A chlorophyll c synthase widely co-opted by phytoplankton.

Marine and terrestrial photosynthesis exhibit a schism in the accessory chlorophyll (Chl) that complements the function of Chl a: Chl b for green plants versus Chl c for most eukaryotic phytoplankton. The enzymes that mediate Chl c biosynthesis have long remained elusive. In this work, we identified the CHLC dioxygenase (Phatr3_J43737) from the marine diatom Phaeodactylum tricornutum as the Chl c synthase. The chlc mutants lacked Chl c, instead accumulating its precursors, and exhibited growth defects. In vitro, recombinant CHLC protein converted these precursors into Chl c, thereby confirming its identity. Phylogenetic evidence demonstrates conserved use of CHLC across phyla but also the existence of distinct Chl c synthases in different algal groups. Our study addresses a long-outstanding question with implications for both contemporary and ancient marine photosynthesis.

Active O-acetylserine-(thiol) lyase A and B confer improved selenium resistance and degrade l-Cys and l-SeCys in Arabidopsis.

The roles of cytosolic O-acetylserine-(thiol)-lyase A (OASTLA), chloroplastic OASTLB, and mitochondrial OASTLC in plant selenate resistance were studied in Arabidopsis. Impairment in OASTLA and OASTLB resulted in reduced biomass, chlorophyll and soluble protein content compared with selenate-treated OASTLC-impaired and wild-type plants. The generally lower total selenium (Se), protein-Se, organic-sulfur and protein-sulfur (S) content in oastlA and oastlB compared with wild-type and oastlC leaves indicated that Se accumulation was not the main cause for the stress symptoms in these mutants. Notably, the application of selenate positively induced S-starvation markers and the OASTLs, followed by increased sulfite reductase, sulfite oxidase activities, and increased sulfite and sulfide concentrations. Taken together, our results indicate a futile anabolic S-starvation response that resulted in lower glutathione and increased oxidative stress symptoms in oastlA and oastlB mutants. In-gel assays of l-cysteine and l-seleno-cysteine, desulfhydrase activities revealed that two of the three OASTL activity bands in each of the oastl single mutants were enhanced in response to selenate, whereas the impaired proteins exhibited a missing activity band. The absence of differently migrated activity bands in each of the three oastl mutants indicates that these OASTLs are major components of desulfhydrase activity, degrading l-cysteine and l-seleno-cysteine in Arabidopsis.

In Vitro Reconstitution of a Five-Step Pathway for Bacterial Ergothioneine Catabolism.

Ergothioneine is a histidine-derived sulfur metabolite that is biosynthesized by bacteria and fungi. Plants and animals absorb ergothioneine as a micronutrient from their environment or nutrition. Several different mechanisms of microbial ergothioneine production have been described in the past ten years. Much less is known about the genetic and structural basis for ergothioneine catabolism. In this report, we describe the in vitro reconstitution of a five-step pathway that degrades ergothioneine to l-glutamate, trimethylamine, hydrogen sulfide, carbon dioxide, and ammonia. The first two steps are catalyzed by the two enzymes ergothionase and thiourocanate hydratase. These enzymes are closely related to the first two enzymes in histidine catabolism. However, the crystal structure of thiourocanate hydratase from the firmicute Paenibacillus sp. reveals specific structural features that strictly differentiate the activity of this enzyme from that of urocanate hydratases. The final two steps are catalyzed by metal-dependent hydrolases that share most homology with the last two enzymes in uracil catabolism. The early and late part of this pathway are connected by an entirely new enzyme type that catalyzes desulfurization of a thiohydantoin intermediate. Homologous enzymes are encoded in many soil-dwelling firmicutes and proteobacteria, suggesting that bacterial activity may have a significant impact on the environmental availability of ergothioneine.

A novel hyperthermophilic methylglyoxal synthase: molecular dynamic analysis on the regional fluctuations.

Two putative methylglyoxal synthases, which catalyze the conversion of dihydroxyacetone phosphate to methylglyoxal, from Oceanithermus profundus DSM 14,977 and Clostridium difficile 630 have been characterized for activity and thermal stability. The enzyme from O. profundus was found to be hyperthermophilic, with the optimum activity at 80 °C and the residual activity up to 59% after incubation of 15 min at 95 °C, whereas the enzyme from C. difficile was mesophilic with the optimum activity at 40 °C and the residual activity less than 50% after the incubation at 55 °C or higher temperatures for 15 min. The structural analysis of the enzymes with molecular dynamics simulation indicated that the hyperthermophilic methylglyoxal synthase has a rigid protein structure with a lower overall root-mean-square-deviation value compared with the mesophilic or thermophilic counterparts. In addition, the simulation results identified distinct regions with high fluctuations throughout those of the mesophilic or thermophilic counterparts via root-mean-square-fluctuation analysis. Specific molecular interactions focusing on the hydrogen bonds and salt bridges in the distinct regions were analyzed in terms of interatomic distances and positions of the individual residues with respect to the secondary structures of the enzyme. Key interactions including specific salt bridges and hydrogen bonds between a rigid beta-sheet core and surrounding alpha helices were found to contribute to the stabilisation of the hyperthermophilic enzyme by reducing the regional fluctuations in the protein structure. The structural information and analysis approach in this study can be further exploited for the engineering and industrial application of the enzyme.

Secretome Analysis of the Banana Fusarium Wilt Fungi Foc R1 and Foc TR4 Reveals a New Effector OASTL Required for Full Pathogenicity of Foc TR4 in Banana.

Banana Fusarium wilt (BFW), which is one of the most important banana diseases worldwide, is mainly caused by Fusarium oxysporum f. sp. cubense tropic race 4 (Foc TR4). In this study, we conducted secretome analysis of Foc R1 and Foc TR4 and discovered a total of 120 and 109 secretory proteins (SPs) from Foc R1 cultured alone or with banana roots, respectively, and 129 and 105 SPs respectively from Foc TR4 cultured under the same conditions. Foc R1 and Foc TR4 shared numerous SPs associated with hydrolase activity, oxidoreductase activity, and transferase activity. Furthermore, in culture with banana roots, Foc R1 and Foc TR4 secreted many novel SPs, of which approximately 90% (Foc R1; 57/66; Foc TR4; 50/55) were unconventional SPs without signal peptides. Comparative analysis of SPs in Foc R1 and Foc TR4 revealed that Foc TR4 not only generated more specific SPs but also had a higher proportion of SPs involved in various metabolic pathways, such as phenylalanine metabolism and cysteine and methionine metabolism. The cysteine biosynthesis enzyme O-acetylhomoserine (thiol)-lyase (OASTL) was the most abundant root inducible Foc TR4-specific SP. In addition, knockout of the OASTL gene did not affect growth of Foc TR4; but resulted in the loss of pathogenicity in banana 'Brazil'. We speculated that OASTL functions in banana by interfering with the biosynthesis of cysteine, which is the precursor of an enormous number of sulfur-containing defense compounds. Overall, our studies provide a basic understanding of the SPs in Foc R1 and Foc TR4; including a novel effector in Foc TR4.

Secreted Osteoclastogenic Factor of Activated T Cells (SOFAT) Is Associated With Rheumatoid Arthritis and Joint Pain: Initial Evidences of a New Pathway.

