Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroA_A.sp. A phylogenetic analysis revealed that AroA_A.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The K_M for PEP and IC₅₀ [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroA_A.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC₅₀ [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroA_A.sp. The specific activity of the wild-type AroA_A.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.     

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i × j) and three-factor (i k × j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1 × i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1–i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1 × i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1 × i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII⁺ recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3–8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants.     

High titer production of tetracenomycins by heterologous expression of the pathway in a Streptomyces cinnamonensis industrial monensin producer strain

Streptomyces cinnamonensis C730.1 and C730.7, are industrially mutagenized strains that produce moderate and high levels of the polyketide polyether antibiotic monensin A, respectively, in an oil-based fermentation medium. The possibility that these strains could be used for high titer production of a heterologous polyketide product was investigated by expression of the entire tetracenomycin (TCM) biosynthetic pathway using an integrative plasmid, pSET154. Expression in C730.1 led to stable production of ~0.44 g/l TCM C (the final biosynthetic product) and ~2.69 g/l TCM A2 (the penultimate biosynthetic product), and resulted in a 40% decrease in monensin production. Expression in the C730.7 led to higher levels of TCMs, ~0.6 g/l TCM C and ~4.35 g/l TCM A2, without any detectable decrease in the higher titer monensin production. Abrogation of monensin production in this strain through deletion of the corresponding biosynthetic genes did not lead to higher levels of TCM products. In the case of the C730.7 host, 85% of the TCM C and virtually all of the TCM A2 were intracellular, suggesting feedback inhibition leads to the accumulation of the final pathway intermediate. These observations contrast those made for the native producer Streptomyces glaucescens where the predominant product is TCM C and TCM titers are significantly lower levels (~0.3 g/l), and demonstrate the potential utility of S. cinnamonensis strains as heterologous hosts for high level expression of a variety of polyketide synthase derived products.     

Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway

Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.4 μg g⁻¹ parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding a costunolide and parthenolide 3β-hydroxylase was identified opening up further options to improve the water solubility of parthenolide and therefore its potential as a drug.     

Does the white clover response to sulphur availability correspond to phenotypic or ontogenetic plasticity?

White clover (Trifolium repens L.) is a key species in grasslands. Its performance in grassland communities is strongly linked to nitrogen (N) availability. A decrease in soil sulphur (S) content has appeared in the last few decades in grasslands in Northern Europe and this could change the behaviour of white clover. S is essential for plants and particularly for legumes through its effect upon nitrogen fixation. The aim of this study was to determine the effects of S deficiency on white clover fitness, analysing its plasticity in a time course of growth.Three concentrations of SO₄^2?, “Low S” (0.009 mM), “Medium S” (0.384 mM) and “High S” (1.509 mM), were used to grow plants in a hydroponic system. S availability modified biomasses significantly only at the end of the experiment (11 weeks). Medium S appeared optimal while Low S induced a lower aboveground dry mass. An appropriate S availability (Medium S) not only increased S content but also increased N content by stimulating N₂ fixation. Plant growth analysis using growth fitted curves and the calculation of RGR revealed that S effects on biomass corresponded to the production of different phenotypes and not to a growth delay. This work shows that the acceleration growth phase (49–56 days) is a key period for the nutritional needs of white clover and should be the best period for a sulphur fertilisation regime that aims to enhance white clover fitness.     

Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae

Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142 g/L with production yield of 0.89 g/g and productivity of 3.55 g L⁻¹ h⁻¹ under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast.     

Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway

Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1 g/L) with a yield of 0.47 mol/mol glucose.     

Ring A-seco limonoids and flavonoids from the Kenyan Vepris uguenensis Engl. and their antioxidant activity

Two A-seco-limonoids, accorded the trivial names, uguenensene and uguenensone and a C-7 prenylated flavonoid, uguenenprenol were isolated from Vepris uguenensis (Rutaceae). In addition, 11 known compounds, niloticin, chisocheton A, kihadalactone A, limonyl acetate, methyl uguenenoate, 7-O-methylaromadenrin, flindersiamine, 8α,11-elemodiol, tricoccin S₁₃ acetate, skimmianine, and lupeol were isolated. The structures of the compounds were elucidated and characterized by spectroscopic analyses (NMR, GC–MS and IR). Antioxidant activity of the isolated compounds showed that uguenenprenol and 7-O-methylaromadenrin are good antioxidant agents. Significantly high antioxidant activity was also exhibited by 8α,11-elemodiol, which was 72% at 250 μg mL⁻¹ and 57% at 15.62 μg mL⁻¹ when tested with the deoxyribose method. The two liminoids fit nicely into the biosynthetic pathway from niloticin to methyl uguenenoate.     

Cloning of Sal1, a scytalone dehydratase gene involved in melanin biosynthesis in Cochliobolus heterostrophus

A melanin biosynthetic gene, Sal1, in the southern corn leaf blight fungus Cochliobolus heterostrophus, was identified by degenerate and inverse PCR. Structural analysis revealed that the open reading frame is 555 bp in length, interrupted by one intron. Melanin deficiency of the sal1 mutant strain, which accumulates the intermediate scytalone, was successfully complemented by introducing the gene fragment. These results suggest that Sal1 is a scytalone dehydratase gene of C. heterostrophus.     

