Systems metabolic engineering of the primary and secondary metabolism of Streptomyces albidoflavus enhances production of the reverse antibiotic nybomycin against multi-resistant Staphylococcus aureus.

Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus, making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 µg L-1 of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common Perm* promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that Perm* failed to drive expression during the product synthesis phase but that use of two synthetic promoters (PkasOP* and P41) enabled strong constitutive expression during the entire process. Using PkasOP*, several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L-1 in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3'-end nyb of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L-1 nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin.

Saccharomyces Cerevisiae-An Interesting Producer of Bioactive Plant Polyphenolic Metabolites.

Secondary phenolic metabolites are defined as valuable natural products synthesized by different organisms that are not essential for growth and development. These compounds play an essential role in plant defense mechanisms and an important role in the pharmaceutical, cosmetics, food, and agricultural industries. Despite the vast chemical diversity of natural compounds, their content in plants is very low, and, as a consequence, this eliminates the possibility of the production of these interesting secondary metabolites from plants. Therefore, microorganisms are widely used as cell factories by industrial biotechnology, in the production of different non-native compounds. Among microorganisms commonly used in biotechnological applications, yeast are a prominent host for the diverse secondary metabolite biosynthetic pathways. Saccharomyces cerevisiae is often regarded as a better host organism for the heterologous production of phenolic compounds, particularly if the expression of different plant genes is necessary.

Increased biomass accumulation in maize grown in mixed nitrogen supply is mediated by auxin synthesis.

The use of mixed nitrate and ammonium as a nitrogen source can improve plant growth. Here, we used metabolomics and transcriptomics to study the underlying mechanisms. Maize plants were grown hydroponically in the presence of three forms of nitrogen (nitrate alone, 75%/25% nitrate/ammonium, and ammonium alone). Plants grown with mixed nitrogen had a higher photosynthetic rate than those supplied only with nitrate, and had the highest leaf area and shoot and root biomass among the three nitrogen treatments. In shoot and root, the concentration of nitrogenous compounds (ammonium, glutamine, and asparagine) and carbohydrates (sucrose, glucose, and fructose) in plants with a mixed nitrogen supply was higher than that with nitrate supply, but lower than that with ammonium supply. The activity of the related enzymes (glutamate synthase, asparagine synthase, phosphoenolpyruvate carboxylase, invertase, and ADP-glucose pyrophosphorylase) changed accordingly. Specifically, the mixed nitrogen source enhanced auxin synthesis via the shikimic acid pathway, as indicated by the higher levels of phosphoenolpyruvate and tryptophan compared with the other two treatments. The expression of corresponding genes involving auxin synthesis and response was up-regulated. Supply of only ammonium resulted in high levels of glutamine and asparagine, starch, and trehalose hexaphosphate. We conclude that, in addition to increased photosynthesis, mixed nitrogen supply enhances leaf growth via increasing auxin synthesis to build a large sink for carbon and nitrogen utilization, which, in turn, facilitates further carbon assimilation and nitrogen uptake.

Pathway engineering for the production of heterologous aromatic chemicals and their derivatives in Saccharomyces cerevisiae: bioconversion from glucose.

Saccharomyces cerevisiae has been extensively engineered for optimising its performance as a microbial cell factory to produce valuable aromatic compounds and their derivatives as bulk and fine chemicals. The production of heterologous aromatic molecules in yeast is achieved via engineering of the aromatic amino acid biosynthetic pathway. This pathway is connected to two pathways of the central carbon metabolism, and is highly regulated at the gene and protein level. These characteristics impose several challenges for tailoring it, and various modifications need to be applied in order to redirect the carbon flux towards the production of the desired compounds. This minireview addresses the metabolic engineering approaches targeting the central carbon metabolism, the shikimate pathway and the tyrosine and phenylalanine biosynthetic pathway of S. cerevisiae for biosynthesis of aromatic chemicals and their derivatives from glucose.

Isolation and dynamic expression of four genes involving in shikimic acid pathway in Camellia sinensis 'Baicha 1' during periodic albinism.

