BUSCADOR RECOLECTA

Microorganisms able to degrade aromatic contaminants constitute potential valuable biocatalysts to deal with a significant reusable carbon fraction suitable for eco-efficient valorization processes. Metabolic engineering of anaerobic pathways for degradation and recycling of aromatic compounds is an almost unexplored field. In this work, we present the construction of a functional bzd cassette encoding the benzoyl-CoA central pathway for the anaerobic degradation of benzoate. The bzd cassette has been used to expand the ability of some denitrifying bacteria to use benzoate as sole carbon source under anaerobic conditions, and it paves the way for future pathway engineering of efficient anaerobic biodegraders of aromatic compounds whose degradation generates benzoyl-CoA as central intermediate. Moreover, a recombinant Azoarcus sp. CIB strain harbouring the bzd cassette was shown to behave as a valuable biocatalyst for anaerobic toluene valorization towards the synthesis of poly-3-hydroxybutyrate (PHB), a biodegradable and biocompatible polyester of increasing biotechnological interest as a sustainable alternative to classical oil-derived polymers., This work was supported by grants BIO2012-39501, BIO2016-
79736-R and PCIN-2014-113 from the Ministry of Economy and
Competitiveness of Spain; European Union FP7 Grant 311815; and
by a grant of Fundación Ramón-Areces XVII CN., Peer reviewed

32 p.-2 fig.-1 tab., Microbial desulfurization or biodesulfurization (BDS) is an attractive low-cost and environmentally friendly complementary technology to the hydrotreating chemical process based on the potential of certain bacteria to specifically remove sulfur from S-heterocyclic compounds of crude fuels that are recalcitrant to the chemical treatments. The 4S or Dsz sulfur specific pathway for dibenzothiophene (DBT) and alkyl-substituted DBTs, widely used as model S-heterocyclic compounds, has been extensively studied at the physiological, biochemical and genetic levels mainly in Gram-positive bacteria. Nevertheless, several Gram-negative bacteria have been also used in BDS because they are endowed with some properties, e.g., broad metabolic versatility and easy genetic and genomic manipulation, that make them suitable chassis for systems metabolic engineering strategies. A high number of recombinant bacteria, many of which are Pseudomonas strains, have been constructed to overcome the major bottlenecks of the desulfurization process, i.e., expression of the dsz operon, activity of the Dsz enzymes, retro-inhibition of the Dsz pathway, availability of reducing power, uptake-secretion of substrate and intermediates, tolerance to organic solvents and metals, and other host-specific limitations. However, to attain a BDS process with industrial applicability, it is necessary to apply all the knowledge and advances achieved at the genetic and metabolic levels to the process engineering level, i.e., kinetic modelling, scale-up of biphasic systems, enhancing mass transfer rates, biocatalyst separation, etc. The production of high-added value products derived from the organosulfur material present in oil can be regarded also as an economically viable process that has barely begun to be explored., Work supported by Ministry of Economy and Competitiveness of Spain Grants BIO2012-39501, BIO2012-39695-C02-01, BIO2015-66960-C3-3-R, BIO2016-79736-R, CTQ2011-12725-E and PCIN2014-113, European UnionGrants FP7-KBBE 6-311815 and H2020-FET-OPEN 686585, and Fundación Ramón-Areces XVII Concurso Nacional., Peer reviewed

60 p.-11 fig., Removal of aromatic compounds, which mostly derive from the polymer lignin, fossil fuel reservoirs, and industrial activities, is very important both for a balanced global carbon budget and to protect natural ecosystems and human health from the toxic effect of some of these environmental pollutants. Whereas the aerobic catabolism of aromatic compounds has been extensively studied for many decades, the anaerobic catabolism is a more recently discovered and so far poorly characterized microbial capacity despite the fact that anoxic conditions dominate in many natural habitats and contaminated sites. The anaerobic catabolism of aromatic compounds by some specialized bacteria becomes, thus, crucial for the biogeochemical cycles and for the sustainable development of the biosphere. Moreover, anaerobic degradation of aromatic compounds involves various intriguing biochemically unprecedented reactions that are also of great biotechnological potential as alternatives to the current synthesis processes and for the anaerobic valorization of aromatic compounds to produce biofuels, biopolymers, and commodity chemicals. In this chapter we summarize the major degradation pathways and the associated cellular responses when bacteria grow anaerobically in the presence of aromatic compounds. The unexplored potential and some biotechnological applications of the anaerobic catabolism of aromatic compounds are also discussed., Work in E. Díaz laboratory was supported by Ministry of Economy and Competitiveness of Spain Grant BIO 2012-39501, BIO 2016-79736-R, and PCIN 2014-113, European Union FP7 Grant 311815,and Fundación Ramón-Areces XVIICN., Peer reviewed

