Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design

Chaves, Julie E.; Wilton, Rosemarie; Gao, Yuqian; Munoz, Nathalie Munoz; Burnet, Meagan C.; Schmitz, Zachary P; Rowan, John; Burdick, Leah H.; Elmore, Joshua R.; Guss, Adam M.; Close, Dan; Magnuson, Jon

doi:10.1016/j.mec.2020.e00139

Cited by 23 publications

(32 citation statements)

References 49 publications

(50 reference statements)

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“…A set of constitutive promoters (P mxaF , P coxB , P fumC , and P tuf ) of different strengths has been reported for the expression of genes in M. extorquens AM1 ( Schada Von Borzyskowski et al, 2015 ). In general, uncontrolled high-level expression or constitutive expression systems are not always desirable because they are detrimental to cells due to uncontrolled production of target protein(s) and structural instability ( Lee and Keasling, 2005 ; Chaves et al, 2020 ). Moreover, cumate- or anhydrotetracycline-inducible hybrid promoters (P R/cmtO and P R/tetO ) with low basal expression and high expression upon induction have been developed ( Chubiz et al, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

Sathesh‐Prabu

Ryu

Lee

2021

Front. Bioeng. Biotechnol.

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Methylorubrum extorquens AM1 is an efficient platform strain possessing biotechnological potential in formate- and methanol-based single carbon (C1) bioeconomy. Constitutive expression or costly chemical-inducible expression systems are not always desirable. Here, several glucose-, xylose-, and levulinic acid (LA)-inducible promoter systems were assessed for the induction of green fluorescent protein (GFP) as a reporter protein. Among them, the LA-inducible gene expression system (HpdR/PhpdH) showed a strong expression of GFP (51-fold) compared to the control. The system was induced even at a low concentration of LA (0.1 mM). The fluorescence intensity increased with increasing concentrations of LA up to 20 mM. The system was tunable and tightly controlled with meager basal expression. The maximum GFP yield obtained using the system was 42 mg/g biomass, representing 10% of the total protein content. The efficiency of the proposed system was nearly equivalent (90%–100%) to that of the widely used strong promoters such as PmxaF and PL/O4. The HpdR/PhpdH system worked equally efficiently in five different strains of M. extorquens. LA is a low-cost, renewable, and sustainable platform chemical that can be used to generate a wide range of products. Hence, the reported system in potent strains of M. extorquens is highly beneficial in the C1-biorefinery industry to produce value-added products and bulk chemicals.

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Section: Introductionmentioning

confidence: 99%

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

Sathesh‐Prabu

Ryu

Lee

2021

Front. Bioeng. Biotechnol.

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“…2C ), quite likely due to a higher transcriptional activity. This is not surprising, as the pedigree of strain P. putida PaW140 involved random chromosomal insertion of TOL genes and selection for the best growth on m- xylene, which plausibly favored insertions into regions of high transcriptional activity ( 37 – 39 ). Yet, when RNA-red signals were compared to those of the DAPI-stained nucleoid, most of them were observed away from the denser DNA regions.…”

Section: Resultsmentioning

confidence: 99%

Subcellular Architecture of the xyl Gene Expression Flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2

Kim

Goñi-Moreno

2021

mBio

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Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs exit from their initiation sites to move to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest the distinct subcellular motion of xyl mRNAs results from both innate properties of the sequences and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed within the background of current knowledge on the three-dimensional organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism. IMPORTANCE The transfer of information between DNA, RNA, and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware, i.e., the nucleoid, the RNA polymerase, and the ribosomes, are habitually taken for granted. In this work, we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida strain KT2440 through the nonhomogeneous milieu of the bacterial cytoplasm. The results expose that—similarly to computers—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code and accompanies the environmental lifestyle of this bacterium.

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“…Recently advances in CRISPR and other molecular biology techniques have allowed the integration of reporter genes into a high number of defined genomic sites. Significant variations in expression levels of reporter proteins by the site of genomic integration have been demonstrated in Saccharomyces [ 258 ], E. coli [ 259 , 260 ], Bacillus subtilis [ 261 ], Pseudomonas putida [ 262 ], and Acinetobacter baylyi [ 263 ]. Generally higher levels of expression are seen for integration at sites closer to the origin of replication, as during replication there is in essence a higher copy number of genes that are on the DNA strands replicated first [ 264 ].…”

Section: Transcription Vs Translation Engineeringmentioning

confidence: 99%

Intelligent host engineering for metabolic flux optimisation in biotechnology

Munro

Kell

2021

Biochemical Journal

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Optimising the function of a protein of length N amino acids by directed evolution involves navigating a ‘search space’ of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is ‘making such biology predictable’. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing kcat in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering.

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Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design

Cited by 23 publications

References 49 publications

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

Subcellular Architecture of the xyl Gene Expression Flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2

Intelligent host engineering for metabolic flux optimisation in biotechnology

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