Flavonoids are the main flavor components and functional ingredients in tea, and the shikimic acid pathway is considered as one of the most important pathways in flavonoid biosynthesis, but little was known about the function of regulatory genes in the metabolism phenolic compounds in tea plant (Camellia sinensis), especially related genes in shikimic acid pathway. The dynamic changes of catechin (predominant flavonoid) contents were analyzed in this study, and four genes (CsPPT, CsDAHPS, CsSDH and CsCS) involving in shikimic acid pathway in C. sinensis albino cultivar 'Baicha 1' were cloned and characterized. The full-length cDNA sequences of these genes were obtained using reverse transcription-PCR and rapid amplification of cDNA ends. At the albinistic stage, the amounts of all catechins decreased to the lowest levels, when epigallocatechin gallate was the highest, whereas gallocatechin-3-O-gallate the lowest. Gene expression patterns analyzed by qRT-PCR showed that CsPPT and CsDAHPS were highly expressed in flowers and buds, while CsSDH and CsCS showed high expression levels in buds and leaves. It was also found that the transcript abundance of shikimic acid biosynthetic genes followed a tightly regulated biphasic pattern, and was affected by albinism. The transcript levels of CsPPT and CsDAHPS were decreased at albinistic stage followed elevated expression, whereas CsSDH and CsCS were increased only at re-greening stage. Taken together, these findings suggested that these four genes in C. sinensis may play different roles in shikimic acid biosynthesis and these genes may have divergent functions.

Volatile Organic Compounds Induced by Herbivory of the Soybean Looper Chrysodeixis includens in Transgenic Glyphosate-Resistant Soybean and the Behavioral Effect on the Parasitoid, Meteorus rubens.

Transgenic soybean plants (RR) engineered to express resistance to glyphosate harbor a variant of the enzyme EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) involved in the shikimic acid pathway, the biosynthetic route of three aromatic amino acids: phenylalanine, tyrosine, and tryptophan. The insertion of the variant enzyme CP4 EPSPS confers resistance to glyphosate. During the process of genetic engineering, unintended secondary effects are likely to occur. In the present study, we quantified volatile organic compounds (VOCs) emitted constitutively or induced in response to herbivory by the soybean looper Chrysodeixis includens in transgenic soybean and its isogenic (untransformed) line. Since herbivore-induced plant volatiles (HIPVs) are known to play a role in the recruitment of natural enemies, we assessed whether changes in VOC profiles alter the foraging behavior of the generalist endoparasitic larval parasitoid, Meteorus rubens in the transgenic line. Additionally, we assessed whether there was a difference in plant quality by measuring the weight gain of the soybean looper. In response to herbivory, several VOCs were induced in both the conventional and the transgenic line; however, larger quantities of a few compounds were emitted by transgenic plants. Meteorus rubens females were able to discriminate between the odors of undamaged and C. includens-damaged plants in both lines, but preferred the odors emitted by herbivore-damaged transgenic plants over those emitted by herbivore-damaged conventional soybean plants. No differences were observed in the weight gain of the soybean looper. Our results suggest that VOC-mediated tritrophic interactions in this model system are not negatively affected. However, as the preference of the wasps shifted towards damaged transgenic plants, the results also suggest that genetic modification affects that tritrophic interactions in multiple ways in this model system.

The volatile profiles of a rare apple (Malus domestica Borkh.) honey: shikimic acid-pathway derivatives, terpenes, and others.

The volatile profiles of rare Malus domestica Borkh. honey were investigated for the first time. Two representative samples from Poland (sample I) and Spain (sample II) were selected by pollen analysis (44-45% of Malus spp. pollen) and investigated by GC/FID/MS after headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). The apple honey is characterized by high percentage of shikimic acid-pathway derivatives, as well as terpenes, norisoprenoids, and some other compounds such as coumaran and methyl 1H-indole-3-acetate. The main compounds of the honey headspace were (sample I; sample II): benzaldehyde (9.4%; 32.1%), benzyl alcohol (0.3%; 14.4%), hotrienol (26.0%, 6.2%), and lilac aldehyde isomers (26.3%; 1.7%), but only Spanish sample contained car-2-en-4-one (10.2%). CH2 Cl2 and pentane/Et2 O 1 : 2 (v/v) were used for USE. The most relevant compounds identified in the extracts were: benzaldehyde (0.9-3.9%), benzoic acid (2.0-11.2%), terpendiol I (0.3-7.4%), coumaran (0.0-2.8%), 2-phenylacetic acid (2.0-26.4%), methyl syringate (3.9-13.1%), vomifoliol (5.0-31.8%), and methyl 1H-indole-3-acetate (1.9-10.2%). Apple honey contained also benzyl alcohol, 2-phenylethanol, (E)-cinnamaldehyde, (E)-cinnamyl alcohol, eugenol, vanillin, and linalool that have been found previously in apple flowers, thus disclosing similarity of both volatile profiles.

