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

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，两个关键障碍阻止伯克霍尔德菌菌株在工业和农业环境中的使用。首先，伯克霍尔德氏菌既是“朋友也是敌人”，因为一些伯克霍尔德氏菌菌株也会导致免疫功能低下的人类感染。其次，遗传操作受到伯克霍尔德氏菌基因组高G+C含量的阻碍，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提高伯克霍尔德氏菌的生物技术潜力。我们的目标是产生伯克霍尔德菌菌株能够可逆地快速增长和非增长状态之间的转换，延长生存期和有效合成所需的产品。从越南伯克霍尔德菌中与生物塑料降解相关的遗传元件的鉴定和表征开始，该计划的5年研究计划将重点关注B的增强。通过减少基因组和广泛操纵基本基因网络作为生物遏制战略，发挥了越南小球藻的生物技术潜力。具体目标是：1）通过大规模遗传筛选揭示伯克霍尔德菌生物塑料降解能力。我们的HQP已经开发了一种表型筛选和大规模转座子诱变方法，以确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/缺失诱变确认基因与功能的联系。 2)通过基因组减少来改善伯克霍尔德氏菌的生物技术特性。通过基因组减少消除非必需基因可以提高生长速度，健身和增加产品产量，以及降低毒力。HQP将使用LoxTnSeq引入大基因组减少，然后进行适应性富集，生长速率评估和靶基因表达定量。3)探索生物遏制中必需基因的平行调控。HQP将在CRISPRi中使用错配的gRNA进行训练，以操纵必需的基因网络。我们将抑制细胞复制，延长生存期，并通过平行沉默细胞分裂与过度表达的保护性和能量产生的遗传回路来增强靶产物的合成。 重要性：由于缺乏有效的遗传工具和无法控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开发。通过解决这些障碍，我们将释放伯克霍尔德菌在工业和农业环境中的潜力。总体而言，该计划将突出合成生物学的力量，以促进经济和环境需求，并为HQP提供培训尖端知识和技术的机会，为他们在行业和学术部门的工作做好准备。

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.