An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

Abstract

Summary Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

“…Furthermore, a dynamic regulation approach can be employed to balance the biochemical activities based on metabolite concentrations: CRISPR-interference [159][160][161] as well as conditional proteolysis systems [162] have been implemented in Pseudomonas and can be used to down-regulate biochemical functions leading up to the toxic intermediate (e.g. by coupling the output to a suitable expression system, biosensor or riboswitch).…”

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

“… TF Promoter Inducer(s) Reported pros, cons and main applications in P. putida Ref. (s) Positively regulated induction systems XylS Pm m -toluate and derivatives + High expression levels + Extensively characterized + Cheap inducers − Catabolite repression − Dose-dependent response in absence of inducer metabolization pathways − Bimodal response − Leaky expression 28 , 49 , 63 , 130 – 139 Applications: Expression of I- sceI , dCas9 , trfA , the λ red operon, and recombinases in genome engineering tools; expression of toxic genes for controlled autolysis; production of recombinant antibody fragments and p -coumaric acid RhaRS P rhaB l -rhamnose + No catabolite repression + No metabolization of rhamnose: dose-dependent response + Tight regulation + Non-toxic inducer − Inhomogeneous response at intermediate inducer levels − Expensive inducers 132 , 138 , 140 , 141 Applications: Expression of dCas9 for CRISPRi and Cre for genomic deletions; production of p -coumaric acid AraC P BAD l -arabinose + Characterization + No catabolite repression + No metabolization of arabinose: dose-dependent response + Tight regulation + Non-toxic inducer − Inhomogeneous response at intermediate inducer levels − Poor arabinose uptake without AraE transporter − Expensive inducers 132 , 142 , 143 Applications: Production of p -coumaric acid Negatively regulated induction systems LacI Plac, PlacUV5, Ptac, Ptrc Lactose, isopropyl β...…”

“…In some applications, the lack of a suitable repressor can be substituted by CRISPR interference (CRISPRi) technology 39 . This technology enables the simultaneous, tunable, and transient repression of multiple genes by expression of gene-specific sgRNAs and a single nuclease-deficient Cas9 (dCas9) and has been successfully applied in P. putida 49 , M. smegmatis 50 , K. pneumoniae 51 , B. subtilis 51 , and L. lactis 52 with up to 98% repression. Although this technique offers great potential, one must note that its efficiency and efficacy remains dependent on (1) the presence of a PAM sequence within or near the promoter region for efficient transcriptional repression, (2) the strengths and weaknesses of the expression system used for dCas9 expression, (3) the burden imposed by accumulation of a large heterologous protein (dCas9), and (4) polar effects such as the repression of downstream operons.…”

Industrial biotechnology of Pseudomonas putida: advances and prospects

“…More recent innovative developments deal with recombineering methods, such as RecET-based markerless recombineering system for deletion and integration of large-sized genes and clusters (Choi and Lee 2020 ), efficient single-stranded recombineering by using a thermoinducible system (Aparicio et al 2020a ), as well as CRISPR/Cas9 technologies. The latter was utilized for efficient curing of helper plasmids (Wirth et al 2020 ), counterselection of infrequent mutations created through recombineering (Aparicio et al 2018 ), metabolic engineering for PHA bioconversion from ferulic acid (Zhou et al 2020 ), as well as CRISPR interference-mediated gene regulation (Batianis et al 2020 ; Kim et al 2020 ). The incredible pace of development of new tools leaves no doubt that there will be precise CRISPR-based technologies soon, speeding up genomic manipulations even further.…”