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

基本信息

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

项目摘要

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.

伯克霍尔德氏菌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。然而，两个关键障碍阻碍了伯克霍尔德杆菌菌株在工业和农业环境中的使用。首先，伯克霍尔德杆菌既是“朋友也是敌人”，因为一些伯克霍尔德杆菌菌株也会引起免疫功能低下的人类感染。其次，伯克霍尔德氏菌基因组的高 G+C 含量阻碍了基因操作，这使得标准重组 DNA 技术变得困难。我的研究计划的长期目标是利用合成生物学工具增强伯克霍尔德杆菌的生物技术潜力。我们的目标是产生能够在快速生长和非生长状态之间可逆切换的伯克霍尔德杆菌菌株，并延长存活时间并有效合成所需产物。该项目的五年研究计划将开始鉴定和表征与越南伯克霍尔德杆菌生物塑料降解相关的遗传元件，重点是通过基因组减少和对基本基因网络的广泛操作作为生物遏制策略来增强越南伯克霍尔德杆菌的生物技术潜力。具体目标是：1）通过大规模遗传筛选揭示伯克霍尔德杆菌的生物塑料降解能力。我们的总部开发了表型筛选和大规模转座子诱变方法，以鉴定生物塑料降解胞外酶和相关的调控遗传元件。学员将筛选新的酶活性及其调控遗传网络，通过插入/删除突变确认基因与功能的联系。 2) 通过基因组缩减来改善伯克霍尔德氏菌的生物技术特性。通过基因组缩减消除非必需基因可以提高生长速度、适应性并提高产品产量，并降低毒力。 HQP 将使用 LoxTnSeq 引入大规模基因组缩减，然后进行适应性富集、生长速率评估和目标基因表达量化。 3）探索生物遏制必需基因的并行控制。 HQP 将使用不匹配的 gRNA 训练 CRISPRi，以操纵重要的基因网络。我们将通过平行沉默细胞分裂和过度表达保护性和能量产生遗传电路来抑制细胞复制，从而延长生存期并增强目标产物合成。意义：由于缺乏有效的遗传工具以及无法遏制环境中的潜在致病菌株，伯克霍尔德氏菌的生物技术潜力尚未得到充分开发。通过解决这些障碍，我们将释放伯克霍尔德杆菌在工业和农业环境中的应用潜力。总体而言，该计划将突出合成生物学在促进经济和环境需求方面的力量，并为总部提供接受尖端知识和技术培训的机会，为他们在行业和学术领域的工作做好准备。