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

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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.