The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas putida </it>KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by <it>P. putida </it>strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.</p> <p>Results</p> <p>We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)^-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other <it>P. putida </it>strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system.</p> <p>Conclusion</p> <p>This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.</p

The role of GlpR repressor in Pseudomonas putida KT2440 growth and PHA production from glycerol

14 pag.- 8 fig.- 5 tab.Pseudomonas putida KT2440 has evolved a tightly regulated system for metabolizing glycerol implying a prolonged growth lag-phase. We have learnt that this fact can be avoided by the addition of small amounts of some growth precursors. The addition of 1mM octanoic acid as co-feeder completely eliminated the lag-phase, resulting in an improvement, in terms of invested time, of both growth and polyhydroxyalkanoates (PHA) accumulation. To investigate this phenomenon, we have followed co-metabolic approaches combined with mutations of the specific and global regulatory networks that connect glycerol catabolism and PHA synthesis. By using mutant strains in metabolic genes from the PHA and tricarboxylic acid (TCA) cycles, we have demonstrated that the co-feeding effect is independent of PHA accumulation, but driven on active glyoxylate shunt and Entner-Doudoroff (ED) routes. These findings suggested that the effect of octanoate on glycerol metabolism could rely, either on a global activation of the cell energy state, or on the generation of specific metabolites or cofactors needed for the activation of glycerol metabolism. Our results addressed GlpR as the key factor controlling the efficient utilization of glycerol as growth precursor in P.putida KT2440. Accordingly, a glpR knockout mutant of P.putida KT2440 showed an elimination of the lag-phase when cultured on glycerol in the absence of co-feeder. Besides, the production of PHA in this strain was increased near twofold, resulting in a higher final yield in terms of PHA accumulation. The repressor activity of the GlpR protein over the glp genes in the absence of glycerol was finally demonstrated by qRT-PCR. This work contributed to unravel the physiological causes of the long lag-phase produced by glycerol in the model strain P.putida KT2440 that hinders its use as carbon source in biotechnological applications for generating valuable products.This work was supported by 597 grants from the Comisión Interministerial de Ciencia y Tecnología and CSIC598 (BIO2010-21049 and 201120E092), and by the European Union Grant (NMP2-CT-599 2007-026515). Isabel F. Escapa is a recipient of CSIC-I3P predoctoral fellowship.Peer reviewe

New insights on the reorganization of gene transcription in Pseudomonas putida KT2440 at elevated pressure

Abstract Background Elevated pressure, elevated oxygen tension (DOT) and elevated carbon dioxide tension (DCT) are readily encountered at the bottom of large industrial bioreactors and during bioprocesses where pressure is applied for enhancing the oxygen transfer. Yet information about their effect on bacteria and on the gene expression thereof is scarce. To shed light on the cellular functions affected by these specific environmental conditions, the transcriptome of Pseudomonas putida KT2440, a bacterium of great relevance for the production of medium-chain-length polyhydroxyalkanoates, was thoroughly investigated using DNA microarrays. Results Very well defined chemostat cultivations were carried out with P. putida to produce high quality RNA samples and ensure that differential gene expression was caused exclusively by changes of pressure, DOT and/or DCT. Cellular stress was detected at 7 bar and elevated DCT in the form of heat shock and oxidative stress-like responses, and indicators of cell envelope perturbations were identified as well.Globally, gene transcription was not considerably altered when DOT was increased from 40 ± 5 to 235 ± 20% at 7 bar and elevated DCT. Nevertheless, differential transcription was observed for a few genes linked to iron-sulfur cluster assembly, terminal oxidases, glutamate metabolism and arginine deiminase pathway, which shows their particular sensitivity to variations of DOT. Conclusions This study provides a comprehensive overview on the changes occurring in the transcriptome of P. putida upon mild variations of pressure, DOT and DCT. Interestingly, whereas the changes of gene transcription were widespread, the cell physiology was hardly affected, which illustrates how efficient reorganization of the gene transcription is for dealing with environmental changes that may otherwise be harmful. Several particularly sensitive cellular functions were identified, which will certainly contribute to the understanding of the mechanisms involved in stress sensing/response and to finding ways of enhancing the stress tolerance of microorganisms.This work was partially supported by the Swiss National Science Foundation grant 315200-116812/1 and by the Spanish grant BIO2010-21049 from the Comisión Interministerial de Ciencia y Tecnología.Peer Reviewe

Dolosigranulum pigrum cooperation and competition in human nasal microbiota

Multiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae Using a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support the idea of a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro, whereas robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together. D. pigrum l-lactic acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and on cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum harbored a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions. These new insights into D. pigrum's functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments.IMPORTANCEStaphylococcus aureus and Streptococcus pneumoniae infections cause significant morbidity and mortality in humans. For both, nasal colonization is a risk factor for infection. Studies of nasal microbiota identify Dolosigranulum pigrum as a benign bacterium present when adults are free of S. aureus or when children are free of S. pneumoniae Here, we validated these in vivo associations with functional assays. We found that D. pigrum inhibited S. aureusin vitro and, together with a specific nasal Corynebacterium species, also inhibited S. pneumoniae Furthermore, genomic analysis of D. pigrum indicated that it must obtain key nutrients from other nasal bacteria or from humans. These phenotypic interactions support the idea of a role for microbe-microbe interactions in shaping the composition of human nasal microbiota and implicate D. pigrum as a mutualist of humans. These findings support the feasibility of future development of microbe-targeted interventions to reshape nasal microbiota composition to exclude S. aureus and/or S. pneumoniae