Rheumatoid arthritis (RA) has an inflammatory milieu in the synovial compartment, which is regulated by a complex cytokine and chemokine network that induces continuously degenerative and inflammatory reactions. The secreted osteoclastogenic factor of activated T cells (SOFAT) is a unique cytokine and represents an alternative pathway for osteoclast activation. In this study, we examined whether SOFAT is able to induce joint pain and investigated the presence of SOFAT in a Collagen-induced Arthritis (CIA) model and in human subjects. Here, we found that an intra-articular stimulation with SOFAT (1, 10, 100, or 1,000 ng/10 µl) in the knee joint significantly decreases the mechanical threshold in the hind paw of mice (p < 0.05). Moreover, after a second injection of SOFAT, the mechanical threshold decrease was sustained for up to 8 days (p < 0.05). In the CIA model, the immunohistochemical assay of knee joint showed positivity stained for SOFAT, and the mRNA and protein expression of SOFAT were significantly higher in the affected-group (p < 0.05). Besides, the mRNA of RANKL, IL-1ß, IL-6, and IL-15 were significantly higher in the affected-group (p < 0.05). Finally, SOFAT was detected in the synovial fluid of RA patients, but not in OA patients (p < 0.05). In conclusion, SOFAT is up regulated in inflammatory milieu such as RA but not in non-inflammatory OA. SOFAT may be a novel molecule in the complex inflammatory phenotype of RA.

Underground isoleucine biosynthesis pathways in E. coli.

The promiscuous activities of enzymes provide fertile ground for the evolution of new metabolic pathways. Here, we systematically explore the ability of E. coli to harness underground metabolism to compensate for the deletion of an essential biosynthetic pathway. By deleting all threonine deaminases, we generated a strain in which isoleucine biosynthesis was interrupted at the level of 2-ketobutyrate. Incubation of this strain under aerobic conditions resulted in the emergence of a novel 2-ketobutyrate biosynthesis pathway based upon the promiscuous cleavage of O-succinyl-L-homoserine by cystathionine γ-synthase (MetB). Under anaerobic conditions, pyruvate formate-lyase enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate. Surprisingly, we found this anaerobic route to provide a substantial fraction of isoleucine in a wild-type strain when propionate is available in the medium. This study demonstrates the selective advantage underground metabolism offers, providing metabolic redundancy and flexibility which allow for the best use of environmental carbon sources.

Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme That Catalyzes C-C Bond Formation.

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze transformations of l-amino acids at α, ß, and γ positions. These enzymes are frequently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S,6S)-6-methyl pipecolate 1, from the biosynthetic pathway of citrinadin. The key enzyme CndF is PLP-dependent and catalyzes the synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs γ-elimination of O-acetyl-l-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new Cγ-Cδ bond in 3 and complete the γ-substitution reaction. CndF displays promiscuity toward different ß-keto carboxylate and esters. With use of an Aspergillus strain expressing CndF and CndE, feeding various alkyl-ß-keto esters led to the biosynthesis of 6-substituted l-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria, using α-humulene as a model compound.

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden-Meyerhof-Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.

Reaction of threonine synthase with the substrate analogue 2-amino-5-phosphonopentanoate: implications into the proton transfer at the active site.

Threonine synthase catalyses the conversion of O-phospho-l-homoserine and a water molecule to l-threonine and has the most complex catalytic mechanism among the pyridoxal 5'-phosphate-dependent enzymes. In order to study the less-characterized earlier stage of the catalytic reaction, we studied the reaction of threonine synthase with 2-amino-5-phosphonopentanoate, which stops the catalytic reaction at the enamine intermediate. The global kinetic analysis of the triphasic spectral changes showed that, in addition to the theoretically expected pathway, the carbanion is rapidly reprotonated at Cα to form an aldimine distinct from the external aldimine directly formed from the Michaelis complex. The Kd for the binding of inhibitor to the enzyme decreased with increasing pH, showing that the 2-amino-group-unprotonated form of the ligand binds to the enzyme. On the other hand, the rate constants for the proton migration steps within the active site are independent of the solvent pH, indicating that protons are shared by the active dissociative groups and are not exchanged with the solvent during the course of catalysis. This gives an insight into the role of the phosphate group of the substrate, which may increase the basicity of the ε-amino group of the catalytic lysine residue in the active site.

The Salmonella type III effector SpvC triggers the reverse transmigration of infected cells into the bloodstream.