Hormetic Effect of Glyphosate on the Morphology, Physiology and Metabolism of Coffee Plants.

Glyphosate is a nonselective herbicide of systemic action that inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase, thus compromising amino acid production and consequently the growth and development of susceptible plants. The objective of this study was to evaluate the hormetic effect of glyphosate on the morphology, physiology, and biochemistry of coffee plants. Coffee seedlings (Coffea arabica cv Catuaí Vermelho IAC-144) were transplanted into pots filled with a mixture of soil and substrate and subjected to ten doses of glyphosate: 0, 11.25, 22.5, 45, 90, 180, 360, 720, 1440, and 2880 g acid equivalent (ae) ha-1. Evaluations were performed using the morphological, physiological, and biochemical variables. Data analysis for the confirmation of hormesis occurred with the application of mathematical models. The hormetic effect of glyphosate on coffee plant morphology was determined by the variables plant height, number of leaves, leaf area, and leaf, stem, and total dry mass. Doses from 14.5 to 30 g ae ha-1 caused the highest stimulation. In the physiological analyses, the highest stimulation was observed upon CO2 assimilation, transpiration, stomatal conductance, carboxylation efficiency, intrinsic water use efficiency, electron transport rate, and photochemical efficiency of photosystem II at doses ranging from 4.4 to 55 g ae ha-1. The biochemical analyses revealed significant increases in the concentrations of quinic acid, salicylic acid, caffeic acid, and coumaric acid, with maximum stimulation at doses between 3 and 140 g ae ha-1. Thus, the application of low doses of glyphosate has positive effects on the morphology, physiology, and biochemistry of coffee plants.

An in-silico approach to unravel the structure of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS): a critical enzyme for sennoside biosynthesis in Cassia angustifolia Vahl.

The laxative properties of senna are attributed to the presence of sennosides produced in the plant. The low production level of sennosides in the plant is an important impediment to their growing demand and utilization. Understanding biosynthetic pathways helps to engineer them in terms of enhanced production. The biosynthetic pathways of sennoside production in plants are not completely known yet. However, attempts to get information on genes and proteins engaged in it have been made which decode involvement of various pathways including shikimate pathway. 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) is a key enzyme involved in sennosides production through the shikimate pathway. Unfortunately, there is no information available on proteomic characterization of DAHPS enzyme of senna (caDAHPS) resulting in lack of knowledge about its role. We for the first time characterized DAHPS enzyme of senna using in-silico analysis. To the best of our knowledge this is the first attempt to identify the coding sequence of caDAHPS by cloning and sequencing. We found Gln179, Arg175, Glu462, Glu302, Lys357 and His420 amino acids in the active site of caDAHPS through molecular docking. followed by molecular dynamic simulation. The amino acid residues, Lys182, Cys136, His460, Leu304, Gly333, Glu334, Pro183, Asp492 and Arg433 at the surface interact with PEP by van der Waals bonds imparting stability to the enzyme-substrate complex. Docking results were further validated by molecular dynamics. The presented in-silico analysis of caDAHPS will generate opportunities to engineer the sennoside biosynthesis in plants.Communicated by Ramaswamy H. Sarma.

Identification of key drivers of the microbial shikimic acid pathway during different materials composting.

Metabolites of shikimic acid (SA) pathway can be used as humic substance (HS) precursors. Due to the complexity of SA anabolism, there were few studies on SA pathway during composting. The aim of this study was to identify the key drivers of SA pathway during different materials composting. During composting, the SA, protocatechuic acid (PA) and gallic acid (GA) decreased by 57.09%, 72.27% and 54.04% on average, respectively. The structural equation model showed that SA had key driving factors (organic matter and pH) during lawn waste composting. In addition, the complexity of material structure was the main factor affecting PA driving factors. The factors and degree of influence on GA varied with different materials. Accordingly, this study provided theoretical support for the improvement of SA metabolic intensity by single material and mixed material composting, and further provided a new direction for future HS research.

