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Microbes and Environments 2023Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they...
Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they potentially differ from the genetically defined "true Pseudomonas putida" because many were classified as P. putida based on their phenotypic and metabolic characteristics. A phylogenetic ana-lysis based on the concatenated sequences of the 16S rRNA and rpoD genes revealed that 46 strains of P. putida deposited in Japanese culture collections were classified into nine operational taxonomic units (OTUs) and eleven singletons. The OTU7 strain produces N-acylhomoserine lactone as a quorum-sensing signal. One of the OTU7 strains, JCM 20066, exhibited a ppuI-rsaL-ppuR quorum-sensing system that controls biofilm formation and motility. The P. putida type strain JCM 13063 and six other strains were classified as OTU4. Classification based on the calculation of whole-genome similarity revealed that three OTU4 strains, JCM 20005, 21368, and 13061, were regarded as the same species as JCM 13063 and defined as true P. putida. When orthologous genes in the whole-genome sequences of true P. putida strains were screened, PP4_28660 from P. putida NBRC 14164 (=JCM 13063) was present in all true P. putida genome sequences. The internal region of PP4_28660 was successfully amplified from all true P. putida strains using the specific primers designed in this study.
Topics: Bacterial Typing Techniques; DNA, Bacterial; Fatty Acids; Genomics; Phylogeny; Pseudomonas putida; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 37286511
DOI: 10.1264/jsme2.ME23019 -
Scientific Reports Sep 2021Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter...
Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/P was also efficiently induced by LA. LvaR/P and XutR/P systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/P showed weak GFP expression. These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.
Topics: Flow Cytometry; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Reporter; Genetic Engineering; Genetic Vectors; Glucose; Lactic Acid; Promoter Regions, Genetic; Pseudomonas putida; Recombinant Proteins
PubMed: 34508142
DOI: 10.1038/s41598-021-97550-7 -
Applied Microbiology and Biotechnology Jun 2024Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene... (Review)
Review
Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene terephthalate) (PET). Recently, the use of EG as a starting material for the production of bio-fuels or bio-chemicals is gaining attention as a sustainable process since EG can be derived from materials not competing with human food stocks including CO, syngas, lignocellulolytic biomass, and PET waste. In order to design and construct microbial process for the conversion of EG to value-added chemicals, microbes capable of catabolizing EG such as Escherichia coli, Pseudomonas putida, Rhodococcus jostii, Ideonella sakaiensis, Paracoccus denitrificans, and Acetobacterium woodii are candidates of chassis for the construction of synthetic pathways. In this mini-review, we describe EG catabolic pathways and catabolic enzymes in these microbes, and further review recent advances in microbial conversion of EG to value-added chemicals by means of metabolic engineering. KEY POINTS: • Ethylene glycol is a potential next-generation feedstock for sustainable industry. • Microbial conversion of ethylene glycol to value-added chemicals is gaining attention. • Ethylene glycol-utilizing microbes are useful as chassis for synthetic pathways.
Topics: Ethylene Glycol; Metabolic Engineering; Metabolic Networks and Pathways; Bacteria; Pseudomonas putida; Biofuels; Escherichia coli
PubMed: 38861200
DOI: 10.1007/s00253-024-13179-2 -
Microbiology (Reading, England) Jan 2023The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive...
The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS-GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.
Topics: Type VI Secretion Systems; Bacterial Proteins; Pseudomonas putida; Sigma Factor; Multigene Family; Gene Expression Regulation, Bacterial
PubMed: 36748579
DOI: 10.1099/mic.0.001295 -
Microbial Cell Factories Jan 2023Pseudomonas putida has received increasing interest as a cell factory due to its remarkable features such as fast growth, a versatile and robust metabolism, an extensive...
BACKGROUND
Pseudomonas putida has received increasing interest as a cell factory due to its remarkable features such as fast growth, a versatile and robust metabolism, an extensive genetic toolbox and its high tolerance to oxidative stress and toxic compounds. This interest is driven by the need to improve microbial performance to a level that enables biologically possible processes to become economically feasible, thereby fostering the transition from an oil-based economy to a more sustainable bio-based one. To this end, one of the current strategies is to maximize the product-substrate yield of an aerobic biocatalyst such as P. putida during growth on glycolytic carbon sources, such as glycerol and xylose. We demonstrate that this can be achieved by implementing the phosphoketolase shunt, through which pyruvate decarboxylation is prevented, and thus carbon loss is minimized.