The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida

15 páginas, 4 figuras, 3 tablas -- PAGS nros. 1049-1063The synthesis and degradation of polyhydroxyalkanoates (PHAs), the storage polymer of many bacteria, is linked to the operation of central carbon metabolism. To rationalize the impact of PHA accumulation on central carbon metabolism of the prototype bacterium Pseudomonas putida, we have revisited PHA production in quantitative physiology experiments in the wild-type strain vs. a PHA negative mutant growing under low nitrogen conditions. When octanoic acid was used as PHA precursor and as carbon and energy source, we have detected higher intracellular flux via acetyl-CoA in the mutant strain than in the wild type, which correlates with the stimulation of the TCA cycle and glyoxylate shunt observed on the transcriptional level. The mutant defective in carbon and energy storage spills the additional resources, releasing CO2 instead of generating biomass. Hence, P. putida operates the metabolic network to optimally exploit available resources and channels excess carbon and energy to storage via PHA, without compromising growth. These findings demonstrate that the PHA metabolism plays a critical role in synchronizing global metabolism to availability of resources in PHA-producing microorganismsThis work was supported by grants from the Comunidad Autónoma de Madrid (P-AMB-259-0505), the Comisión Interministerial de Ciencia y Tecnología (BIO2007-67304-C02, CSD2007-00005, BIO2010-21049) and by European Union Grants (GEN 2006-27750-C5-3-E and NMP2-CT-2007-026515). Isabel F. Escapa is a recipient of CSIC-I3P predoctoral fellowship. This work was supported by the German Ministry of Science and Education (BMBF, Project ERA-NET SysMO, no. 0313980A) (VAPMdS)Peer reviewe

Expression profile of pha gene cluster of Pseudomonas putida KT2442

3 páginas, 3 figuras -- PAGS nros. 8-10Polyhydroxyalkanoates (PHAs) are biodegradable polymers that many bacteria accumulate as carbon and energy storage when growth conditions are unbalanced. Pseudomonas strains belonging to the rRNA homology group I such as P. putida can accumulate medium-chain-length-PHA from monomers in the C8 to C10 range. Regulation of PHA synthesis and degradation in P. putida KT2442 has been studied using different molecular approaches. In this study six promoter regions located upstream of each pha gene were identified. The expression of the pha cluster have been analysed in the presence of octanoic acid versus glucose in the culture medium. Results demonstrated that the system is activated in the presence of octanoic acid as PHA precursorThis work is being supported by the projects BIO2007-67304-CO2, GEN2006-27750-C5-3-E and BIOPRODUCTION (NMP2-CT-2007-026515)Peer reviewe

Disruption of β-oxidation pathway in Pseudomonas putida KT2442 to produce new functionalized PHAs with thioester groups

This work describes the generation of novel PHAs (named PHACOS) with a new monomer composition containing thioester groups in the side chain, which confers new properties and made them suitable for chemical modifications after their biosynthesis. We have analyzed the PHACOS production abilities of the wild-type strain Pseudomonas putida KT2442 vs. its derived strain P. putida KT42FadB, mutated in the fadB gene from the central metabolic β-oxidation pathway involved in the synthesis of medium-chain-length PHA (mcl-PHA). Different fermentation strategies based on one- or two-stage cultures have been tested resulting in PHACOS with different monomer composition. Using decanoic acid as inducer of the growth and polymer synthesis and 6-acetylthiohexanoic acid as PHA precursor in a two-stage strategy, the maximum yield was obtained by culturing the strain KT42FadB. Nuclear magnetic resonance and gas chromatography coupled to mass spectrometry showed that polymers obtained from the wild-type and KT42FadB strains, included 6-acetylthio-3-hydroxyhexanoic acid (OH-6ATH) and the shorter derivative 4-acetylthio-3-hydroxybutanoic acid (OH-4ATB) in their composition, although in different ratios. While the polymer obtained from KT42FadB strain contained mainly OH-6ATH monomer units, mcl-PHA produced by the wild-type strain contained OH-6ATH and OH-4ATB. Furthermore, polyesters showed differences in the OH-alkyl derivates moiety. The strain KT42FadB overproduced PHACOS when compared to the production rate of the control strain in one- and two-stage cultures. Thermal properties obtained by differential scanning calorimetry indicated that both polymers have different glass transition temperatures related to their composition.We thank Dr. E. Díaz for helpful discussions. We are indebted to Marta Tortajada from Biopolis S.L. for sending us the standard mcl-PHA. The technical works of A. Valencia are greatly appreciated. This work was supported by grants from the Ministry of Science and Innovation (BIO2007-67304, BIO2010-21049, CSD2007-00005) and by European Union Grants (GEN 2006- 27750-C5-3-E and NMP2-CT-2007-026515). Isabel F. Escapa is a recipient of CSIC-I3P predoctoral fellowshipPeer Reviewe

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (PC1, PZ, PC2, PF and PI respectively). PC1 and PI are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of PF are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the PC1 and PI promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at PC1 promoter (named OPRc1) and a 29 bp region at PI promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid β-oxidation.Ministerio de Ciencia e Innovación (MCIN)Unión EuropeaComunidad de MadridDepto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (PC1, PZ, PC2, PF and PI respectively). PC1 and PI are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of PF are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the PC1 and PI promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at PC1 promoter (named OPRc1) and a 29 bp region at PI promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid β-oxidation.Ministerio de Ciencia e Innovación (MCIN)Unión EuropeaComunidad de MadridDepto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEunpu