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12- cis -oxo-phytodienoic acid (OPDA), a precursor of jasmonoyl-isoleucine (JA-Ile), is known to have distinct signaling roles in Arabidopsis, as shown in studies using the opr3 mutant, which lacks OPDA REDUCTASE3 (OPR3). This mutant, however, accumulates low levels of JA-Ile through an OPR2-mediated bypass. To investigate OPDA signaling, the wound-induced transcriptome of the opr2opr3 mutant is compared to that of wild-type and allene oxide synthase mutant. Endogenous OPDA shows no unique transcriptional signature under control or wounding conditions, and previously identified OPDA-responsive genes are wound-induced independently of OPDA. Applying OPDA to opr2opr3 triggers a distinct response suggesting compartmentalization of endogenously formed OPDA. Trans-organellar complementation reveals that expression of OPR3 or OPR2 in opr2opr3 restores JA-Ile production regardless of localization, whereas mitochondrial targeted OPR1 exhibiting low OPDA/4,5-ddh-JA conversion activity does not. Our findings show that OPDA primarily serves as a JA precursor with limited independent signaling functions in the early wound response. Jasmonates are mediators of plant wound-responses. Using OPDA and JA-deficient mutants, the authors show that OPDA produced in wounded Arabidopsis seedlings does not confer signaling capacity, whereas application of OPDA triggers a distinct response.

Vitamins are essential components of the diet of animals and humans. Vitamins are thus important targets for biotechnological production. While efficient fermentation processes have been developed for several vitamins, this is not the case for vitamin B5 (pantothenate), the precursor of coenzyme A. We have elucidated the complete pathway for coenzyme A biosynthesis in the biotechnological workhorse Bacillus subtilis . Moreover, a salvage pathway for coenzyme A synthesis was found in this study. Normally, this pathway depends on pantetheine; however, we observed activity of the salvage pathway on complex medium in mutants lacking the pantothenate biosynthesis pathway even in the absence of supplemented pantetheine. This required rewiring of metabolism by expressing a cystine transporter due to acquisition of mutations affecting the regulation of cysteine metabolism. This shows how the hidden “underground metabolism” can give rise to the rapid formation of novel metabolic pathways.

Promiscuous enzymes often serve as the starting point for the evolution of novel functions. Yet, the extent to which the promiscuity of an individual enzyme can be harnessed several times independently for different purposes during evolution is poorly reported. Here, we present a case study illustrating how NAD(P) + -dependent succinate semialdehyde dehydrogenase of Escherichia coli (Sad) is independently recruited through various evolutionary mechanisms for distinct metabolic demands, in particular vitamin biosynthesis and central carbon metabolism. Using adaptive laboratory evolution (ALE), we show that Sad can substitute for the roles of erythrose 4-phosphate dehydrogenase in pyridoxal 5’-phosphate (PLP) biosynthesis and glyceraldehyde 3-phosphate dehydrogenase in glycolysis. To recruit Sad for PLP biosynthesis and glycolysis, ALE employs various mechanisms, including active site mutation, copy number amplification, and (de)regulation of gene expression. Our study traces down these different evolutionary trajectories, reports on the surprising active site plasticity of Sad, identifies regulatory links in amino acid metabolism, and highlights the potential of an ordinary enzyme as innovation reservoir for evolution. Enzyme promiscuity seeds evolutionary innovation, but how flexible a single enzyme can be (re-)used during evolution remains unclear. Here, the authors show that various evolutionary trajectories applied to succinate semialdehyde dehydrogenase can compensate for the loss of two different functions in E. coli .

