Exploring Rhodospirillum rubrum response to high doses of carbon monoxide under light and dark conditions.

Environmental concerns about residues and the traditional disposal methods are driving the search for more environmentally conscious processes, such as pyrolysis and gasification. Their main final product is synthesis gas (syngas) composed of CO, CO2, H2, and methane. Syngas can be converted into various products using CO-tolerant microorganisms. Among them, Rhodospirillum rubrum is highlighted for its biotechnological potential. However, the extent to which high doses of CO affect its physiology is still opaque. For this reason, we have studied R. rubrum behavior under high levels of this gas (up to 2.5 bar), revealing a profound dependence on the presence or absence of light. In darkness, the key variable affected was the lag phase, where the highest levels of CO retarded growth to more than 20 days. Under light, R. rubrum ability to convert CO into CO2 and H2 depended on the presence of an additional carbon source, such as acetate. In those conditions where CO was completely exhausted, CO2 fixation was unblocked, leading to a diauxic growth. To enhance R. rubrum tolerance to CO in darkness, a UV-accelerated adaptive laboratory evolution (UVa-ALE) trial was conducted to isolate clones with shorter lag phases, resulting in the isolation of clones 1.4-2B and 1.7-2A. The adaptation of 1.4-2B was mainly based on mutated enzymes with a metabolic function, while 1.7-3A was mostly affected at regulatory genes, including the anti-repressor PpaA/AerR. Despite these mutations having slight effects on biomass and pigment levels, they successfully provoked a significant reduction in the lag phase (-50%). KEYPOINTS: • CO affects principally R. rubrum lag phase (darkness) and growth rate (light) • CO is converted to CO2/H2 during acetate uptake and inhibits CO2 fixation (light) • UVa-ALE clones showed a 50% reduction in the lag phase (darkness).

A singular PpaA/AerR-like protein in Rhodospirillum rubrum rules beyond the boundaries of photosynthesis in response to the intracellular redox state.

IMPORTANCE: Rhodospirillum rubrum vast metabolic versatility places it as a remarkable model bacterium and an excellent biotechnological chassis. The key component of photosynthesis (PS) studied in this work (HP1) stands out among the other members of PpaA/AerR anti-repressor family since it lacks the motif they all share: the cobalamin B-12 binding motif. Despite being reduced and poorly conserved, HP1 stills controls PS as the other members of the family, allowing a fast response to changes in the redox state of the cell. This work also shows that HP1 absence affects genes from relevant biological processes other than PS, including nitrogen fixation and stress response. From a biotechnological perspective, HP1 could be manipulated in approaches where PS is not necessary, such as hydrogen or polyhydroxyalkanoates production, to save energy.

Nature-inspired material binding peptides with versatile polyester affinities and binding strengths.

Biodegradable polyesters, such as polyhydroxyalkanoates (PHAs), are having a tremendous impact on biomedicine. However, these polymers lack functional moieties to impart functions like targeted delivery of molecules. Inspired by native GAPs, such as phasins and their polymer-binding and surfactant properties, we generated small material binding peptides (MBPs) for polyester surface functionalization using a rational approach based on amphiphilicity. Here, two peptides of 48 amino acids derived from phasins PhaF and PhaI from Pseudomonas putida, MinP and the novel-designed MinI, were assessed for their binding towards two types of PHAs, PHB and PHOH. In vivo, fluorescence studies revealed selective binding towards PHOH, whilst in vitro binding experiments using the Langmuir-Blodgett technique coupled to ellipsometry showed KD in the range of nM for all polymers and MBPs. Marked morphological changes of the polymer surface upon peptide adsorption were shown by BAM and AFM for PHOH. Moreover, both MBPs were successfully used to immobilize cargo proteins on the polymer surfaces. Altogether, this work shows that by redesigning the amphiphilicity of phasins, a high affinity but lower specificity to polyesters can be achieved in vitro. Furthermore, the MBPs demonstrated binding to PET, showing potential to bind cargo molecules also to synthetic polyesters.

Aerobic-anaerobic transition boosts poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Rhodospirillum rubrum: the key role of carbon dioxide.

BACKGROUND: Microbially produced bioplastics are specially promising materials since they can be naturally synthesized and degraded, making its end-of-life management more amenable to the environment. A prominent example of these new materials are polyhydroxyalkanoates. These polyesters serve manly as carbon and energy storage and increase the resistance to stress. Their synthesis can also work as an electron sink for the regeneration of oxidized cofactors. In terms of biotechnological applications, the co-polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), or PHBV, has interesting biotechnological properties due to its lower stiffness and fragility compared to the homopolymer poly(3-hydroxybutyrate) (P3HB). In this work, we explored the potentiality of Rhodospirillum rubrum as a producer of this co-polymer, exploiting its metabolic versatility when grown in different aeration conditions and photoheterotrophically. RESULTS: When shaken flasks experiments were carried out with limited aeration using fructose as carbon source, PHBV production was triggered reaching 29 ± 2% CDW of polymer accumulation with a 75 ± 1%mol of 3-hydroxyvalerate (3HV) (condition C2). Propionate and acetate were secreted in this condition. The synthesis of PHBV was exclusively carried out by the PHA synthase PhaC2. Interestingly, transcription of cbbM coding RuBisCO, the key enzyme of the Calvin-Benson-Bassham cycle, was similar in aerobic and microaerobic/anaerobic cultures. The maximal PHBV yield (81% CDW with 86%mol 3HV) was achieved when cells were transferred from aerobic to anaerobic conditions and controlling the CO2 concentration by adding bicarbonate to the culture. In these conditions, the cells behaved like resting cells, since polymer accumulation prevailed over residual biomass formation. In the absence of bicarbonate, cells could not adapt to an anaerobic environment in the studied lapse. CONCLUSIONS: We found that two-phase growth (aerobic-anaerobic) significantly improved the previous report of PHBV production in purple nonsulfur bacteria, maximizing the polymer accumulation at the expense of other components of the biomass. The presence of CO2 is key in this process demonstrating the involvement of the Calvin-Benson-Bassham in the adaptation to changes in oxygen availability. These results stand R. rubrum as a promising producer of high-3HV-content PHBV co-polymer from fructose, a PHBV unrelated carbon source.

