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Journal of Microbiology and... Apr 2023Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production...
Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an strain and was found to be non-functional. When the host of the LA biosensor was switched from to KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.
Topics: Bacterial Proteins; Escherichia coli; Pseudomonas putida; Biosensing Techniques
PubMed: 36775859
DOI: 10.4014/jmb.2301.01021 -
MicrobiologyOpen Dec 2014Molecular chaperones function in various important physiological processes. Null mutants of genes for the molecular chaperone ClpB (Hsp104), and those that encode...
Molecular chaperones function in various important physiological processes. Null mutants of genes for the molecular chaperone ClpB (Hsp104), and those that encode J-domain proteins (DnaJ, CbpA, and DjlA), which may act as Hsp40 co-chaperones of DnaK (Hsp70), were constructed from Pseudomonas putida KT2442 (KT) to elucidate their roles. The KTΔclpB mutant showed the same heat shock response (HSR) as the wild-type, both in terms of heat-shock protein (Hsp) synthesis (other than ClpB) and in hsp gene expression; however, the mutant was quite sensitive to high temperatures and was unable to disaggregate into thermo-mediated protein aggregates, indicating that ClpB is important for cell survival after heat stress and essential for solubilization of protein aggregates. On the other hand, the KTΔdnaJ mutant was temperature-sensitive, and formed more protein aggregates (especially of high molecular weight) upon heat stress than did KT. P. putida CbpA, a probable Hsp, partially substituted the functions of DnaJ in cell growth and solubilization of thermo-mediated protein aggregates, and might be involved in the HSR which was regulated by a fine-tuning system(s) that could sense subtle changes in the ambient temperature and control the levels of σ(32) activity and quantity, as well as the mRNA levels of hsp genes.
Topics: Bacterial Proteins; Genotype; Heat-Shock Proteins; Heat-Shock Response; Hot Temperature; Phenotype; Pseudomonas putida
PubMed: 25303383
DOI: 10.1002/mbo3.217 -
Microbial Biotechnology Sep 2021Understanding metabolism is fundamental to access and harness bacterial physiology. In most bacteria, nutrient utilization is hierarchically optimized according to their...
Understanding metabolism is fundamental to access and harness bacterial physiology. In most bacteria, nutrient utilization is hierarchically optimized according to their energetic potential and their availability in the environment to maximise growth rates. Low-throughput methods have been largely used to characterize bacterial metabolic profiles. However, in-depth analysis of large collections of strains across several conditions is challenging since high-throughput approaches are still limited - especially for non-traditional hosts. Here, we developed a high-throughput dilution-resolved cultivation method for metabolic footprinting of Pseudomonas putida and Pseudomonas aeruginosa. This method was benchmarked against a conventional low-throughput time-resolved cultivation approach using either a synthetic culture medium (where a single carbon source is present) for P. putida or a complex nutrient mixture for P. aeruginosa. Dynamic metabolic footprinting, either by sugar quantification or by targeted exo-metabolomic analyses, revealed overlaps between the bacterial metabolic profiles irrespective of the cultivation strategy, suggesting a certain level of robustness and flexibility of the high-throughput dilution-resolved method. Cultivation of P. putida in microtiter plates imposed a metabolic constraint, dependent on oxygen availability, which altered the pattern of secreted metabolites at the level of sugar oxidation. Deep-well plates, however, constituted an optimal cultivation set-up yielding consistent and comparable metabolic profiles across conditions and strains. Altogether, the results illustrate the usefulness of this technological advance for high-throughput analyses of bacterial metabolism for both biotechnological applications and automation purposes.
Topics: Biological Transport; Culture Media; Metabolomics; Pseudomonas aeruginosa; Pseudomonas putida
PubMed: 34327837
DOI: 10.1111/1751-7915.13905 -
PloS One 2019Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant...
Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant growth promotion and bioremediation. Here we compared a set of 19 P. putida isolates obtained from sugarcane rhizosphere or bulk soil using a population genomics approach aiming to assess genomic and metabolic differences between populations from these habitats. Phylogenomics placed rhizosphere versus bulk soil strains in separate clades clustering with different type strains of the P. putida group. Multivariate analyses indicated that the rhizosphere and bulk soil isolates form distinct populations. Comparative genomics identified several genetic functions (GO-terms) significantly different between populations, including some exclusively present in the rhizosphere or bulk soil strains, such as D-galactonic acid catabolism and cellulose biosynthesis, respectively. The metabolic profiles of rhizosphere and bulk soil populations analyzed by Biolog Ecoplates also differ significantly, most notably by the higher oxidation of D-galactonic/D-galacturonic acid by the rhizosphere population. Accordingly, D-galactonate catabolism operon (dgo) was present in all rhizosphere isolates and absent in the bulk soil population. This study showed that sugarcane rhizosphere and bulk soil harbor different populations of P. putida and identified genes and functions potentially associated with their soil niches.