Background Antimicrobial peptides (AMPs) are promising candidates for the development of novel antibiotics, but it is difficult to produce sufficient quantities for preclinical and clinical studies due to their toxicity towards microbial expression hosts. To avoid laborious trial-and-error testing for the identification of suitable expression constructs, we have developed a small-scale expression screening platform based on a combinatorial plasmid library. Results The combinatorial library is based on the Golden Gate cloning system. In each reaction, six donor plasmids (each containing one component: a promoter, fusion partner 1, fusion partner 2, protease cleavage site, gene of interest, or transcriptional terminator) were combined with one acceptor plasmid to yield the final expression construct. As a proof of concept, screening was carried out in Escherichia coli and Pichia pastoris to study the expression of three different model AMPs with challenging characteristics, such as host toxicity or multiple disulfide bonds. The corresponding genes were successfully cloned in 27 E. coli and 18 P. pastoris expression plasmids, each in a one-step Golden Gate reaction. After transformation, small-scale expression screening in microtiter plates was followed by AMP quantification using a His 6 tag-specific ELISA. Depending on the plasmid features and the expression host, the protein yields differed by more than an order of magnitude. This allowed the identification of high producers suitable for larger-scale protein expression. Conclusions The optimization of recombinant protein production is best achieved from first principles by initially optimizing the genetic construct. The unrestricted combination of multiple plasmid features yields a comprehensive library of expression strains that can be screened for optimal productivity. The availability of such a platform could benefit all laboratories working in the field of recombinant protein expression.

The straightforward synthesis of a urea polymer network is presented. Commercially available monomers are polymerized using light-induced polymerization, resulting in networks crosslinked by hindered urea molecules. These moieties are reversible and, thus, can be converted into the starting compounds (that is, isocyanate and amine) by a simple thermal treatment. This process is monitored using differential scanning calorimetry as well as Raman and infrared spectroscopy. Furthermore, the self-healing ability of these polymer networks is investigated using scratch-healing tests as well as bulk-healing investigations using tensile testing. The resultant materials have a high E -modulus, are able to heal scratches at temperatures above 70 °C multiple times and their mechanical properties can be partially regenerated. The underlying healing mechanism is based on the reversible opening of the urea bonds and exchange reactions between two functional groups, which were confirmed from a spectroscopic analysis. In summary, these new materials are a new type of intrinsically healable polymers and provide a first step toward hard and healable polymers. Self-healing polymers: Learning the hard stuff One of the hardest self-healing polymers ever reported has been prepared using the reversible bonds of sterically hindered urea groups. Polymers that can re-form internal chemical links after being scratched or cracked are usually subject to design constraints that lower their mechanical strength. To overcome these constraints, Martin D. Hager and colleagues from Friedrich Schiller University Jena, Germany, created a series of poly(methacrylate) polymers bearing reversible urea units. By simply exposing the starting reagents to brief flashes of light, they prepared a cross-linked polymer featuring the urea units on the poly(methacrylate) chains resulting in mechanically tough materials. After optimizing the cross-link density, the team deliberately scratched the polymer and then heated it to begin the self-healing process. Temperatures of about 100 degree Celsius were sufficient to open urea bonds up and initiate material repair. New intrinsic self-healing polymers with outstanding mechanical performance are presented. For this purpose, sterically hindered amines were utilized to crosslink isocyanate containing poly(methacrylates) resulting in urea crosslinked networks. The reversibility of the urea bond during thermal treatment could be utilized to induce self-healing ability and could be proven using various techniques.

Reproductive isolation among locally adapted populations may arise when immigrants from foreign habitats are selected against via natural or (inter-)sexual selection (female mate choice). We asked whether also intrasexual selection through male-male competition could promote reproductive isolation among populations of poeciliid fishes that are locally adapted to extreme environmental conditions [i.e., darkness in caves and/or toxic hydrogen sulphide (H2S)]. We found strongly reduced aggressiveness in extremophile Poecilia mexicana, and darkness was the best predictor for the evolutionary reduction of aggressiveness, especially when combined with presence of H2S. We demonstrate that reduced aggression directly translates into migrant males being inferior when paired with males from nonsulphidic surface habitats. By contrast, the phylogenetically old sulphur-endemic P. sulphuraria from another sulphide spring area showed no overall reduced aggressiveness, possibly indicating evolved mechanisms to better cope with H2S.

