这一目标，我们将筛选黑芥子酶、分泌信号和酵母菌的文库 启动子以确定在胃肠道中最有效地产生SFN的遗传成分 条件。为了建立原则证明并推动进一步的研究，第一阶段将寻求设计 每天生产200微克分子SFN的布氏链霉菌，作为管理这个数量的小型临床试验 已经显示出令人鼓舞的结果。在第二阶段的研究中，我们将进一步改造布拉第链霉菌，使其包含 整个SFN生物合成途径，并从可发酵的糖产生SFN。由此产生的菌株将是 在前列腺癌小鼠模型中测试安全性和防癌特性。该项目直接在 与NCI的使命一致，因为它将促进我们对前景光明的癌症治疗分子的理解， 同时也为癌症治疗和预防提供了一种直接的方式。

Project Summary / Abstract for WS00393217 Principal Investigator: Charles Denby, PhD (BFS) Engineering probiotic yeast for efficient sulforaphane delivery Consumption of cruciferous vegetables such as broccoli, brussels sprouts, and cauliflower has been shown to reduce the risk of developing multiple forms of cancer. Recent research has suggested that these health benefits result in part from the biochemical activity of isothiocyanates, a class of natural products found in these vegetables. Sulforaphane (SFN), one such isothiocyanate produced in abundance by crucifers like broccoli and kale, has shown especially impressive potential as a chemo-preventative and cancer therapeutic in in vitro and animal model studies. It has been challenging to rigorously assess the health benefits of SFN in humans, however. These challenges result from SFN’s inherent instability, as well as the varying effects that different food preparation methods and personal microbiome compositions have on SFN bioavailability. In this STTR, we propose to engineer a probiotic yeast that will biosynthesize SFN from within the human gastrointestinal tract. This probiotic yeast will provide a consistent and direct source of SFN that readily diffuses into the bloodstream, and is not dependent on specific microbiome compositions, food preparation methods, or drug regimens. Given the impressive body of research detailing the potential for SFN to treat prostate cancer in cell culture and animal models, we will develop this probiotic for potential use as a prostate cancer therapeutic. The probiotic microbe we will use as a host for our engineering efforts is the safe, and genetically tractable probiotic yeast, S. boulardii. The engineered S. boulardii strain will provide a means to directly assess the health benefits of SFN in prostate cancer therapy, while also providing a mechanism to efficiently deliver SFN for cancer therapeutic and preventative purposes. Phase I experiments will develop a S. boulardii strain that secretes a highly active myrosinase enzyme that is able to perform the terminal step in SFN biosynthesis. To achieve this goal, we will screen libraries of myrosinases, secretion signals, and yeast promoters to identify the genetic components that most effectively produce SFN under gastrointestinal conditions. To establish a proof-of-principle and motivate further research, Phase I will seek to engineer S. boulardii for production of 200 µmoles SFN per day, as small clinical trials that administered this quantity have shown encouraging results. In Phase II research, we will further engineer S. boulardii such that it contains the entire SFN biosynthesis pathway and produces SFN from fermentable sugars. Resultant strains will be tested in prostate cancer mouse models for safety and cancer-preventive properties. This project is directly in line with the NCI mission, as it will advance our understanding of a promising cancer therapeutic molecule, while also providing a direct means of delivering this potential therapeutic for cancer therapy and prevention.