Development of advanced genetic engineering tools and a biological platform for upcycling depolymerized biomass and plastic waste streams

An organism with robust catabolic capabilities and high tolerance to many industrial bioprocess stressors is highly desirable for applications in industrial biotechnology. Therefore, Pseudomonas putida KT2440 is a promising host for the biological conversion of depolymerized lignocellulosic biomass and plastic waste streams. Advances in synthetic biology have greatly increased the rate at which novel DNA elements can be synthesized; however, in most non-model organisms, stable genetic integration of these elements into the chromosome is a limiting factor. Previously, a highly efficient and accurate genomic integration system was demonstrated using Serine recombinase-Assisted Genome Engineering (SAGE), which can facilitate numerous engineering strategies. In this work I have expanded the capabilities of SAGE to include multiplexing (mSAGE) to further accelerate strain engineering. This technology was applied to rapidly screen a collection of orthogonal genes to assemble a non-native catabolic pathway in P. putida and establish optimal combinations for growth. With the mSAGE tools, I constructed a gene ortholog strain library that was used to determine optimal genetic composition following a growth-based selection process. Beyond optimization of catabolic pathways, I sought to establish P. putida as a robust host for production of industrial chemicals. I identified a combination of genes that encode a biosynthetic pathway to drive the accumulation of medium-chain length (mcl) alcohols. I achieved the first demonstration of mcl-alcohol production from a model lignin substrate with this base pathway. The initial pathway produced total mcl-alcohol titers in the range of 80-90 mg/L. To increase production, investigation of alternate pathway orthologs was pursued. In silico tools were utilized to identify orthologs that may possess improved functionality, efficiency, or substrate specificity. I constructed an mSAGE-based plasmid library to integrate eighty ortholog combinations into P. putida and evaluated strains for variations in titers, yields, and chain length composition. Experimental assessment of the ortholog library revealed combinations that delivered increases in total mcl-alcohol titers as well as shifts in chain length composition. Together, these results and the established utility of the mSAGE tools continue the expansion of P. putida as a robust industrial biotechnology host organism.