Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the β-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of β-alanine and its subsequent conversion into 3HP using a novel β-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.

Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions worldwide. A major factor contributing to success of this bacterium is its outstanding ability to survive on dry surfaces. The molecular basis for desiccation resistance is not completely understood. This study focused on growth under osmotic stress and aimed to identify the pool of compatible solutes synthesized in response to these low water activity conditions. A. baumannii produced mannitol as compatible solute, but in contrast to Acinetobacter baylyi, also trehalose was accumulated in response to increasing NaCl concentrations. The genome of A. baumannii encodes a trehalose-6-phosphate phosphatase (OtsB) and a trehalose-6-phosphate synthase (OtsA). Deletion of otsB abolished trehalose formation, demonstrating that otsB is essential for trehalose biosynthesis. Growth of the mutant was neither impaired at low salt nor at 500 mM NaCl, but it did not grow at high temperatures, indicating a dual function of trehalose in osmo- and thermoprotection. This led us to analyse temperature dependence of trehalose formation. Indeed, expression of otsB was not only induced by high osmolarity but also by high temperature. Concurrently, trehalose was accumulated in cells grown at high temperature. Taken together, these data point to an important role of trehalose in A. baumannii beyond osmoprotection.

The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and D-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and "Dehalococcoides ethenogenes." GpgS had maximal activity at 50°C. The maximal activity of GpgP was at 50°C with GPG as the substrate and at 60°C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.Most microorganisms capable of osmotic adjustment accumulate low-molecular-weight organic compounds, commonly designated compatible solutes, which can be taken up from the environment or synthesized de novo (6). Although the uptake of organic solutes such as glycine betaine and trehalose, among others, by microorganisms under salt stress is preferred because it is energetically favorable (10), many organisms synthesize specific compatible solutes because these are not present in the environment or those that are present do not fulfill the specific requirements of the organisms.Ectoine, glycine betaine, trehalose, and glutamate are among the most common compatible solutes of bacteria and archaea, where they normally accumulate in response to stress imposed by salt (10). Mannosylglycerate (MG), di-myo-inositol-phosphate, and diglycerol-phosphate have a more restricted distribution, being found primarily in (hyper)thermophilic prokaryotes. Mannosylglycerate is widespread in (hyper)thermophilic bacteria and archaea and is also found in some red algae (21, 35). Cyclic 2,3-bisphosphoglycerate has only been encountered in methanogens (20,25,36).The rare solute glucosylglycerate (GG) was originally identified in the cyanobacterium Agmenellum quadruplicatum strain PCC7002 grown under nitrogen-limiting conditions (22). This solute was also detected in the archaeon Methanohalophilus portucalensis strain FDF...

Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.One of the most striking characteristics of extremophiles is their ability to thrive under environmental conditions that would be lethal to most organisms. In particular, hyperthermophiles, having optimal growth temperatures above 80°C (4), pose intriguing questions regarding the biochemical strategies used for the adaptation of their cellular structures to withstand such high temperatures. Maintaining protein performance at high temperature could be accomplished by a number of mechanisms: (i) intrinsic thermostability, (ii) increased turnover, (iii) improved action of molecular chaperones, and (iv) extrinsic stabilization by compatible solutes (13). Although most enzymes from thermophilic sources show an intrinsic thermostability higher than that of their mesophilic counterparts, several enzymes derived from hyperthermophilic sources show an unexpectedly low intrinsic stability in vitro (13,14). Therefore, extrinsic factors, such as compatible solutes, may play a role in the thermostabilization of these cellular components.Some compatible solutes, namely, glutamate, betaine, and trehalose, are widespread in mesophilic organisms, but compatible solutes unique to thermophiles and hyperthermophiles have also been identified in recent years (8; H. Santos and M. S. da Costa, submitted for publication). Newly discovered solutes from thermophilic and hyperthermophilic organisms include cyclic-2,3-bisphosphoglycerate (17), two isomers of dimyo-inositol phosphate (25, 31), mannosylglycerate and mannosylglyceramide (24, 28, 36), di-mannosyl-di-myo-inosito...