Salmonella can appear in the bloodstream within CD18 expressing phagocytes following oral ingestion in as little as 15 minutes. Here, we provide evidence that the process underlying this phenomenon is reverse transmigration. Reverse transmigration is a normal host process in which dendritic cells can reenter the bloodstream by traversing endothelium in the basal to apical direction. We have developed an in vitro reverse transmigration assay in which dendritic cells are given the opportunity to cross endothelial monolayers in the basal to apical direction grown on membranes with small pores, modeling how such cells can penetrate the bloodstream. We demonstrate that exposing dendritic cells to microbial components negatively regulates reverse transmigration. We propose that microbial components normally cause the host to toggle between positively and negatively regulating reverse transmigration, balancing the need to resolve inflammation with inhibiting the spread of microbes. We show that Salmonella in part overcomes this negative regulation of reverse transmigration with the Salmonella pathogenicity island-2 encoded type III secretion system, which increases reverse transmigration by over an order of magnitude. The SPI-2 type III secretion system does this in part, but not entirely by injecting the type III effector SpvC into infected cells. We further demonstrate that SpvC greatly promotes early extra-intestinal dissemination in mice. This result combined with the previous observation that the spv operon is conserved amongst strains of non-typhoidal Salmonella capable of causing bacteremia in humans suggests that this pathway to the bloodstream could be important for understanding human infections.

Threonine synthase CoTHR4 is involved in infection-related morphogenesis during the pre-penetration stage in Colletotrichum orbiculare.

Upon recognition of host plants, Colletotrichum orbiculare, an anthracnose disease fungus of cucurbitaceous plants, initiates morphological differentiation, including conidial germination and appressorium formation on the cuticle layer. The series of infection processes of C. orbiculare requires enormous nutrient and energy, but the surface of the cucurbitaceous hosts is hardly nutrient-rich. Hence, C. orbiculare must exert tight management of its intracellular nutrients in order to properly induce infection-related morphogenesis. Here, we carried out a large-scale insertional mutagenesis screen using Agrobacterium tumefaciens-mediated transformation to identify novel genes involved in the pathogenicity of C. orbiculare and found that CoTHR4-encoded threonine synthase, a homolog of Saccharomyces cerevisiae THR4, is required for pathogenicity and conidiation in C. orbiculare. Threonine supplementation allowed the cothr4 mutant to produce conidia to a level equivalent to that of the wild-type. The conidia produced from the threonine-treated cothr4 mutant failed to germinate in the absence of threonine, but retained the ability to germinate and to form appressoria in the presence of threonine. However, the conidia produced from the threonine-treated cothr4 mutant remained attenuated in pathogenicity on cucumber cotyledons even in the presence of threonine. Cytorrhysis assays revealed that appressoria of the cothr4 mutant induced by exogenous threonine treatment showed low turgor generation. Taken together, these results showed that threonine synthase CoThr4 plays a pivotal role in infection-related morphogenesis during the pre-penetration stage of C. orbiculare.

Identification of O-acetylhomoserine sulfhydrylase, a putative enzyme responsible for methionine biosynthesis in Clostridioides difficile: Gene cloning and biochemical characterizations.

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue Nγ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with Ki = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.

Rhizobium spp exopolysaccharides production and xanthan lyase use on its structural modification.

Enzymes can be very useful on exopolysaccharides (EPS) research, can be used at elucidation and also to modify the polysaccharides' structure in order to alter their physical properties. Thus, the reduction of the molecular mass could increase applications of these biopolymers. Therefore, the EPS production of different rhizobia and the action of xanthan lyase on its structures were evaluated. The strains produced significant amounts of EPS, and it was noticed that are heteropolysaccharides, composed galactose and glucose. Both EPS and xanthan were modified on ß-glycosidic bonds, the mannose was removed of xanthan had but the EPS was affected in the CO stretching vibration, where the glucuronic acid removed from of your structure. The ester/carboxylic acid portions affected functional groups of the acetate/succinate, methyl carbons of the O-acetyl and pyruvate methyl groups in addition to affect the carbons the main pyranoid. The Resistance to temperature increase of the EPS was observed, made possible by the activity of the lyase. EPS has the ability to form stable gels at higher temperatures and anionic feature can be used on solubilization and controlled release of substances. Modified EPS knowledge will presently facilitate future investigations relating the structure of the rhizobia polysaccharide against rheological properties.