Metabolic engineering of Corynebacterium glutamicum for the de novo production of ethylene glycol from glucose

Development of sustainable biological process for the production of bulk chemicals from renewable feedstock is an important goal of white biotechnology. Ethylene glycol (EG) is a large-volume commodity chemical with an annual production of over 20 million tons, and it is currently produced exclusively by petrochemical route. Herein, we report a novel biosynthetic route to produce EG from glucose by the extension of serine synthesis pathway of Corynebacterium glutamicum. The EG synthesis is achieved by the reduction of glycoaldehyde derived from serine. The transformation of serine to glycoaldehyde is catalyzed either by the sequential enzymatic deamination and decarboxylation or by the enzymatic decarboxylation and oxidation. We screened the corresponding enzymes and optimized the production strain by combinatorial optimization and metabolic engineering. The best engineered C. glutamicum strain is able to accumulate 3.5 g/L of EG with the yield of 0.25 mol/mol glucose in batch cultivation. This study lays the basis for developing an efficient biological process for EG production.     

Multilocus phylogeography of the Wedge-billed Woodcreeper Glyphorynchus spirurus (Aves,Furnariidae) in lowland Amazonia: Widespread cryptic diversity and paraphyly reveal a complex diversification pattern

Amazonian rivers function as important barriers to dispersal of Amazonian birds. Studying population genetics of lineages separated by rivers may help us to uncover the dynamics of biological diversification in the Amazon. We reconstructed the phylogeography of the Wedge-billed Woodcreeper, Glyphorynchus spirurus (Furnariidae) in the Amazon basin. Sampling included 134 individuals from 63 sites distributed in eight Amazonian areas of endemism separated by major Amazonian rivers. Nucleotide sequences were generated for five genes: two mtDNA genes (1047 bp for cyt b and 1002 bp for ND2) and three nuclear genes (647 bp from the sex-linked gene ACO, 319 bp from the intron of G3PDH, and 619 bp from intron 2 of MYO). In addition, 37 individuals were randomly selected from the Rondônia and Inambari areas of endemism for genomic fingerprinting, using five ISSR primers. Our results reveal allopatric and well-supported lineages within G. spirurus with high levels of genetic differentiation (p-distances 0.9–6.3%) across opposite banks of major Amazonian rivers. The multilocus phylogenetic reconstructions obtained reveal several incongruences with current subspecies taxonomy. Within currently recognized subspecies, we found high levels of both paraphyly and genetic differentiation, indicating deep divergences and strong isolation consistent with species-level differences. ISSR fingerprinting supports the existence of genetically differentiated populations on opposite sides of the Madeira River. Molecular dating suggests an initial vicariation event isolating populations from the Guiana center of endemism during the Late Miocene/Early Pliocene, while more recent events subdivided Brazilian Shield populations during the Lower Pleistocene.     

Ethylene- and dark-induced flower abscission in potted Plectranthus: Sensitivity,prevention by 1-MCP,and expression of ethylene biosynthetic genes

Prevention of ethylene- and shipping-induced flower abscission is necessary to maintain the quality of both cut flowers and potted plants during handling, transport and retail display. The aims of the present work were to determine the sensitivity of Plectranthus cultivars to applied ethylene, to alleviate ethylene- and shipping-induced flower abscission in intact potted plants using 1-methylcyclopropene (1-MCP), and to investigate the possible causes of dark-induced flower abscission. All cultivars were sensitive to ethylene in a concentration-dependent manner, and complete abscission occurred within 24 h with 1 and 2 μl l^− 1 ethylene. Unopened buds were more sensitive to applied ethylene, and exhibited greater abscission than open flowers. Ethylene synthesis remained below detection limits at all time points under control and continuous dark conditions. Dark treatment significantly increased flower abscission in Plectranthus cultivars, and like ethylene-induced flower abscission, this could be prevented by continuous 1-MCP treatment. Gene expression of ethylene biosynthetic enzymes ACS and ACO was examined as possible causes for the accelerated flower abscission observed in plants kept in continuous darkness. Expression patterns of ACS and ACO varied between different cultivars of Plectranthus. In some cases, increased expression of ACS and ACO led to increased flower abscission. Gene expression was higher in open flowers when compared to unopened flowers suggesting a cause for the observed preferential shedding of open flowers in some cultivars. Although the cause of dark-induced abscission in Plectranthus remains elusive, it can be effectively controlled by treatment with 1-MCP.     

Triterpenoid biosynthesis and the transcriptional response elicited by nitric oxide in submerged fermenting Ganoderma lucidum

Ganoderic triterpenoid (GT) is a promising anti-tumour constituent in Ganoderma lucidum. The aim of this study was to investigate induction by and a possible signalling mechanism of nitric oxide (NO) for GT synthesis. Compared to the control, the biomass decreased by 43.5% at 120 h and the GT yield increased by 40.94% at 72 h in the presence of a 5 mM NO donor sodium nitroprusside supplement. The gene expression profiles of G. lucidum in response to NO were investigated by RNA-sequencing. Functional annotation and an enrichment analysis of the differentially expressed genes indicated that NO inhibited mycelial growth probably via the suppression of the glycolysis genes involved in carbohydrate metabolism. NO may function directly as a regulator of gene expression in the mevalonate pathway to induce GT biosynthesis, and the hyper-production of GT in response to NO could also be accomplished by a signalling function involving Ca²⁺ and a reactive oxygen species (ROS) pathway. The results of this study are useful for large-scale GT production and can facilitate further studies on the endogenous signalling pathways involved in the GT biosynthetic pathway.