The environment-dependent multiexponential behavior of N-acetyltyrosinamide (NAYA) and other tyrosine derivatives is revisited, aiming for a better understanding of tyrosine as an intrinsic fluorescent probe for protein microenvironment changes during conformational changes. The effects of solvent polarity, viscosity, and temperature on the fluorescence decay of NAYA were evaluated using dioxane-water mixtures and pure solvents. Double-exponential decays were observed in dioxane-water mixtures above 70% (v/v) water concentration including pure water, for temperatures below 50 °C. However, at higher temperatures, or in dioxane-water mixtures with lower water concentrations, NAYA shows single-exponential decays. Singleexponential decays were also generally observed in pure solvents (dioxane, acetonitrile, methanol, ethanol, DMSO). The exception was the strong hydrogen-bond donor trifluorethanol, in which NAYA decays as a double exponential. The results are consistent with a solvent-modulated excited-state intramolecular electron transfer from the phenol to the amide moiety occurring in one of the three rotamers of NAYA. On the basis of a full kinetic analysis of the data, it is shown that experimental observation of double-exponential decays depends on three factors: the ground-state population of rotamers, their rates of interconversion (k r ) 4.4 × 10 8 s -1 and k r ′ ) 5.2 × 10 8 s -1 , in water at 23 °C, E r ) 4.9 ( 0.1 kcal mol -1 and E r ′) 5.2 ( 0.3 kcal mol -1 ), and the electron-transfer rate constant (k ET ) 2.0 × 10 9 s -1 , in water at 23 °C, E ET ) 1.8 ( 0.2 kcal mol -1 ). Solvent viscosity controls the interconversion rate constants while solvent polarity and hydrogen-bonding ability determines the Gibbs energy of electron transfer and the magnitude of its rate constant. Consequently, the nature of tyrosine decays in proteins is determined from a delicate balance between the interconversion and electron-transfer rate constants.

Designing small peptides that are capable of binding Cu(2+) ions mainly through the side-chain functionalities is a hard task because the amide nitrogen atoms strongly compete for Cu(2+) ion coordination. However, the design of such peptides is important for obtaining biomimetic small systems of metalloenyzmes as well as for the development of artificial systems. With this in mind, a cyclic decapeptide, C-Asp, which contained three His residues and one Asp residue, and its linear derivative, O-Asp, were synthesized. The C-Asp peptide has two Pro-Gly β-turn-inducer units and, as a result of cyclization, and as shown by CD spectroscopy, its backbone is constrained into a more defined conformation than O-Asp, which is linear and contains a single Pro-Gly unit. A detailed potentiometric, mass spectrometric, and spectroscopic study (UV/Vis, CD, and EPR spectroscopy) showed that at a 1:1 Cu(2+)/peptide ratio, both peptides formed a major [CuHL](2+) species in the pH range 5.0-7.5 (C-Asp) and 5.5-7.0 (O-Asp). The corrected stability constants of the protonated species (log K*(CuH(O-Asp))=9.28 and log K*(CuH(C-Asp))=10.79) indicate that the cyclic peptide binds Cu(2+) ions with higher affinity. In addition, the calculated value of K(eff) shows that this higher affinity for Cu(2+) ions prevails at all pH values, not only for a 1:1 ratio but even for a 2:1 ratio. The spectroscopic data of both [CuHL](2+) species are consistent with the exclusive coordination of Cu(2+) ions by the side-chain functionalities of the three His residues and the Asp residue in a square-planar or square-pyramidal geometry. Nonetheless, although these data show that, upon metal coordination, both peptides adopt a similar fold, the larger conformational constraints that are present in the cyclic scaffold results in different behaviour for both [CuHL](2+) species. CD and NMR analysis revealed the formation of a more rigid structure and a slower Cu(2+)-exchange rate for [CuH(C-Asp)](2+) compared to [CuH(O-Asp](2+). This detailed comparative study shows that cyclization has a remarkable effect on the Cu(2+)-coordination properties of the C-Asp peptide, which binds Cu(2+) ions with higher affinity at all pH values, stabilizes the [CuHL](2+) species in a wider pH range, and has a slower Cu(2+)-exchange rate compared to O-Asp.

Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO 2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the 'green' Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P rnpB , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.

No abstract