Enhancement of 5-aminolevulinic acid production by metabolic engineering of the glycine biosynthesis pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Objective

To construct a strain of Corynebacterium glutamicum capable of efficiently producing 5-aminolevulinic acid (5-ALA) via the C4 pathway by modification of serine and glycine pathway using glucose as sole carbon source.

Results

The recombinant C. glutamicum strain AP2 harboring a codon-optimized hemA gene from Rhodobacter sphaeroides was used as host strain for 5-ALA production. A plasmid harboring the serine operon, which contained serB, serC and the site-specific mutant serA ^Δ197 , was constructed and introduced into C. glutamicumAP2, leading to an increase of 70% in 5-ALA production. Further overexpression of the glyA gene increased production of 5-ALA by 150% over the control. 5-ALA production was thus significantly enhanced by engineering the glycine biosynthetic pathway. C.glutamicum AG3 produced 3.4 ± 0.2 g 5-ALA/l in shake-flask cultures in CGIIIM medium with the addition of 7.5 g glycine/l.

Conclusion

This is the first report of remodeling the serine and glycine biosynthetic pathway to improve the production of 5-ALA in C. glutamicum.

     

Development of leaffolder resistant transgenic rice expressing <Emphasis Type="Italic">cry2AX1</Emphasis> gene driven by green tissue-specific <Emphasis Type="Italic">rbcS</Emphasis> promoter

The insecticidal cry genes of Bacillus thuringiensis (Bt) have been successfully used for development of insect resistant transgenic rice plants. In this study, a novel cry2AX1 gene consisting a sequence of cry2Aa and cry2Ac gene driven by rice rbcS promoter was introduced into a rice cultivar, ASD16. Among 27 putative rice transformants, 20 plants were found to be positive for cry2AX1 gene. The expression of Cry2AX1 protein in transgenic rice plants ranged from 5.95 to 122.40 ng/g of fresh leaf tissue. Stable integration of the transgene was confirmed in putative transformants of rice by Southern blot hybridization analysis. Insect bioassay on T₀ transgenic rice plants against rice leaffolder (Cnaphalocrosis medinalis) recorded larval mortality up to 83.33 %. Stable inheritance and expression of cry2AX1 gene in T₁ progenies was demonstrated using Southern and ELISA. The detached leaf bit bioassay with selected T₁ plants showed 83.33–90.00 % mortality against C. medinalis. The whole plant bioassay for T₁ plants with rice leaffolder showed significant level of resistance even at a lower level of Cry2AX1 expression varying from 131 to 158 ng/g fresh leaf tissue during tillering stage.     

<Superscript>15</Superscript>N photo-CIDNP MAS NMR analysis of reaction centers of <Emphasis Type="Italic">Chloracidobacterium thermophilum</Emphasis>

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by ¹⁵N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical ¹⁵N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 8¹-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from <Emphasis Type="Italic">Citreicella</Emphasis> sp. SE45

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS–polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including l-alanine amide and l-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K _m and V _max values for d- and l-ACL were 5.3 and 2.17 mM, and 769 and 558 μmol/min.mg protein, respectively. Moreover, the turn over number (K _cat) and catalytic efficiency (K _cat/K _m ) of purified ACL racemase from Citreicella sp. SE45 using l-ACL as a substrate were 465 S^?1 and 214 S^?1mM^?1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.     

Enhanced <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> immune responses caused by heterologous plant genes from <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Background

Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F₁ hybrid was proposed.

Results

By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F₁ hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance.

Conclusions

Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F₁ hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.

     

A new source of resistance to 2-furaldehyde from <Emphasis Type="Italic">Scheffersomyces</Emphasis> (<Emphasis Type="Italic">Pichia</Emphasis>) <Emphasis Type="Italic">stipitis</Emphasis> for sustainable lignocellulose-to-biofuel conversion

Aldehyde inhibitory compounds derived from lignocellulosic biomass pretreatment have been identified as a major class of toxic chemicals that interfere with microbial growth and subsequent fermentation for advanced biofuel production. Development of robust next-generation biocatalyst is a key for a low-cost biofuel production industry. Scheffersomyces (Pichia) stipitis is a naturally occurring C-5 sugar utilization yeast; however, little is known about the genetic background underlying its potential tolerance to biomass conversion inhibitors. We investigated and identified five uncharacterized putative aryl-alcohol dehydrogenase genes (SsAADs) from this yeast as a new source of resistance against biomass fermentation inhibitor 2-furaldehyde (furfural) by gene expression, gene cloning, and direct enzyme assay analysis using partially purified proteins. All five proteins from S. stipitis showed furfural reduction using cofactor NADH. An optimum active temperature was observed at 40 °C for SsAad1p; 30 °C for SsAad3p, SsAad4p, and SsAad5p; and 20 °C for SsAad2p. SsAad2p, SsAad3p, and SsAad4p showed tolerance to a wide range of pH from 4.5 to 8, but SsAad1p and SsAad5p were sensitive to pH changes beyond 7. Genes SsAAD2, SsAAD3, and SsAAD4 displayed significantly enhanced higher levels of expression in response to the challenge of furfural. Their encoding proteins also showed higher levels of specific activity toward furfural and were suggested as core functional enzymes contributing aldehyde resistance in S. stipitis.     

<Emphasis Type="Italic">Pm62</Emphasis>, an adult-plant powdery mildew resistance gene introgressed from <Emphasis Type="Italic">Dasypyrum villosum</Emphasis> chromosome arm 2VL into wheat

Key message

Pm62, a novel adult-plant resistance (APR) gene against powdery mildew, was transferred from D. villosum into common wheat in the form of Robertsonian translocation T2BS.2VL#5.

Abstract

Powdery mildew, which is caused by the fungus Blumeria graminis f. sp. tritici, is a major disease of wheat resulting in substantial yield and quality losses in many wheat production regions of the world. Introgression of resistance from wild species into common wheat has application for controlling this disease. A Triticum durum-Dasypyrum villosum chromosome 2V#5 disomic addition line, N59B-1 (2n?=?30), improved resistance to powdery mildew at the adult-plant stage, which was attributable to chromosome 2V#5. To transfer this resistance into bread wheat, a total of 298 BC₁F₁ plants derived from the crossing between N59B-1 and Chinese Spring were screened by combined genomic in situ hybridization and fluorescent in situ hybridization, 2V-specific marker analysis, and reaction to powdery mildew to confirm that a dominant adult-plant resistance gene, designated as Pm62, was located on chromosome 2VL#5. Subsequently, the 2VL#5 (2D) disomic substitution line (NAU1825) and the homozygous T2BS.2VL#5 Robertsonian translocation line (NAU1823), with normal plant vigor and full fertility, were identified by molecular and cytogenetic analyses of the BC₁F₂ generation. The effects of the T2BS.2VL#5 recombinant chromosome on agronomic traits were also evaluated in the F₂ segregation population. The results suggest that the translocated chromosome may have no distinct effect on plant height, 1000-kernel weight or flowering period, but a slight effect on spike length and seeds per spike. The translocation line NAU1823 has being utilized as a novel germplasm in breeding for powdery mildew resistance, and the effects of the T2BS.2VL#5 recombinant chromosome on yield-related and flour quality characters will be further assessed.

     

Another look at the interaction between mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> and flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>

Yeast flavocytochrome b ₂ tranfers reducing equivalents from lactate to oxygen via cytochrome c and cytochrome c oxidase. The enzyme catalytic cycle includes FMN reduction by lactate and reoxidation by intramolecular electron transfer to heme b ₂. Each subunit of the soluble tetrameric enzyme consists of an N terminal b ₅-like heme-binding domain and a C terminal flavodehydrogenase. In the crystal structure, FMN and heme are face to face, and appear to be in a suitable orientation and at a suitable distance for exchanging electrons. But in one subunit out of two, the heme domain is disordered and invisible. This raises a central question: is this mobility required for interaction with the physiological acceptor cytochrome c, which only receives electrons from the heme and not from the FMN? The present review summarizes the results of the variety of methods used over the years that shed light on the interactions between the flavin and heme domains and between the enzyme and cytochrome c. The conclusion is that one should consider the interaction between the flavin and heme domains as a transient one, and that the cytochrome c and the flavin domain docking areas on the heme b ₂ domain must overlap at least in part. The heme domain mobility is an essential component of the flavocytochrome b ₂ functioning. In this respect, the enzyme bears similarity to a variety of redox enzyme systems, in particular those in which a cytochrome b ₅-like domain is fused to proteins carrying other redox functions.     

Changes in photosynthesis of alpine plant <Emphasis Type="Italic">Saussurea superba</Emphasis> during leaf expansion

The native alpine plant Saussurea superba is widely distributed in Qinghai–Tibetan Plateau regions. The leaves of S. superba grow in whorled rosettes, and are horizontally oriented to maximize sunlight exposure. Experiments were conducted in an alpine Kobresia humilis meadow near Haibei Alpine Meadow Ecosystem Research Station (37°29′–37°45′N, 101°12′–101°33′E; alt. 3200 m). Leaf growth, photosynthetic pigments and chlorophyll fluorescence parameters were measured in expanding leaves of S. superba. The results indicate that leaf area increased progressively from inner younger leaves to outside fully expanded ones, and then slightly decreased in nearly senescent leaves, due to early unfavorable environmental conditions, deviating from the ordinary growth pattern. The specific leaf area decreased before leaves were fully expanded, and the leaf thickness was largest in mature leaves. There were no significant changes in the content of chlorophylls (Chl) and carotenoids (Car), but the ratios of Chl a/b and Car/Chl declined after full expansion of the leaves. The variation of Chl a/b coincided well with changes in photochemical quenching (q _P) and the fraction of open PSII reaction centers (q _L). The maximum quantum efficiency of PSII photochemistry after 5 min dark relaxation (F _(v)/F _(m)) continuously increased from younger leaves to fully mature leaves, suggesting that mature leaves could recover more quickly from photoinhibition than younger leaves. The light-harvesting capacity was relatively steady during leaf expansion, as indicated by the maximum quantum efficiency of open PSII centers (\(F_{\text{v}}^{{\prime }}\)/\(F_{\text{m}}^{{\prime }}\)). UV-absorbing compounds could effectively screen harmful solar radiation, and are a main protection way on the photosynthetic apparatus. The decline of q _P and q _L during maturation, together with limitation of quantum efficiency of PSII reaction centers (L _(PFD)), shows a decrease of oxidation state of Q_A in PSII reaction centers under natural sunlight. Furthermore, light-induced (Φ _NPQ) and non-light-induced quenching (Φ _NO) were consistent with variation of L _(PFD). It is concluded that the leaves of S. superba could be classified into four functional groups: young, fully expanded, mature, and senescent. Quick recovery from photoinhibition was correlated with protection by screening pigments, and high level of light energy trapping was correlated with preservation of photosynthetic pigments. Increasing of Φ _NPQ and Φ _NO during leaves maturation indicates that both thermal dissipation of excessive excitation energy in safety and potential threat to photosynthetic apparatus were strengthened due to the declination of q _P and q _L, and enhancement of L _(PFD).     