16 p.-8 fig.-1 tab., We have identified and characterized the AccS multidomain sensor kinase that mediates the activation of the AccR master regulator involved in carbon catabolite repression (CCR) of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB. A truncated AccS protein that contains only the soluble C-terminal autokinase module (AccS′) accounts for the succinate-dependent CCR control. In vitro assays with purified AccS′ revealed its autophosphorylation, phosphotransfer from AccS′∼P to the Asp60 residue of AccR, and the phosphatase activity toward its phosphorylated response regulator, indicating that the equilibrium between the kinase and phosphatase activities of AccS′ may control the phosphorylation state of the AccR transcriptional regulator. Oxidized quinones, e.g., ubiquinone 0 and menadione, switched the AccS′ autokinase activity off, and three conserved Cys residues, which are not essential for catalysis, are involved in such inhibition. Thiol oxidation by quinones caused a change in the oligomeric state of the AccS′ dimer resulting in the formation of an inactive monomer. This thiol-based redox switch is tuned by the cellular energy state, which can change depending on the carbon source that the cells are using. This work expands the functional diversity of redox-sensitive sensor kinases, showing that they can control new bacterial processes such as CCR of the anaerobic catabolism of aromatic compounds. The AccSR two-component system is conserved in the genomes of some betaproteobacteria, where it might play a more general role in controlling the global metabolic state according to carbon availability., This work was supported by grants BIO2016-79736-R and PCIN-2014-113 from the Ministry of Economy and Competitiveness of Spain; by a grant of Fundación Ramón-Areces XVII CN; by grant CSIC 2016 2 0E 093 from the CSIC; and by European Union H2020 grant 760994., Peer reviewed

9 p.-6 fig.-1 tab, Selenium and selenium nanoparticles (SeNPs) are extensively used in biomedicine, electronics and
some other industrial applications. The bioproduction of SeNPs is gaining interest as a green method
to manufacture these biotechnologically relevant products. Several microorganisms have been used
for the production of SeNPs either under aerobic or anaerobic conditions. Vibrio natriegens is a nonpathogenic
fast-growing bacterium, easily cultured in different carbon sources and that has recently
been engineered for easy genetic manipulation in the laboratory. Here we report that V. natriegens
was able to perfectly grow aerobically in the presence of selenite concentrations up to 15 mM with a
significant survival still observed at concentrations as high as 100 mM selenite. Electron microscopy
and X-ray spectroscopy analyses demonstrate that V. natriegens cells growing aerobically in selenitecontaining
LB medium at 30 °C produced spherical electron-dense SeNPs whose size ranged from
100–400 nm. Selenite reduction just started at the beginning of the exponential growth phase and the
release of SeNPs was observed after cell lysis. Remarkably, V. natriegens produced SeNPs faster than
other described microorganisms that were proposed as model bioreactors for SeNPs production. Thus,
the fast-growing V. natriegens bacterium becomes a suitable biocatalyst for bioremediation of selenite
and for speeding-up the eco-friendly synthesis of SeNPs., This work was supported by Ministry of Economy, Industry and Competitiveness of Spain Grants BIO2016-79736-R, PCIN-2014-113 and MAT2014-50222R, Fundación Ramón-Areces XVII CN., Peer reviewed

56 p.-7 fig.-1 tab.+8 p.( 4 fig. supl.-1 tab. supl.), ortho ‐Phthalate derives from industrially produced phthalate esters, which are massively used as plasticizers and constitute major emerging environmental pollutants. The pht pathway for the anaerobic bacterial biodegradation of o ‐phthalate involves its activation to phthaloyl‐CoA followed by decarboxylation to benzoyl‐CoA. Here, we have explored further the pht peripheral pathway in denitrifying bacteria and shown that it requires also an active transport system for o ‐phthalate uptake that belongs to the poorly characterized class of TAXI‐TRAP transporters. The construction of a fully functional pht cassette combining both catabolic and transport genes allowed to expand the o ‐phthalate degradation ecological trait to heterologous hosts. Unexpectedly, the pht cassette also allowed the aerobic conversion of o ‐phthalate to benzoyl‐CoA when coupled to a functional box central pathway. Hence, the pht pathway may constitute an evolutionary acquisition for o ‐phthalate degradation by bacteria that thrive either in anoxic environments or in environments that face oxygen limitations and that rely on benzoyl‐CoA, rather than on catecholic central intermediates, for the aerobic catabolism of aromatic compounds. Finally, the recombinant pht cassette was used both to screen for functional aerobic box pathways in bacteria and to engineer recombinant biocatalysts for o ‐phthalate bioconversion into sustainable bioplastics, e.g., polyhydroxybutyrate, in plastic recycling industrial processes., Support was provided by grants BIO2016-79736-R and PCIN-2014-113 from the Ministry of Economy and Competitiveness of Spain; by a grant from the Fundación Ramón Areces XVII CN; by Grant CSIC 2016 2 0E 093; and by European Union H2020 Grant 760994., Peer reviewed