Microbial Cell Factories à la Carte: Elimination of Global Regulators Cra and ArcA Generates Metabolic Backgrounds Suitable for the Synthesis of Bioproducts in Escherichia coli.

Manipulation of global regulators is one of the strategies used for the construction of bacterial strains suitable for the synthesis of bioproducts. However, the pleiotropic effects of these regulators can vary under different conditions and are often strain dependent. This study analyzed the effects of ArcA, CreC, Cra, and Rob using single deletion mutants of the well-characterized and completely sequenced Escherichia coli strain BW25113. Comparison of the effects of each regulator on the synthesis of major extracellular metabolites, tolerance to several compounds, and synthesis of native and nonnative bioproducts under different growth conditions allowed the discrimination of the particular phenotypes that can be attributed to the individual mutants and singled out Cra and ArcA as the regulators with the most important effects on bacterial metabolism. These data were used to identify the most suitable backgrounds for the synthesis of the reduced bioproducts succinate and 1,3-propanediol (1,3-PDO). The Δcra mutant was further modified to enhance succinate synthesis by the addition of enzymes that increase NADH and CO2 availability, achieving an 80% increase compared to the parental strain. Production of 1,3-PDO in the ΔarcA mutant was optimized by overexpression of PhaP, which increased more than twice the amount of the diol compared to the wild type in a semidefined medium using glycerol, resulting in 24 g · liter-1 of 1,3-PDO after 48 h, with a volumetric productivity of 0.5 g · liter-1 h-1IMPORTANCE Although the effects of many global regulators, especially ArcA and Cra, have been studied in Escherichia coli, the metabolic changes caused by the absence of global regulators have been observed to differ between strains. This scenario complicates the identification of the individual effects of the regulators, which is essential for the design of metabolic engineering strategies. The genome of Escherichia coli BW25113 has been completely sequenced and does not contain additional mutations that could mask or interfere with the effects of the global regulator mutations. The uniform genetic background of the Keio collection mutants enabled the characterization of the physiological consequences of altered carbon and redox fluxes caused by each global regulator deletion, eliminating possible strain-dependent results. As a proof of concept, Δcra and ΔarcA mutants were subjected to further manipulations to obtain large amounts of succinate and 1,3-PDO, demonstrating that the metabolic backgrounds of the mutants were suitable for the synthesis of bioproducts.

About how to capture and exploit the CO2 surplus that nature, per se, is not capable of fixing.

Human activity has been altering many ecological cycles for decades, disturbing the natural mechanisms which are responsible for re-establishing the normal environmental balances. Probably, the most disrupted of these cycles is the cycle of carbon. In this context, many technologies have been developed for an efficient CO2 removal from the atmosphere. Once captured, it could be stored in large geological formations and other reservoirs like oceans. This strategy could present some environmental and economic problems. Alternately, CO2 can be transformed into carbonates or different added-value products, such as biofuels and bioplastics, recycling CO2 from fossil fuel. Currently different methods are being studied in this field. We classified them into biological, inorganic and hybrid systems for CO2 transformation. To be environmentally compatible, they should be powered by renewable energy sources. Although hybrid systems are still incipient technologies, they have made great advances in the recent years. In this scenario, biotechnology is the spearhead of ambitious strategies to capture CO2 and reduce global warming.

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations.

The CreBC (carbon source-responsive) two-component regulation system of Escherichia coli affects a number of functions, including intermediary carbon catabolism. The impacts of different creC mutations (a ΔcreC mutant and a mutant carrying the constitutive creC510 allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carrying creC510 and shown to be affected by oxygen availability. The ΔcreC strain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P)(+) ratios showed that the creC510 strain had a more reduced intracellular redox state, while the opposite was observed for the ΔcreC mutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase from E. coli and a NADH-forming formate dehydrogenase from Candida boidinii in the ΔcreC mutant further increased the yield of succinate on glucose. Interestingly, the elimination of ackA and adhE did not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network of E. coli make it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate.

Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals.

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level.

Elimination of D-lactate synthesis increases poly(3-hydroxybutyrate) and ethanol synthesis from glycerol and affects cofactor distribution in recombinant Escherichia coli.

The effect of eliminating D-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP(+) ratio was higher than the NADH/NAD(+) ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP(+). In 60-h fed-batch cultures, K24KL reached 41.9 g·liter⁻¹ biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g·g⁻¹, the highest reported using this substrate.