- Vitor A.P. Martins dos Santos (1)
- J. Becker (2)
- D. Binger (1)
- Eduardo Castro-Nallar (1)
- K. Dohnt (1)
- I. Escapa (1)
- Izabook Gutierrez-Urrutia (1)
- Matthieu J. Miossec (1)
- C. Jager (1)
- W. Kessler (1)
- Sandro L. Valenzuela (1)
- M.C. Lam (2)
- V.A.P. Martins Dos Santos (3)
- Claudio Meneses (1)
- Ignacio Poblete-Castro (1)
- I. Poblete-Castro (4)
- M. Prieto (1)
- J. Puchalka (1)
- A. Rodrigues (1)
- A.L. Rodriguez (1)
- D. Schomburg (1)
- C. Wittmann (2)
Genome sequence of two members of the chloroaromatic-degrading MT community : Pseudomonas reinekei MT1 and Achromobacter xylosoxidans MT3
Gutierrez-Urrutia, Izabook ; Miossec, Matthieu J. ; Valenzuela, Sandro L. ; Meneses, Claudio ; Martins dos Santos, Vitor A.P. ; Castro-Nallar, Eduardo ; Poblete-Castro, Ignacio - \ 2018
Journal of Biotechnology 275 (2018). - ISSN 0168-1656 - p. 13 - 16.
Biodegradation - Chloro- and nitro-aromatic compounds - Genome sequence - MT bacterial community
We describe the genome sequence of Pseudomonas reinekei MT1 and Achromobacter xylosoxidans MT3, the most abundant members of a bacterial community capable of degrading chloroaromatic compounds. The MT1 genome contains open reading frames encoding enzymes responsible for the catabolism of chlorosalicylate, methylsalicylate, chlorophenols, phenol, benzoate, p-coumarate, phenylalanine, and phenylacetate. On the other hand, the MT3 strain genome possesses no ORFs to metabolize chlorosalicylates; instead the bacterium is capable of metabolizing nitro-phenolic and phenolic compounds, which can be used as the only carbon and energy source by MT3. We also confirmed that MT3 displays the genetic machinery for the metabolism of chlorocathecols and chloromuconates, where the latter are toxic compounds secreted by MT1 when degrading chlorosalicylates. Altogether, this work will advance our fundamental understanding of bacterial interactions.
Improved production of medium-chain-length Polyhydroxyalkanotes in glucose-based fed-batch cultivations of metabolically engineered Pseudomonas putida strains
Poblete-Castro, I. ; Rodriguez, A.L. ; Lam, M.C. ; Kessler, W. - \ 2014
Journal of Microbiology and Biotechnology 24 (2014)1. - ISSN 1017-7825 - p. 59 - 69.
cell-density cultivation - mcl-pha - catabolite repression - unsaturated monomers - nonanoic acid - biosynthesis - kt2442 - poly(3-hydroxyalkanoates) - poly(hydroxyalkanoates) - accumulation
One of the major challenges in metabolic engineering for enhanced synthesis of value-added chemicals is to design and develop new strains which can be translated into well-controlled fermentation processes using bioreactors. The aim of this study was to assess the influence of various fed-batch strategies in the performance of metabolically-engineered Pseudomonas putida strains, ¿gcd and ¿gcd-pgl, for improving production of medium-chain-length poly-hydroxyalkanoates (mcl-PHAs) using glucose as the only carbon source. First we developed a fed-batch process which comprised an initial phase of biomass accumulation based on an exponential feeding carbon-limited strategy. For the mcl-PHA accumulation stage, three induction techniques were tested under nitrogen limitation. The substrate-pulse feeding was more efficient than the constant-feeding approach to promote the accumulation of the desirable product. Nonetheless, the most efficient approach for maximum PHA synthesis was the application of a dissolved-oxygen-stat feeding strategy (DO-stat), where P. putida ¿gcd mutant strain showed a final PHA content and specific PHA productivity of 67%, and 0.83 [g•L-1•h-1], respectively. To our knowledge this mcl-PHA titer is the highest value that has been ever reported using glucose as the solely carbon and energy source. Our results also highlighted the effect of different fed-batch strategies upon the extent of realization of the intended metabolic modification of the mutant strains
In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates
Poblete-Castro, I. ; Binger, D. ; Rodrigues, A. ; Becker, J. ; Martins Dos Santos, V.A.P. ; Wittmann, C. - \ 2013
Metabolic Engineering 15 (2013). - ISSN 1096-7176 - p. 113 - 123.
chain-length polyhydroxyalkanoates - gram-negative bacteria - fed-batch culture - genome sequence - escherichia-coli - kt2440 - pathways - acid - poly(3-hydroxyalkanoates) - biosynthesis
Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism of P. putida was used to predict genetic targets for strain engineering. Among a set of priority ranked targets, glucose dehydrogenase (encoded by gcd) was predicted as most promising deletion target. The mutant P. putida ¿gcd, generated on basis of the computational design, exhibited 100% increased PHA accumulation as compared to the parent wild type, maintained a high specific growth rate and exhibited an almost unaffected gene expression profile, which excluded detrimental side effects of the modification. A second mutant strain, P. putida ¿pgl, that lacked 6-phosphogluconolactonase, exhibited a substantially decreased PHA synthesis, as was also predicted by the model. The production potential of P. putida ¿gcd was assessed in batch bioreactors. The novel strain showed an increase of the PHA yield (+80%), the PHA titer (+100%) and cellular PHA content (+50%) and revealed almost unaffected growth and diminished by-product formation. It was thus found superior in all relevant criteria towards industrial production. Beyond the contribution to more efficient PHA production processes at reduced costs that might replace petrochemical plastics in the future, the study illustrates the power of computational prediction to tailor microbial strains for enhanced biosynthesis of added-value compounds
Industrial biotechnology of Pseudomonas putida and related species
Poblete-Castro, I. ; Becker, J. ; Dohnt, K. ; Martins Dos Santos, V.A.P. ; Wittmann, C. - \ 2012
Applied Microbiology and Biotechnology 93 (2012)6. - ISSN 0175-7598 - p. 2279 - 2290.
aromatic catabolic pathways - complete genome sequence - fada knockout mutant - transcriptome analysis - lignocellulosic biomass - nitrogen availability - proteomic analysis - p-hydroxybenzoate - gene-expression - stress-response
Since their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.
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
Poblete-Castro, I. ; Escapa, I. ; Jager, C. ; Puchalka, J. ; Lam, M.C. ; Schomburg, D. ; Prieto, M. ; Martins Dos Santos, V.A.P. - \ 2012
Microbial Cell Factories 11 (2012). - ISSN 1475-2859
chain-length polyhydroxyalkanoates - aeruginosa outer-membrane - fada knockout mutant - acidic amino-acids - pseudomonas-putida - escherichia-coli - continuous cultures - proteomic analysis - terminal oxidases - monomer content
Background - Pseudomonas putida 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 P. putida 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. Results - 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 P. putida 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. Conclusion - 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