Robustness of Pseudomonas 1 putida KT2440 as a host for ethanol 2 biosynthesis

Expansion of the burgeoning biofuels agenda involves not only the design of suitable genetic and metabolic devices but also their deployment into suitable hosts that can endure the stress brought about by the products themselves. The microorganisms easiest to genetically manipulate for these endeavours (e.g., Escherichia coli) are often afflicted by an undesirable sensitivity to the very product that they are engineered to synthesize. In this context, we have examined the resistance to the stress arising from ethanol synthesis and/or its addition to cultures of recombinant Pseudomonas putida, using as a benchmark the same trait in an E. coli strain. To this end, ethanologenic strains of these two species were constructed by functionally expressing pdc (pyruvate decarboxylase) and adhB (alcohol dehydrogenase) from Zymomonas mobilis. Recombinants were compared under anoxic conditions as ethanol producers, and cell survival, stress resistance, and phenotypic stability were quantified in each case. P. putida consistently outperformed E. coli in every ethanol tolerance test conducted – whether the alcohol was produced endogenously or added exogenously. These results highlight the value of such bacterium as a microbial cell factory for the production of biofuels owing to its naturally preevolved ability to withstand different kinds of chemical stresses.This study was supported by the BIO Program of the Spanish Ministry of Economy and Competitiveness, the ST-FLOW and ARIAYS Contracts of the EU, and the PROMT Project of the CAM.Peer reviewe

Pyridine nucleotide transhydrogenases enable redox balance of Pseudomonas putida during biodegra dation of aromatic compounds

Supporting informationThe metabolic versatility of the soil bacterium Pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. Biodegradation of aromatics occurs via the pathway encoded in the archetypal TOL plasmid pWW0, yet the effect of running such oxidative route on redox balance against the background metabolism of P. putida remains unexplored. To answer this question, the activity of pyridine nucleotide transhydrogenases (that catalyze the reversible interconversion of NADH and NADPH) was inspected under various physiological and oxidative stress regimes. The genome of P. putida KT2440 encodes a soluble transhydrogenase (SthA) and a membrane-bound, proton-pumping counterpart (PntAB). Mutant strains, lacking sthA and/or pntAB, were subjected to a panoply of genetic, biochemical, phenomic and functional assays in cells grown on customary carbon sources (e.g., citrate) versus difficult-to-degrade aromatic substrates. The results consistently indicated that redox homeostasis is compromised in the transhydrogenases-defective variant, rendering the mutant sensitive to oxidants. This metabolic deficiency was, however, counteracted by an increase in the activity of NADP+ -dependent dehydrogenases in central carbon metabolism. Taken together, these observations demonstrate that transhydrogenases enable a redox-adjusting mechanism that comes into play when biodegradation reactions are executed to metabolize unusual carbon compounds.This work was supported by the EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797) and EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536), Contracts of the European Union, and the CAMBIOS (RTC-2014-1777-3) and CONTIBUGS (PCIN-2013-040) projects of the Spanish Ministry of Economy and Competitiveness.Peer reviewe

High-Performance Biocomputing in Synthetic Biology–Integrated Transcriptional and Metabolic Circuits

Biocomputing uses molecular biology parts as the hardware to implement computational devices. By following pre-defined rules, often hard-coded into biological systems, these devices are able to process inputs and return outputs—thus computing information. Key to the success of any biocomputing endeavor is the availability of a wealth of molecular tools and biological motifs from which functional devices can be assembled. Synthetic biology is a fabulous playground for such purpose, offering numerous genetic parts that allow for the rational engineering of genetic circuits that mimic the behavior of electronic functions, such as logic gates. A grand challenge, as far as biocomputing is concerned, is to expand the molecular hardware available beyond the realm of genetic parts by tapping into the host metabolism. This objective requires the formalization of the interplay of genetic constructs with the rest of the cellular machinery. Furthermore, the field of metabolic engineering has had little intersection with biocomputing thus far, which has led to a lack of definition of metabolic dynamics as computing basics. In this perspective article, we advocate the conceptualization of metabolism and its motifs as the way forward to achieve whole-cell biocomputations. The design of merged transcriptional and metabolic circuits will not only increase the amount and type of information being processed by a synthetic construct, but will also provide fundamental control mechanisms for increased reliability

The private life of environmental bacteria: pollutant biodegradation at the single cell level

Bacteria display considerable cell-to-cell heterogeneity in a number of genetic and physiological traits. Stochastic differences in regulatory patterns (e.g., at the transcriptional level) propagate into the metabolic and physiological status of otherwise isogenic cells, which ultimately results in appearance of sub-populations within the community. As new technologies emerge and because novel single cell strategies are constantly being refined, our knowledge on microbial individuality is in burgeoning and constant expansion. These approaches encompass not only molecular biology tools (e.g., fluorescent protein based reporters) but also a suite of sophisticated, non-invasive technologies to gain insight into the metabolic state of individual cells. Defining the role of individual heterogeneities is thus instrumental for the population-level understanding of macroscopic processes, in both environmental and industrial setups. The present article reviews the state-of-the-art methodologies for the investigation of single bacteria at both the genetic and metabolic level, and places the application of currently available tools in the context of microbial ecology and environmental microbiology. As a case example, we examine the stochastic and multi-stable behaviour of the TOL-encoded pathway of Pseudomonas putida mt-2 for the biodegradation of aromatic compounds. Bet-hedging strategies and division of labour are considered as factors pushing forward the evolution of environmental microorganisms.This study was supported by the BIO and FEDER CONSOLIDER INGENIO Program of the Spanish Ministry of Science and Innovation, the MICROME, ST-FLOW and ARYSIS Contracts of the EU, the ERANET-IB Program and the PROMT Project of the CAM.Peer reviewe

Engineering Gram-Negative Microbial Cell Factories Using Transposon Vectors

The construction of microbial cell factories à la carte largely depends on specialized molecular biology and synthetic biology tools needed to reprogram bacteria for modifying their existing functions or for bestowing them with new-to-Nature tasks. In this chapter, we document the use of a series of broad-host-range mini-Tn5 vectors for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for the generation of saturated, random mutagenesis libraries for studies of gene function. The application of these tailored mini-transposon vectors, which could also be used for chromosomal engineering of a wide variety of Gram-negative microorganisms, is demonstrated in the platform environmental bacterium Pseudomonas putida KT2440.The work described in this protocol was supported by the CAMBIOS Project of the Spanish Ministry of Economy and Competitiveness (RTC-2014-1777-3), the ST-FLOW (FP7- KBBE- 2011-5-289326), EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797), and EmPowerPutida (EU-H2020-BIOTEC 2014-2015-6335536) Contracts of the European Union, and the PROMPT Project of the Autonomous Community of Madrid (CAM-S2010/BMD-2414).CAM-S2010/BMD-2414Peer reviewe

Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts

A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacte sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources. [Int Microbiol 2005; 8(4):243-250

New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories

Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena, but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5 vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5 vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes

Industrial biotechnology of Pseudomonas putida: advances and prospects

Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory