Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440

Abstract

Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli, the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while use of homologous recombination for insertion of DNA into the chromosome is slow and laborious. Further, most heterologous expression efforts to date typically rely on overexpression of exogenous pathways using a handful of poorly characterized promoters. To improve the P. putida toolkit, we developed a rapid genome integration system using the site-specific recombinase from bacteriophage Bxb1 to enable rapid, high efficiency integration of DNA into the P. putida chromosome. We also developed a library of synthetic promoters with various UP elements, −35 sequences, and −10 sequences, as well as different ribosomal binding sites. We tested these promoters using a fluorescent reporter gene, mNeonGreen, to characterize the strength of each promoter, and identified UP-element-promoter-ribosomal binding sites combinations capable of driving a ~150-fold range of protein expression levels. An additional integrating vector was developed that confers more robust kanamycin resistance when integrated at single copy into the chromosome. This genome integration and reporter systems are extensible for testing other genetic parts, such as examining terminator strength, and will allow rapid integration of heterologous pathways for metabolic engineering.

The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria

“…Homologous recombinationbased allelic exchange relies upon endogenous DNA-repair machinery -which is typically inefficient 27 , precluding the construction of even moderately-sized strain libraries. Integrase-mediated recombination technologies [28][29][30][31][32][33][34][35][36][37] have the potential overcome many of the limitations faced by the above genetic tools. Phage integrases (or site-specific recombinases) are enzymes that catalyze recombination between two specific sequences of DNA 34,38 .…”

“…Furthermore, serine integrases, such as Bxb1 and FC31, have been used across the tree of life and thus are likely to work in the vast majority of organisms. Phage integrase-based tools are often limited to integration of a single DNA fragment 28,29,31,32,35,36,39 or function in a limited range of hosts 30,33,40 . To address these issues and limitations, in this work we developed the Serine-integrase Assisted Genome Engineering (SAGE) system.…”

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

“…Chinese hamster ovary (CHO) cells, used for the manufacture of a wide range of heterologous proteins, require a demanding selection process to generate production clones efficiently . This is because the expression level of the heterologous gene is determined by multiple factors, including the strength and efficiency of regulatory sequences directing its transcription and processing into messenger RNA (mRNA), the chromosomal integration site, the copy number, the efficiency of translation as well as specific requirements of the particular protein such as post‐translational assembly and modification steps, and finally, secretion . In this context, promoters and their genetic control elements such as enhancers play an important role in integrating and processing gene expression …”

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

“…Due to the physicochemical stresses that prevail in the niches in which the soil bacterium Pseudomonas putida thrives (it is typically abundant in sites contaminated by industrial pollutants), this microorganism is endowed with a large number of traits desirable in hosts of harsh biotransformations of industrial interest (Nikel et al, 2014). The P. putida strain KT2440 is a saprophytic, non-pathogenic, GRAS-certified (Generally Recognized as Safe) bacterium; as the most thoroughly characterized laboratory pseudomonad, it has an expanding catalogue of available systems and synthetic biology tools (Aparicio et al, 2017; Elmore et al, 2017; Martínez-García and de Lorenzo, 2017, p.; Nogales et al, 2017). This bacterium is becoming a laboratory workhorse as well as a valued cell factory (Benedetti et al, 2016; Loeschcke and Thies, 2015; Nikel and de Lorenzo, 2013; Poblete-Castro et al, 2012).…”