The level of threonine in tobacco seeds is limited by substrate availability, while the level of methionine is limited also by the activity of cystathionine γ-synthase.

Methionine and threonine are two essential amino acids whose low levels limit the nutritional quality of seeds. The current objective was to define factors that regulate and might increase their levels in seeds. Feeding experiments carried out on receptacles of developing tobacco (Nicotiana tabacum) capsules showed that 1 mM of S-methylmethionine increased the level of methionine to contents similar to 2.5 mM of homoserine, an intermediate metabolite of the aspartate family of amino acids. The latter also increased the level of threonine. Based on these findings, we generated tobacco seeds that expressed a combination of bacterial feedback-insensitive aspartate kinase (bAK), which was previously reported to have a high level of threonine/methionine, and feedback-insensitive cystathionine γ-synthase (CGS), the regulatory enzyme of the methionine biosynthesis pathway. Plants expressing this latter gene previously showed having higher levels of methionine. The results of total amino acids analysis showed that the level of threonine was highest in the bAK line, which has moderate levels of methionine and lysine, while the highest level of methionine was found in seeds expressing both heterologous genes. The results suggest that the level of threonine in tobacco seeds is limited by the substrate, while that of methionine is limited also by the activity of CGS.

Identification of an Enzyme Catalyzing the Conversion of Sulfoacetaldehyde to 2-Mercaptoethanesulfonic Acid in Methanogens.

Coenzyme M is an essential coenzyme for the biochemical production of methane. This Communication reports on the identification of an enzyme catalyzing the last step in the biosynthesis of coenzyme M in methanogens. Data presented here show that the enzyme, derived from mj1681, catalyzes the conversion of the aldehyde functional group of sulfoacetaldehyde into the thiol group of 2-mercaptoethanesulfonic acid. Thus, a putative coenzyme M synthase (comF) has similarities in sequence with both MJ0100 and MJ0099 proteins previously shown to be involved in the biosynthesis of homocysteine [Allen, K. D., et al. (2015) Biochemistry 54, 3129-3132], and both reactions likely proceed by the same mechanism. In the MJ0100-catalyzed reaction, Rauch has proposed [Rauch, B. L. (2017) Biochemistry 56, 1051-1061] that MJ1526 and its homologues in other methanogens likely supply the sulfane sulfur required for the reaction.

Mechanism of a Standalone ß-Lactone Synthetase: New Continuous Assay for a Widespread ANL Superfamily Enzyme.

Enzyme-catalyzed ß-lactone formation from ß-hydroxy acids is a crucial step in bacterial biosynthesis of ß-lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for ß-lactone synthetase activity using synthetic ß-hydroxy acid substrates with alkene or alkyne moieties. ß-Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real-time evaluation by UV/Vis spectroscopy. The assay was used to determine steady-state kinetics of a long-chain ß-lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site-directed mutagenesis was used to test the involvement of conserved active site residues in Mg2+ and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the ß-lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC-MS. Stereoisomers of 2-hexyl-3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo-preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the ß-lactone synthetases to map their functional diversity within the acyl-CoA synthetase, NRPS adenylation domain, and luciferase superfamily.

Molecular Basis of Bacillus subtilis ATCC 6633 Self-Resistance to the Phosphono-oligopeptide Antibiotic Rhizocticin.

