Protocols for yTREX/Tn5‐based gene cluster expression in Pseudomonas putida

Abstract

SummaryBacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5‐based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX‐based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine‐1‐carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putida KT2440 as well as the use of β‐galactosidase‐encoding lacZ as a reporter of production levels.

SEVAtile: a standardised DNA assembly method optimised for Pseudomonas

“…Although these data clearly indicate that the T7 transcription system functions satisfactorily in our three‐vector system in Pseudomonas , further optimisations may be envisioned. The well‐known toxicity of both the T7 RNAP and the T7 lysozyme and the high leakiness of the system have restricted the broad implementation of the T7 transcriptional scheme in Pseudomonas thus far (Szafranski et al ., 1997; Shis and Bennett, 2013; Weihmann et al ., 2020). Interestingly, recent studies where the T7 lysozyme is replaced with an asRNA or a feedback‐loop to repress the unwanted basal expression of T7 RNAP show promising results in Pseudomonas and would enable the use of this widely used system in Pseudomonas hosts (Kushwaha and Salis, 2015; Liang et al ., 2018).…”

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida

“…Construction of P. putida KT2440 sal mRL E SK40 was performed as follows: DNA fragments with homology arms of ca. 30 bp as building blocks for the subsequent assembly cloning were obtained by PCR as described before ( Weihmann et al, 2020 ). Linearized plasmid backbones obtained by restriction endonuclease digestion with I-SceI or MauBI were dephosphorylated with FastAP (NEB, Frankfurt a. M, Germany, and Thermo Fisher Scientific GmbH, Waltham, MA, United States, respectively) according to the manufacturer’s instructions.…”

“…Vector assembly by recombinational cloning in uracil deficient yeast cells was performed as described before ( Domröse et al, 2017 ; Weihmann et al, 2020 ). Competent cells of Saccharomyces cerevisiae VL6-48 were prepared according to Gietz and Schiestl (2007) .…”

Marine Biosurfactants: Biosynthesis, Structural Diversity and Biotechnological Applications

“…Besides conventional restriction-ligation cloning, diverse “smart” protocols are established including type II restriction enzyme-based cloning strategies [223,224] and restriction-independent methods like phage enzyme-mediated recombination in E. coli and in vitro homology-based methods [225,226,227]. In addition, yeast recombination has been increasingly used for successful cloning and engineering of biosynthetic genes, for example, encoding biosurfactant synthesis [228,229,230].…”