Evaluating differences in humic substances formation based on the shikimic acid pathway during different materials composting.

This study aimed to evaluate differences in humic substance (HS) formation based on the shikimic acid pathway (SAP) during five different materials composting. The results showed that compared with other three materials, gallic acid, protocatechuic acid and shikimic acid of the SAP products in lawn waste (LW) and garden waste (GW) compost decreased significantly. Furthermore, as important indicators for evaluating humification, humic acid and degree of polymerization increased by 39.4%, 79.5% and 21.8%, 87.9% in LW and GW, respectively. Correlation analysis showed that SAP products were strongly correlated with HS fractions in LW and GW. Meanwhile, network analysis indicated that more core bacteria associated with both SAP products and HS were identified in LW and GW. Finally, the structural equation model proved that SAP had more significant contribution to humification improvement in LW and GW. These findings provided theoretical foundation and feasible actions to improve compost quality by the SAP.

How glyphosate affects plant disease development: it is more than enhanced susceptibility.

Glyphosate has been shown to affect the development of plant disease in several ways. Plants utilize phenolic and other shikimic acid pathway-derived compounds as part of their defense against pathogens, and glyphosate inhibits the biosynthesis of these compounds via its mode of action. Several studies have shown a correlation between enhanced disease and suppression of phenolic compound production after glyphosate. Glyphosate-resistant crop plants have also been studied for changes in resistance as a result of carrying the glyphosate resistance trait. The evidence indicates that neither the resistance trait nor application of glyphosate to glyphosate-resistant plants increases susceptibility to disease. The only exceptions to this are cases where glyphosate has been shown to reduce rust diseases on glyphosate-resistant crops, supporting a fungicidal role for this chemical. Finally, glyphosate treatment of weeds or volunteer crops can cause a temporary increase in soil-borne pathogens that may result in disease development if crops are planted too soon after glyphosate application. © 2017 Society of Chemical Industry.

Combined effect of water loss and wounding stress on gene activation of metabolic pathways associated with phenolic biosynthesis in carrot.

The application of postharvest abiotic stresses is an effective strategy to activate the primary and secondary metabolism of plants inducing the accumulation of antioxidant phenolic compounds. In the present study, the effect of water stress applied alone and in combination with wounding stress on the activation of primary (shikimic acid) and secondary (phenylpropanoid) metabolic pathways related with the accumulation of phenolic compound in plants was evaluated. Carrot (Daucus carota) was used as model system for this study, and the effect of abiotic stresses was evaluated at the gene expression level and on the accumulation of metabolites. As control of the study, whole carrots were stored under the same conditions. Results demonstrated that water stress activated the primary and secondary metabolism of carrots, favoring the lignification process. Likewise, wounding stress induced higher activation of the primary and secondary metabolism of carrots as compared to water stress alone, leading to higher accumulation of shikimic acid, phenolic compounds, and lignin. Additional water stress applied on wounded carrots exerted a synergistic effect on the wound-response at the gene expression level. For instance, when wounded carrots were treated with water stress, the tissue showed 20- and 14-fold increases in the relative expression of 3-deoxy-D-arabino-heptulosanate synthase and phenylalanine ammonia-lyase genes, respectively. However, since lignification was increased, lower accumulation of phenolic compounds was detected. Indicatively, at 48 h of storage, wounded carrots treated with water stress showed ~31% lower levels of phenolic compounds and ~23% higher lignin content as compared with wounded controls. In the present study, it was demonstrated that water stress is one of the pivotal mechanism of the wound-response in carrot. Results allowed the elucidation of strategies to induce the accumulation of specific primary or secondary metabolites when plants are treated with water stress alone or when additional water stress is applied on wounded tissue. If the accumulation of a specific primary or secondary metabolite were desirable, it would be recommended to apply both stresses to accelerate their biosynthesis. However, strategies such as the use of enzymatic inhibitors to block the carbon flux and enhance the accumulation of specific compounds should be designed.