Rhizocticins are phosphono-oligopeptide antibiotics that contain a toxic C-terminal ( Z) -l -2-amino-5-phosphono-3-pentenoic acid (APPA) moiety. APPA is an irreversible inhibitor of threonine synthase (ThrC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-phospho-l-homoserine to l-threonine. ThrCs are essential for the viability of bacteria, plants, and fungi and are a target for antibiotic development, as de novo threonine biosynthetic pathway is not found in humans. Given the ability of APPA to interfere in threonine metabolism, it is unclear how the producing strain B. subtilis ATCC 6633 circumvents APPA toxicity. Notably, in addition to the housekeeping APPA-sensitive ThrC ( BsThrC), B. subtilis encodes a second threonine synthase (RhiB) encoded within the rhizocticin biosynthetic gene cluster. Kinetic and spectroscopic analyses show that PLP-dependent RhiB is an authentic threonine synthase, converting O-phospho-l-homoserine to threonine with a catalytic efficiency comparable to BsThrC. To understand the structural basis of inhibition, we determined the crystal structure of APPA bound to the housekeeping BsThrC, revealing a covalent complex between the inhibitor and PLP. Structure-based sequence analyses reveal structural determinants within the RhiB active site that contribute to rendering this ThrC homologue resistant to APPA. Together, this work establishes the self-resistance mechanism utilized by B. subtilis ATCC 6633 against APPA exemplifying one of many ways by which bacteria can overcome phosphonate toxicity.

Cellular physiology controls photoautotrophic production of 1,2-propanediol from pools of CO2 and glycogen.

Synechocystis sp. PCC 6803 PG is a cyanobacterial strain capable of synthesizing 1,2-propanediol from carbon dioxide (CO2 ) via a heterologous three-step pathway and a methylglyoxal synthase (MGS) originating from Escherichia coli as an initial enzyme. The production window is restricted to the late growth and stationary phase and is apparently coupled to glycogen turnover. To understand the underlying principle of the carbon partitioning between the Calvin-Benson-Bassham (CBB) cycle and glycogen in the context of 1,2-propanediol production, experiments utilizing 13 C labeled CO2 have been conducted. Carbon fluxes and partitioning between biomass, storage compounds, and product have been monitored under permanent illumination as well as under dark conditions. About one-quarter of the carbon incorporated into 1,2-propanediol originated from glycogen, while the rest was derived from CO2 fixed in the CBB cycle during product formation. Furthermore, 1,2-propanediol synthesis was depending on the availability of photosynthetic active radiation and glycogen catabolism. We postulate that the regulation of the MGS from E. coli conflicts with the heterologous reactions leading to 1,2-propanediol in Synechocystis sp. PCC 6803 PG. Additionally, homology comparison of the genomic sequence to genes encoding for the methylglyoxal bypass in E. coli suggested the existence of such a pathway also in Synechocystis sp. PCC 6803. These findings are critical for all heterologous pathways coupled to the CBB cycle intermediate dihydroxyacetone phosphate via a MGS and reveal possible engineering targets for rational strain optimization.

Phosphothreonine Lyase Promotes p65 Degradation in a Mitogen-Activated Protein Kinase/Mitogen- and Stress-Activated Protein Kinase 1-Dependent Manner.

Bacterial phosphothreonine lyases have been identified to be type III secretion system (T3SS) effectors that irreversibly dephosphorylate host mitogen-activated protein kinase (MAPK) signaling to promote infection. However, the effects of phosphothreonine lyase on nuclear factor κB (NF-κB) signaling remain largely unknown. In this study, we detected significant phosphothreonine lyase-dependent p65 degradation during Edwardsiella piscicida infection in macrophages, and this degradative effect was blocked by the protease inhibitor MG132. Further analysis revealed that phosphothreonine lyase promotes the dephosphorylation and ubiquitination of p65 by inhibiting the phosphorylation of mitogen- and stress-activated protein kinase-1 (MSK1) and by inhibiting the phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2), p38α, and c-Jun N-terminal kinase (JNK). Moreover, we revealed that the catalytic active site of phosphothreonine lyase plays a critical role in regulating the MAPK-MSK1-p65 signaling axis. Collectively, the mechanism described here expands our understanding of the pathogenic effector in not only regulating MAPK signaling but also regulating p65. These findings uncover a new mechanism by which pathogenic bacteria overcome host innate immunity to promote pathogenesis.