RESULTS
In this study, we introduced the phosphoketolase shunt in the metabolism of P. putida KT2440. To maximize the effect of this pathway, we first tested and selected a phosphoketolase (Xfpk) enzyme with high activity in P. putida. Results of the enzymatic assays revealed that the most efficient Xfpk was the one isolated from Bifidobacterium breve. Using this enzyme, we improved the P. putida growth rate on glycerol and xylose by 44 and 167%, respectively, as well as the biomass yield quantified by OD by 50 and 30%, respectively. Finally, we demonstrated the impact on product formation and achieved a 38.5% increase in mevalonate and a 25.9% increase in flaviolin yield from glycerol. A similar effect was observed on the mevalonate-xylose and flaviolin-xylose yields, which increased by 48.7 and 49.4%, respectively.
CONCLUSIONS
Pseudomonas putida with the implemented Xfpk shunt grew faster, reached a higher final OD and provided better product-substrate yields than the wild type. By reducing the pyruvate decarboxylation flux, we significantly improved the performance of this important workhorse for industrial applications. This work encompasses the first steps towards full implementation of the non-oxidative glycolysis (NOG) or the glycolysis alternative high carbon yield cycle (GATCHYC), in which a substrate is converted into products without CO loss These enhanced properties of P. putida will be crucial for its subsequent use in a range of industrial processes.
Topics: Pseudomonas putida; Xylose; Glycerol; Mevalonic Acid; Pyruvates; Carbon
PubMed: 36658566
DOI: 10.1186/s12934-022-02015-9 -
Metabolic Engineering Sep 2021Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling...
Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling technologies. Adipic acid and other medium-chain-length dicarboxylates are key components of many plastics including polyamides, polyesters, and polyurethanes. This study endows Pseudomonas putida KT2440 with efficient metabolism of these dicarboxylates. The dcaAKIJP genes from Acinetobacter baylyi, encoding initial uptake and activation steps for dicarboxylates, were heterologously expressed. Genomic integration of these dca genes proved to be a key factor in efficient and reliable expression. In spite of this, adaptive laboratory evolution was needed to connect these initial steps to the native metabolism of P. putida, thereby enabling growth on adipate as sole carbon source. Genome sequencing of evolved strains revealed a central role of a paa gene cluster, which encodes parts of the phenylacetate metabolic degradation pathway with parallels to adipate metabolism. Fast growth required the additional disruption of the regulator-encoding psrA, which upregulates redundant β-oxidation genes. This knowledge enabled the rational reverse engineering of a strain that can not only use adipate, but also other medium-chain-length dicarboxylates like suberate and sebacate. The reverse engineered strain grows on adipate with a rate of 0.35 ± 0.01 h, reaching a final biomass yield of 0.27 ± 0.00 g g. In a nitrogen-limited medium this strain produced polyhydroxyalkanoates from adipate up to 25% of its CDW. This proves its applicability for the upcycling of mixtures of polymers made from fossile resources into biodegradable counterparts.
Topics: Acinetobacter; Adipates; Metabolic Engineering; Polyhydroxyalkanoates; Pseudomonas putida
PubMed: 33965615
DOI: 10.1016/j.ymben.2021.05.001 -
Biomolecules Aug 2020-Muconic acid (MA) is a valuable C6 dicarboxylic acid platform chemical that is used as a starting material for the production of various valuable polymers and drugs,... (Review)
Review
-Muconic acid (MA) is a valuable C6 dicarboxylic acid platform chemical that is used as a starting material for the production of various valuable polymers and drugs, including adipic acid and terephthalic acid. As an alternative to traditional chemical processes, bio-based MA production has progressed to the establishment of de novo MA pathways in several microorganisms, such as , , , and . Redesign of the metabolic pathway, intermediate flux control, and culture process optimization were all pursued to maximize the microbial MA production yield. Recently, MA production from biomass, such as the aromatic polymer lignin, has also attracted attention from researchers focusing on microbes that are tolerant to aromatic compounds. This paper summarizes recent microbial MA production strategies that involve engineering the metabolic pathway genes as well as the heterologous expression of some foreign genes involved in MA biosynthesis. Microbial MA production will continue to play a vital role in the field of bio-refineries and a feasible way to complement various petrochemical-based chemical processes.
Topics: Amycolatopsis; Biomass; Biosynthetic Pathways; Corynebacterium glutamicum; Escherichia coli; Industrial Microbiology; Metabolic Engineering; Pseudomonas putida; Saccharomyces cerevisiae; Shikimic Acid; Sorbic Acid; Stereoisomerism
PubMed: 32854378
DOI: 10.3390/biom10091238 -
Synthetic Biology (Oxford, England) 2021Boolean NOR gates have been widely implemented in as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial...
