High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering

Abstract

Application of single-stranded DNA recombineering for genome editing of species other than enterobacteria is limited by the efficiency of the recombinase and the action of endogenous mismatch repair (MMR) systems. In this work we have set up a genetic system for entering multiple changes in the chromosome of the biotechnologically relevant strain EM42 of Pseudomononas putida. To this end high-level heat-inducible co-transcription of the rec2 recombinase and P. putida's allele mutL E36KPP was designed under the control of the P L /cI857 system. Cycles of short thermal shifts followed by transformation with a suite of mutagenic oligos delivered different types of genomic changes at frequencies up to 10% per single modification. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segmentse.g., ribosomal-binding sites. These results enabled multiplexing of genome engineering of P. putida, as required for metabolic reprogramming of this important synthetic biology chassis.

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

“…Similar fold repression of targeted proteins by CRISPRi/dCas9 was recently reported 38 , suggestive of a limitation for existing CRISPR systems in P. putida . The plasmid-based CRISPRi system retained stable phenotype for 6 days, but can be further stabilized using genomic integration of the dCpf1/CRISPRi system or by developing multiplex gene deletion strategies 54 , 55 . Directly targeting proteins for degradation in a multiplex format 56 could eventually be applied to prokaryotes 57 and would sidestep the reliance on variable protein turnover kinetics.…”

Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

“…2. It has been shown that poor ssDNA uptake hampers the efficiency and multiplexing of recombineering-based techniques like MAGE and DIvERGE in P. putida 21 , L. lactis 22 , and M. smegmatis 23 . Optimization of transformation methods is therefore essential.…”

“…Superior ssDNA recombinases have been identified from prophages for P. putida 89 , K. pneumoniae 89 , B. subtilis 154 , L. lactis 22 , and M. smegmatis 153 , but the recombination frequencies remain well below those reported for E. coli , which in turn limits large-scale, multiplexed engineering efforts. Moreover, the use of alternative recombinases requires time-consuming optimization of recombinase expression levels and oligonucleotide design 21 , 89 , 153 , 154 . 4.…”

“…5 and 6. Inhibition of the native MMR system has been shown to increase homologous recombination frequencies in multiple non-traditional hosts 21 , 22 , 159 , 160 . Despite its success in E. coli , (transient) inhibition of the MMR system to improve recombineering has not been widely implemented, probably due to the fact that mechanistic details of MMR are still largely unknown.…”

“…This method is specifically designed to generate resistant mutants with no prior knowledge of possible resistance mechanisms. High-efficiency multi-site genomic editing ( HEMSE ) is a multiplexed recombineering method optimized for P. putida 21 . Due to lower recombination efficiencies compared to MAGE for E. coli , the automation aspect is not (yet) possible, hence the use of the novel HEMSE terminology.…”