Accumulation of homocysteine and S-adenosylhomocysteine in bone has been shown to be associated with reduced bone quality in rats. The aim of the present study was to investigate whether high bone concentrations of homocysteine and S-adenosylhomocysteine as well as a low methylation capacity are related to an impaired bone morphology in humans. Concentrations of homocysteine and its precursors S-adenosylhomocysteine and S-adenosylmethionine were measured in femoral bone samples of eighty-two males and females (age 71 (sd 8) years) who underwent elective hip arthroplasty. Cancellous bone structure was analysed by histomorphometry. In addition, blood was sampled to measure serum concentrations of homocysteine. Results of bone and serum analyses were grouped for individuals with high or low bone concentrations of homocysteine, S-adenosylhomocysteine and S-adenosylmethionine, as well as for individuals with a high or a low methylation capacity, which is indicated by a low or a high S-adenosylhomocysteine:S-adenosylmethionine ratio (n 41, each). Histomorphometry showed a higher trabecular separation and a lower trabecular thickness, trabecular number and trabecular area in individuals with high bone concentrations of homocysteine and S-adenosylhomocysteine compared with individuals with low bone concentrations of homocysteine and S-adenosylhomocysteine. There was no association between the S-adenosylhomocysteine:S-adenosylmethionine ratio and bone morphology. It was found that 48 % of bone homocysteine was bound to the collagen of the extracellular bone matrix. Blood analyses demonstrated a significant correlation between serum and bone homocysteine. The results of the present study indicate an association between altered bone morphology and elevated bone concentrations of homocysteine and S-adenosylhomocysteine, but not between altered bone morphology and methylation capacity.

12-cis-Oxo-phytodienoic acid (OPDA), the main precursor of the key plant growth and defense hormone jasmonoyl-isoleucine (JA-Ile), is believed to have distinct signaling roles in plant’s responses to stress. In Arabidopsis, insights into OPDA functions have been gained from studying a mutant, which is affected in the conversion of OPDA by missing OPDA REDUCTASE3 (OPR3). opr3 mutants, however, accumulate JA-Ile through a cytosolic bypass mediated by OPR2. Therefore, wound-induced transcriptome of opr2opr3 in comparison to wild-type and allene oxide synthase mutant was analyzed to unravel OPDA signaling. Results showed that OPDA lacked a distinct transcriptional signature, whereas known OPDA-response genes were wound-induced independently of OPDA. The application of OPDA to opr2opr3 resulted in a distinct transcriptional response compared to the endogenous rise of OPDA in the same mutant, with the activation of the sulfur assimilation pathway genes occurring only with the external application of the compound. These findings suggested a compartmentalization of endogenously produced OPDA, investigated further through trans-organellar complementation. OPR3 complemented opr2opr3 mutants in fertility and wound-induced JA-Ile production regardless of its localization. Since in vitro enzymatic studies revealed OPR3’s activity on both OPDA and 4,5-ddh-JA, conclusions on translocation of OPDA were not unequivocal. Dissecting the conversion of either OPDA or 4,5-ddh-JA by OPR2 and OPR1 organelle variants pointed, however, to a strong OPDA compartmentalization supporting its lacking signaling function.

The Gram-positive model bacterium Bacillus subtilis is used for many biotechnological applications, including the large-scale production of vitamins. For vitamin B5, a precursor for coenzyme A synthesis, there is so far no established fermentation process available, partly due to the incomplete knowledge on the metabolic pathways that involve this vitamin. In this study, we have elucidated the complete pathways for the biosynthesis pantothenate and coenzyme A in B. subtilis. We have identified the enzymes involved in the pathway and have identified a salvage pathway for coenzyme A acquisition that acts on complex medium even in the absence of pantothenate synthesis. This pathway requires rewiring of sulfur metabolism resulting in the expression of a cysteine transporter. In the salvage pathway, the bacteria import cysteinopantetheine, a novel naturally occurring metabolite, using the cystine transport system TcyJKLMN. This work lays the foundation for the development of effective processes for vitamin B5 production.