Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

Abstract

CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing a widely-applicable genome engineering toolset for Gram-negative bacteria. The challenge is addressed by tailoring a CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly supports multiplex base editing with >85% efficiency. The toolset is adopted to construct and deconstruct complex phenotypes in the soil bacterium Pseudomonas putida. Single-step engineering of an aromatic-compound production phenotype and multi-step deconstruction of the intricate redox metabolism illustrate the versatility of multiplex base editing afforded by our toolbox. Hence, this approach overcomes typical limitations of previous technologies and empowers engineering programs in Gram-negative bacteria that were out of reach thus far.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

“…The elimination of GlcB, GltA, and PrpC did not affect the growth of SCA18 on citrate. Hence, there appear to be unknown enzymatic functions in P. putida that establish a biochemical route from citrate to AcCoA, highlighting the role of 'underground' metabolic functions that come into play in the absence of the main reactions that supply biomass precursors or redox equivalents [50].…”

“…E. coli DH5α λpir was employed for cloning purposes. Chemically-competent E. coli cells were prepared and transformed with plasmids according to well-established protocols [46][47][48][49][50][51]. P. putida was rendered electro-competent by treatment Choi et al [52].…”

Challenges and opportunities in bringing nonbiological atoms to life with synthetic metabolism

“…Blombach and colleagues [117] provide a superb overview of selection criteria for no-traditional microbial hosts. For an in-depth review of industrially relevant chassis (both established and emerging), the reader is referred to a series of recent publications on the topic [118][119][120][121][122][123][124].…”

Integrated rational and evolutionary engineering of genome-reducedPseudomonas putidastrains empowers synthetic formate assimilation

“…The trimming procedure included (i) adapters using presets for Illumina adapters, (ii) read ends based on quality (Q>20), (iii) adapters based on paired read overhangs and (iv) short reads (<20 bp). Mutations with a frequency above 50% and excluded from low coverage regions were identified after comparison with the pre-evolved strains as described previously (46,47).…”