Development of synthetic biology tools for Pseudomonas putida

Biotechnological applications become a forward-looking alternative to chemical production of fine and bulk chemicals. The envisaged circular (bio)economy is aiming to replace fossil resources by renewable biomass, CO2, and other carbon-rich streams like plastic. Microbial hosts with an expanded range of substrates are used to produce all required products. Here, synthetic biology is used for the tailoring of hyperproducing strains, starting with the prominent chassis strain Pseudomonas putida. P. putida KT2440 has a versatile metabolism and high resistance towards oxidative stress. Biotechnological applications become a forward-looking alternative to chemical production of fine and bulk chemicals. The envisaged circular (bio)economy is aiming to replace fossil resources by renewable biomass, CO2, and other carbon-rich streams like plastic. Microbial hosts with an expanded range of substrates are used to produce all required products. Here, synthetic biology is used for the tailoring of hyperproducing strains, starting with the prominent chassis strain Pseudomonas putida. P. putida KT2440 has a versatile metabolism and high resistance towards oxidative stress. This thesis aimed to increase the number of rationally designed and well-characterized genetic tools for P. putida KT2440 to foster synthetic biology. This thesis contributes to the reliability of synthetic biology that often proved as a major issue. We constructed and characterized several sigma-70 factor dependent synthetic promoters and combinations for heterologous gene expression. A library was constructed with single nucleotide exchanges, which reveals that the -35 and -10 regions are crucial for efficient promoter activity. Combined promoters, so-called stacked promoters, allowed, after detailed characterization of the genetic context, prediction of the resulting expression strength. Stable genomic integration is often used for metabolic engineering, but characterized sites, so-called landing pads, across the genome of P. putida KT2440 are missing. We analyzed RNA-Seq data towards regions that are equally expressed to identify suitable integration sites across the genome of P. putida KT2440. These landing pads enabled high heterologous expression with low variability. With well-characterized promoters and landing pads, fine tuning of gene expression can be conducted on two levels, promoter and integration site. Additionally, the thesis delivers tools for marker recycling and an alternative sigma dependent promoter. In summary, this thesis contributes to P. putida KT2440 synthetic biology. The increased number of well-characterized tools will further support the many efforts to establish this microbe as a workhorse in the bioeconomy and biotechnological applications. Getting the tough challenges, like the use of aromatic compounds from plastic or lignin as substrates, will distinguish P. putida KT2440 from the well-established microbes