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

伯克氏菌属的革兰氏阴性细菌以其生物技术潜力、大基因组和多种代谢而闻名。然而，有两个主要障碍阻碍了伯克霍尔德菌菌株在工业和农业环境中的使用。首先，伯克氏菌既是“朋友也是敌人”，因为一些伯克氏菌菌株也可以在免疫功能低下的人身上引起感染。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，这使得标准的重组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菌菌株，延长生存时间并有效合成所需产品。从越南伯克霍尔德菌生物塑料降解相关基因的鉴定和鉴定开始，本项目的5年研究计划将集中于通过基因组还原和广泛操纵必要基因网络来增强越南伯克霍尔德菌的生物技术潜力，作为一种生物控制策略。具体目标是：1)通过大规模遗传筛选揭示伯克霍尔德菌的生物塑料降解能力。我们的HQP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细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基因网络，通过插入/删除突变确认基因与功能之间的联系。2)通过基因组还原提高伯克霍尔德菌的生物技术特性。通过基因组还原去除非必需基因可以提高生长速度、适应性和提高产品产量，并降低毒力。HQP将使用LoxTnSeq引入大基因组缩减，随后进行适应度富集、生长速率评估和靶基因表达量化。3)探索生物防控必要基因的平行控制。HQP将在CRISPRi中训练不匹配的grna来操纵必要的基因网络。我们将通过抑制细胞分裂和过度表达保护性和产生能量的基因回路来延长细胞的存活时间，并增强靶产物的合成。意义：由于缺乏有效的遗传工具和无法在环境中控制潜在致病性菌株，伯克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使用的潜力。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HQP提供尖端知识和技术培训的机会，为他们在工业和学术领域的工作做好准备。

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.