Boolean NOR gates have been widely implemented in as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial hosts/chassis is generally hampered by frequent changes in the parameters of the INPUT/OUTPUT response functions brought about by new genetic and biochemical contexts. Here, we have used the circuit design tool CELLO for assembling a NOR gate in the soil bacterium and the metabolic engineering platform with components tailored for To this end, we capitalized on the functional parameters of 20 genetic inverters for each host and the resulting compatibility between NOT pairs. Moreover, we added to the gate library three inducible promoters that are specific to , thus expanding cross-platform assembly options. While the number of potential connectable inverters decreased drastically when moving the library from to , the CELLO software was still able to find an effective NOR gate in the new chassis. The automated generation of the corresponding DNA sequence and experimental verification accredited that some genetic modules initially optimized for can indeed be reused to deliver NOR logic in as well. Furthermore, the results highlight the value of creating host-specific collections of well-characterized regulatory inverters for the quick assembly of genetic circuits to meet complex specifications.
PubMed: 34712846
DOI: 10.1093/synbio/ysab024 -
Frontiers in Bioengineering and... 2022The demand for non-petroleum-based, especially biodegradable plastics has been on the rise in the last decades. Medium-chain-length polyhydroxyalkanoate (mcl-PHA) is a...
The demand for non-petroleum-based, especially biodegradable plastics has been on the rise in the last decades. Medium-chain-length polyhydroxyalkanoate (mcl-PHA) is a biopolymer composed of 6-14 carbon atoms produced from renewable feedstocks and has become the focus of research. In recent years, researchers aimed to overcome the disadvantages of single strains, and artificial microbial consortia have been developed into efficient platforms. In this work, we reconstructed the previously developed microbial consortium composed of engineered KT∆ABZF (p2-a-J) and ∆4D (ACP-SCLAC). The maximum titer of mcl-PHA reached 3.98 g/L using 10 g/L glucose, 5 g/L octanoic acid as substrates by the engineered KT∆ABZF (p2-a-J). On the other hand, the maximum synthesis capacity of the engineered ∆4D (ACP-SCLAC) was enhanced to 3.38 g/L acetic acid and 0.67 g/L free fatty acids (FFAs) using 10 g/L xylose as substrate. Based on the concept of "nutrient supply-detoxification," the engineered ∆4D (ACP-SCLAC) provided nutrient for the engineered KT∆ABZF (p2-a-J) and it acted to detoxify the substrates. Through this functional division and rational design of the metabolic pathways, the engineered - microbial consortium could produce 1.30 g/L of mcl-PHA from 10 g/L glucose and xylose. Finally, the consortium produced 1.02 g/L of mcl-PHA using lignocellulosic hydrolysate containing 10.50 g/L glucose and 10.21 g/L xylose as the substrate. The consortium developed in this study has good potential for mcl-PHA production and provides a valuable reference for the production of high-value biological products using inexpensive carbon sources.
PubMed: 36338139
DOI: 10.3389/fbioe.2022.1023325 -
Biotechnology For Biofuels 2019Efficient conversion of plant biomass to commodity chemicals is an important challenge that needs to be solved to enable a sustainable bioeconomy. Deconstruction of...
BACKGROUND
Efficient conversion of plant biomass to commodity chemicals is an important challenge that needs to be solved to enable a sustainable bioeconomy. Deconstruction of biomass to sugars and lignin yields a wide variety of low molecular weight carbon substrates that need to be funneled to product. KT2440 has emerged as a potential platform for bioconversion of lignin and the other components of plant biomass. However, is unable to natively utilize several of the common sugars in hydrolysate streams, including galactose.
RESULTS
In this work, we integrated a De Ley-Doudoroff catabolic pathway for galactose catabolism into the chromosome of KT2440, using genes from several different organisms. We found that the galactonate catabolic pathway alone (DgoKAD) supported slow growth of on galactose. Further integration of genes to convert galactose to galactonate and to optimize the transporter expression level resulted in a growth rate of 0.371 h. Additionally, the best-performing strain was demonstrated to co-utilize galactose with glucose.
CONCLUSIONS
We have engineered to catabolize galactose, which will allow future engineered strains to convert more plant biomass carbon to products of interest. Further, by demonstrating co-utilization of glucose and galactose, continuous bioconversion processes for mixed sugar streams are now possible.
PubMed: 31890023
DOI: 10.1186/s13068-019-1627-0