Synthetic Biology Tools to unleash the Biotechnological Potential of the genus Burkholderia

释放伯克霍尔德氏菌属生物技术潜力的合成生物学工具

• 批准号: RGPIN-2022-04119
• 负责人: Cardona, Silvia
• 金额: $ 3.5万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760957
• 关键词: Synthetic; Biology; Tools; unleash; Biotechnological

Gram-negative bacteria of the genus Burkholderia are known for their biotechnological potential, large genomes, and versatile metabolism. However, two key barriers prevent the use of Burkholderia strains in industrial and agricultural settings. First, Burkholderia is both "friend and foe" as some Burkholderia strains can also cause infections in immunocompromised humans. Second, genetic manipulation is hampered by Burkholderia genomes' high G+C content, which makes standard recombinant DNA techniques difficult. The long-term goal of my research program is to enhance Burkholderia biotechnological potential using synthetic biology tools. We aim to generate Burkholderia strains able to reversibly switch between fast-growing and non-growing states with extended survival and efficient synthesis of the desired product. Starting the identification and characterization of genetic elements related to bioplastic degradation in Burkholderia vietnamiensis, the 5-year research plan of this program will focus on the enhancement of B. vietnamiensis biotechnological potential by genome reductions and extensive manipulation of essential gene networks as a biocontainment strategy. The specific objectives are: 1) To uncover Burkholderia bioplastic degradation capacity by large-scale genetic screens. Our HQP has developed a phenotypic screen and a large-scale transposon mutagenesis method to identify bioplastic degrading extracellular enzymes and related regulatory genetic elements. Trainees will screen for novel enzymatic activities and their regulatory genetic network, confirming gene-to-function links by insertional/deletion mutagenesis. 2) To improve Burkholderia biotechnological properties by genome reduction. Elimination of nonessential genes via genome reduction can enhance growth rate, fitness and increase product yields, as well as decreasing virulence. HQP will use LoxTnSeq to introduce large genome reductions, followed by fitness enrichment, growth rate evaluation and target gene expression quantification. 3) To explore parallel control of essential genes for biological containment. HQP will train in CRISPRi with mismatched gRNAs to manipulate the essential gene network. We will inhibit cell replication with extended survival and enhanced target product synthesis by parallel silencing of cell division with overexpression of protective and energy-generating genetic circuits. Significance: Burkholderia biotechnological potential is underdeveloped due to a lack of efficient genetic tools and the inability to contain potentially pathogenic strains in the environment. By solving these obstacles, we will unleash Burkholderia's potential for its use in industrial and agricultural settings. Overall, this program will highlight the power of synthetic biology to advance economic and environmental needs and provide HQP with opportunities to train in cutting-edge knowledge and techniques, preparing them well for jobs in the industry and the academic sector.

Burkholderia属的革兰氏阴性细菌以其生物技术潜力，大基因组和多功能代谢而闻名。但是，两个关键障碍阻止了在工业和农​​业环境中使用伯克霍尔德菌株。首先，Burkholderia既是“朋友又是敌人”，因为一些Burkholderia菌株也可能引起免疫功能低下的人类感染。其次，Burkholderia Genomes的高G+C含量阻碍了遗传操作，这使得标准的重组DNA技术变得困难。 我的研究计划的长期目标是使用合成生物学工具来增强Burkholderia生物技术潜力。我们旨在生成能够在快速增长和非生长状态之间可逆切换的Burkholderia菌株，并具有延长的生存和有效合成所需产品。开始鉴定和表征与Burkholderia越南语中与生物塑性降解有关的遗传因素，该计划的5年研究计划将重点介绍越南芽孢杆菌生物技术潜力的增强基因组减少和广泛的基因网络作为生物植物策略的广泛操纵。具体目标是：1）通过大规模的遗传筛选揭示Burkholderia生物塑性降解能力。我们的HQP开发了一种表型筛选和大规模的转座子诱变方法，以鉴定生物塑性降解细胞外酶和相关的调节遗传元件。受训者将筛选新的酶活性及其调节性遗传网络，从而通过插入/缺失诱变确认基因对功能的联系。 2）通过降低基因组来改善Burkholderia生物技术特性。通过减少基因组消除非必要基因可以提高生长速率，适应性和增加产物产量，并降低毒力。 HQP将使用LoxtNSEQ引入大型基因组减少，然后进行适应性富集，生长速率评估和靶基因表达定量。 3）探索对生物遏制基本基因的平行控制。 HQP将与不匹配的GRNA在CRISPRI进行训练，以操纵基本基因网络。我们将通过延长的生存率抑制细胞复制，并通过平行地静音细胞分裂，并过表达保护性和能量生成的遗传回路，从而抑制细胞复制。 意义：由于缺乏有效的遗传工具和无法在环境中含有潜在的致病菌株，Burkholderia生物技术潜力欠发达。通过解决这些障碍，我们将释放Burkholderia在工业和农​​业环境中使用的潜力。总体而言，该计划将强调合成生物学能够促进经济和环境需求，并为HQP提供培训最先进知识和技术的机会，为行业和学术领域的工作做好准备。