16 p.-4 fig., Environmental pollutants can generate stress in plants causing increased ethylene production that leads to the inhibition of plant growth. Ethylene production by the stressed plant may be lowered by Plant Growth-Promoting Bacteria (PGPB) that metabolizes the immediate precursor of ethylene 1-aminocyclopropane-1-carboxylate (ACC). Thus, engineering PGPB with ACC deaminase activity can be a promising alternative to mitigate the harmful effects of pollutants and thus enhance plant production. Here we show that the aromatics-degrading and metal-resistant Azoarcus sp. CIB behaves as a PGP-bacterium when colonizing rice as an endophyte, showing a 30% increment in plant weight compared to non-inoculated plants. The cloning and expression of an acdS gene led to a recombinant strain, Azoarcus sp. CIB (pSEVA237acdS), possessing significant ACC deaminase activity (6716 nmol mg−1 h−1), constituting the first PGPB of the Rhodocyclaceae family equipped with this PGP trait. The recombinant CIB strain acquired the ability to protect inoculated rice plants from the stress induced by cadmium (Cd) exposure and to increase the Cd concentration in rice seedlings. The observed decrease of the levels of reactive oxygen species levels in rice roots confirms such a protective effect. The broad-host-range pSEVA237acdS plasmid paves the way to engineer PGPB with ACC deaminase activity to improve the growth of plants that might face stress conditions., This work was supported by grants BIO2016-79736-R and PCIN-2014-113 from the Ministry of Economy and Competitiveness of Spain; by a grant of Fundación Ramón-Areces XVII CN; and by Grants CSIC 2016 2 0E 093 and 2017 2 0I 015;. We also acknowledge the UHasselt Methusalem project 08M03VGRJ., Peer reviewed

15 p.-4 fig.-2 tab., Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di‐GMP (c‐di‐GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels., This study was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014‐53530‐R and BIO2017‐83035‐R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I. Lasa and C. Solano and grants BIO2016‐79736‐R, PCIN‐2014‐113 and PCI2019‐111833‐2 to E. Díaz. P. Dorado‐Morales was supported by a F.P.I. (BES‐2015‐072859) contract from the Spanish Ministry of Science, Innovation and Universities., Peer reviewed

17 p.-6 fig.-2 tab., Proficient crop production is needed to ensure the feeding of a growing global population. The association of bacteria with plants plays an important role in the health state of the plants contributing to the increase of agricultural production. Endophytic bacteria are ubiquitous in most plant species providing, in most cases, plant promotion properties. However, the knowledge on the genetic determinants involved in the colonization of plants by endophytic bacteria is still poorly understood. In this work we have used a genetic approach based on the construction of fliM, pilX and eps knockout mutants to show that the motility mediated by a functional flagellum and the pili type IV, and the adhesion modulated by exopolysaccarides are required for the efficient colonization of rice roots by the endophyte Azoarcus sp. CIB. Moreover, we have demonstrated that expression of an exogenous diguanylate cyclase or phophodiesterase, which causes either an increase or decrease of the intracellular levels of the second messenger cyclic di-GMP (c-di-GMP), respectively, leads to a reduction of the ability of Azoarcus sp. CIB to colonize rice plants. Here we present results demonstrating the unprecedented role of the universal second messenger cyclic-di-GMP in plant colonization by an endophytic bacterium, Azoarcus sp. CIB. These studies pave the way to further strategies to modulate the interaction of endophytes with their target plant hosts., This research was funded by grants BIO2016-79736-R, PCIN-2014-113, and PCI2019-111833-2 from the Ministry of Economy and Competitiveness of Spain; by Grant CSIC 2017 2 0I 015; and by European Union H2020 Grant 760994., Peer reviewed

13 p.-8 fig., Arsenic is a toxic element widely distributed in nature, but numerous bacteria are able to resist its toxicity mainly through the ars genes encoding an arsenate reductase and an arsenite efflux pump. Some “arsenotrophic” bacteria are also able to use arsenite as energy supplier during autotrophic growth by coupling anaerobic arsenite oxidation via the arx gene products to nitrate respiration or photosynthesis. Here, we have demonstrated that Azoarcus sp. CIB, a facultative anaerobic β-proteobacterium, is able to resist arsenic oxyanions both under aerobic and anaerobic conditions. Genome mining, gene expression, and mutagenesis studies revealed the presence of a genomic island that harbors the ars and arx clusters involved in arsenic resistance in strain CIB. Orthologous ars clusters are widely distributed in the genomes of sequenced Azoarcus strains. Interestingly, genetic and metabolic approaches showed that the arx cluster of the CIB strain encodes an anaerobic arsenite oxidase also involved in the use of arsenite as energy source. Hence, Azoarcus sp. CIB represents the prototype of an obligate heterotrophic bacterium able to use arsenite as an extra-energy source for anaerobic cell growth. The arsenic island of strain CIB supports the notion that metabolic and energetic skills can be gained by genetic mobile elements., This work was supported by grants BIO2016-79736-R and PCIN-2014-113 from the Ministry of Economy and Competitiveness of Spain; by a grant of Fundación Ramón-Areces XVII CN; by Grants CSIC 2016 2 0E 093 and 2017 2 0I 015; and by European Union H2020 Grant 760994., Peer reviewed

Incluye material complementario, Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels., This study was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014‐53530‐R and BIO2017‐83035‐R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I. Lasa and C. Solano and grants BIO2016‐79736‐R, PCIN‐2014‐113 and PCI2019‐111833‐2 to E. Díaz. P. Dorado‐Morales was supported by a F.P.I. (BES‐2015‐072859) contract from the Spanish Ministry of Science, Innovation and Universities.