Topics: Antibiosis; Genetics, Population; Genome, Bacterial; Genomics; Metabolomics; Phylogeny; Pseudomonas putida; Rhizosphere; Saccharum; Soil Microbiology
PubMed: 31581220
DOI: 10.1371/journal.pone.0223269 -
Metabolic Engineering Jan 2023In recent years branched short-chain dicarboxylates (BSCD) such as itaconic acid gained increasing interest in both medicine and biotechnology. Their use as building...
In recent years branched short-chain dicarboxylates (BSCD) such as itaconic acid gained increasing interest in both medicine and biotechnology. Their use as building blocks for plastics urges for developing microbial upcycling strategies to provide sustainable end-of-life solutions. Furthermore, many BSCD exhibit anti-bacterial properties or exert immunomodulatory effects in macrophages, indicating a medical relevance for this group of molecules. For both of these applications, a detailed understanding of the microbial metabolism of these compounds is essential. In this study, the metabolic pathway of BSCD degradation from Pseudomonas aeruginosa PAO1 was studied in detail by heterologously transferring it to Pseudomonas putida. Heterologous expression of the PA0878-0886 itaconate metabolism gene cluster enabled P. putida KT2440 to metabolize itaconate, (S)- and (R)-methylsuccinate, (S)-citramalate, and mesaconate. The functions of the so far uncharacterized genes PA0879 and PA0881 were revealed and proven to extend the substrate range of the core degradation pathway. Furthermore, the uncharacterized gene PA0880 was discovered to encode a 2-hydroxyparaconate (2-HP) lactonase that catalyzes the cleavage of the itaconate derivative 2-HP to itatartarate. Interestingly, 2-HP was found to inhibit growth of the engineered P. putida on itaconate. All in all, this study extends the substrate range of P. putida to include BSCD for bio-upcycling of high-performance polymers, and also identifies 2-HP as promising candidate for anti-microbial applications.
Topics: Metabolic Networks and Pathways; Pseudomonas; Pseudomonas aeruginosa; Pseudomonas putida; Carboxylic Acids
PubMed: 36581064
DOI: 10.1016/j.ymben.2022.12.008 -
Applied and Environmental Microbiology Apr 2017Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of KT2440...
Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. This study maps the complete transcriptional response of KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization.
Topics: Anti-Bacterial Agents; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Imipenem; Molecular Sequence Annotation; Osmotic Pressure; Oxidative Stress; Pseudomonas putida; RNA, Antisense; RNA, Bacterial; RNA, Messenger; RNA, Small Untranslated; Sequence Analysis, RNA
PubMed: 28130298
DOI: 10.1128/AEM.03236-16 -
Scientific Reports Mar 2020The CbrA/CbrB system is a two-component signal transduction system known to participate in the regulation of the cellular carbon/nitrogen balance and to play a central...
The CbrA/CbrB system is a two-component signal transduction system known to participate in the regulation of the cellular carbon/nitrogen balance and to play a central role in carbon catabolite repression in Pseudomonas species. CbrA is composed of a domain with similarity to proteins of the solute/sodium symporter family (SLC5) and domains typically found in bacterial sensor kinases. Here, the functional properties of the sensor kinase CbrA and its domains are analyzed at the molecular level using the system of the soil bacterium P. putida KT2440 as a model. It is demonstrated that CbrA can bind and transport L-histidine. Transport is specific for L-histidine and probably driven by an electrochemical proton gradient. The kinase domain is not required for L-histidine uptake by the SLC5 domain of CbrA, and has no significant impact on transport kinetics. Furthermore, it is shown that the histidine kinase can autophosphorylate and transfer the phosphoryl group to the response regulator CbrB. The SLC5 domain is not essential for these activities but appears to modulate the autokinase activity. A phosphatase activity of CbrA is not detected. None of the activities is significantly affected by L-histidine. The results demonstrate that CbrA functions as a L-histidine transporter and sensor kinase.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Histidine; Histidine Kinase; Phosphorylation; Pseudomonas putida; Transcription Factors
PubMed: 32214184
DOI: 10.1038/s41598-020-62337-9 -
MSystems Jun 2023The soil bacterium is a robust biomanufacturing host that assimilates a broad range of substrates while efficiently coping with adverse environmental conditions. is...
The soil bacterium is a robust biomanufacturing host that assimilates a broad range of substrates while efficiently coping with adverse environmental conditions. is equipped with functions related to one-carbon (C1) compounds (e.g. methanol, formaldehyde, and formate) oxidation-yet pathways to assimilate these carbon sources are largely absent. In this work, we adopted a systems-level approach to study the genetic and molecular basis of C1 metabolism in . RNA sequencing identified two oxidoreductases, encoded by and , transcriptionally active in the presence of formate. Quantitative physiology of deletion mutants revealed growth defects at high formate concentrations, pointing to an important role of these oxidoreductases in C1 tolerance. Moreover, we describe a concerted detoxification process for methanol and formaldehyde, the C1 intermediates upstream formate. Alcohol oxidation to highly-reactive formaldehyde by PedEH and other broad-substrate-range dehydrogenases underpinned the (apparent) suboptimal methanol tolerance of . Formaldehyde was mostly processed by a glutathione-dependent mechanism encoded in the operon, and thiol-independent FdhAB and AldB-II overtook detoxification at high aldehyde concentrations. Deletion strains were constructed and characterized towards unveiling these biochemical mechanisms, underscoring the worth of for emergent biotechnological applications-e.g. engineering synthetic formatotrophy and methylotrophy. IMPORTANCE C1 substrates continue to attract interest in biotechnology, as their use is both cost-effective and ultimately expected to mitigate the impact of greenhouse gas emissions. However, our current understanding of bacterial C1 metabolism remains relatively limited in species that cannot grow on (i.e., assimilate) these substrates. , a model Gram-negative environmental bacterium, constitutes a prime example of this sort. The biochemical pathways active in response to methanol, formaldehyde, and formate have been largely overlooked-although the ability of to process C1 molecules has been previously alluded to in the literature. By using a systems-level strategy, this study bridges such knowledge gap through the identification and characterization of mechanisms underlying methanol, formaldehyde, and formate detoxification-including hitherto unknown enzymes that act on these substrates. The results reported herein both expand our understanding of microbial metabolism and lay a solid foundation for engineering efforts toward valorizing C1 feedstocks.
Topics: Pseudomonas putida; Methanol; Carbon; Formaldehyde; Formates; Oxidoreductases
PubMed: 37273222
DOI: 10.1128/msystems.00004-23 -
Applied and Environmental Microbiology Jun 2020S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic...
S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies revealed that S12 contains the single-copy 583-kbp megaplasmid pTTS12. pTTS12 carries several important operons and gene clusters facilitating S12 survival and growth in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from S12 and conclusively confirmed that the SrpABC efflux pump is the major determinant of solvent tolerance on the megaplasmid pTTS12. In addition, we identified a novel toxin-antitoxin module (proposed gene names and respectively) encoded on pTTS12 which contributes to the solvent tolerance phenotype and is important for conferring stability to the megaplasmid. Chromosomal introduction of the operon in combination with the AT gene pair created a solvent tolerance phenotype in non-solvent-tolerant strains, such as KT2440, TG1, and BL21(DE3). Sustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during the bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates, or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant S12 for molecular adaptation to an organic solvent. In addition to the solvent extrusion pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to short-term tolerance in moderate solvent concentrations, as well as to the stability of pTTS12. These two gene clusters were successfully expressed in non-solvent-tolerant strains of and strains to confer and enhance solvent tolerance.
Topics: Bacterial Toxins; Plasmids; Pseudomonas putida; Solvents
PubMed: 32358012
DOI: 10.1128/AEM.00686-20 -
Essays in Biochemistry Nov 2016The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to... (Review)
Review
The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to express the genes encoded in it. Despite being an essential element for the appropriate function of genetic circuits, the chassis is rarely considered in their design phase. Consequently, the circuits are transferred to model organisms commonly used in the laboratory, such as Escherichia coli, that may be suboptimal for a required function. In this review, we discuss some of the properties desirable in a versatile chassis and summarize some examples of alternative hosts for synthetic biology amenable for engineering. These properties include a suitable life style, a robust cell wall, good knowledge of its regulatory network as well as of the interplay of the host components with the exogenous circuits, and the possibility of developing whole-cell models and tuneable metabolic fluxes that could allow a better distribution of cellular resources (metabolites, ATP, nucleotides, amino acids, transcriptional and translational machinery). We highlight Pseudomonas putida, widely used in many different biotechnological applications as a prominent organism for synthetic biology due to its metabolic diversity, robustness and ease of manipulation.
Topics: Genetic Engineering; Metabolic Networks and Pathways; Pseudomonas putida; Synthetic Biology
PubMed: 27903818
DOI: 10.1042/EBC20160015