Isolating and confirming the <Emphasis Type="Italic">MuDR</Emphasis>-inserted flanking sequences of maize

MuDR exhibits the highest transposition activity and insertional mutagenesis frequency in Mutator (Mu) family. If we isolate the MuDR-insertion-specific flanking sequences (MuDRFs), it will be crucial for using Mu element-mediated mutants. The MuDR-TAIL-PCR system was constructed and optimized using a combination of MuDR-TIR-nested specific primers and 12 arbitrary degenerate (AD) primers, modified reaction system and procedure and mutant DNA templates of 87 genotypes from M₂ or М_2:3 families created by crossing the W22::Mu line (active MuDR donor parent) from the UniformMu population with the Zong31 (Z31) line (recipient parent). Here 129 different MuDRFs were acquired by MuDR-TAIL-PCR, accounting for 86.60% of the total mutant-specific agarose gel bands. In addition, we confirmed the authenticity of the non-redundant flanking sequence amplifications. The amplified non-redundant flanking sequences accounted for 65.12% of the total MuDRFs, and 88.00% of the non-redundant MuDRFs were inserted inside the genes. These results show that the MuDR-TAIL-PCR system that we developed can be used for specifically isolating MuDRFs.     

Molecular mapping of powdery mildew resistance gene <Emphasis Type="Italic">PmSGD</Emphasis> in Chinese wheat landrace Shangeda using RNA-seq with bulk segregant analysis

Wheat powdery mildew, caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is one of the most devastating diseases of wheat in China and causes serious yield losses. Resistance genes are urgently needed by wheat breeding programs to combat this disease. In the present study, genetic analysis of powdery mildew resistance was conducted on segregated F₂ and F_2:3 populations derived from the cross of Shangeda (providing good resistance to powdery mildew) and Chancellor (susceptible to powdery mildew). The results showed that the resistance of Shangeda to E09 was controlled by a single recessive gene, tentatively designated as PmSGD. In addition, RNA sequencing of the parental lines Shangeda and Chancellor and the corresponding bulked pools derived from homozygous resistant or susceptible F_2:3 lines was implemented to identify single-nucleotide polymorphisms (SNPs). The PmSGD gene was estimated to be located in the 240–250-Mb region of chromosome 7B based on the characteristics of putative SNP loci distributed on 21 wheat chromosomes. Among the developed SNP markers, 17 (57%) markers were linked to PmSGD flanked by SNP2-57 and SNP2-46, with genetic distances of 0.4 and 0.8 cM, respectively. The reaction patterns of Shangeda and cultivars (lines) carrying the Pm5e, Pmhym, mlxbd, and PmTm4 genes to 22 Bgt isolates indicated that PmSGD may be allelic or very closely linked to those genes. All of the SNP loci linked to PmSGD were used to test 38 cultivars with known Pm gene(s), and the results suggested that these SNP loci are useful for pyramiding PmSGD by marker-assisted selection.     

Xylem anatomy and calculated hydraulic conductance of four <Emphasis Type="Italic">Nothofagus</Emphasis> species with contrasting distribution in South-Central Chile

Nothofagus obliqua, N. dombeyi, N. alpina and N. antarctica are characteristic tree species of the temperate forests on the western slopes of the Andes with centres of distribution that differ in their temperature and moisture regimes. We tested branch wood from co-occurring specimens of these species for the inherent differences in xylem anatomy and theoretical hydraulic conductance to evaluate their resistance to drought or frost. The hydraulic conductivity of the xylem was calculated using a modified Hagen–Poiseuille equation and related to wood density. Conduit dimensions were used to predict the water potential that would cause 50 % loss of hydraulic conductivity (Ψ ₅₀). Nothofagus alpina, which mainly grows at sites with low frost frequency, exhibited the largest conduits and the highest mean values for conduit area, fraction of conduit area in the cross-section and hydraulic conductivity, but the lowest wood density. Opposite relationships were found in the plastic N. antarctica, whose xylem seems to be least vulnerable to freezing-induced, but also to drought-induced embolism. Calculated Ψ ₅₀ was highest (least negative) in N. alpina, indicating a relatively high susceptibility to cavitation. The xylem of the thermophilic N. obliqua and of N. dombeyi, which mainly occurs under oceanic climate, but can also survive at sporadically dry and warm sites, is not particularly adapted to periods of drought stress. Across all species, wood density was negatively correlated with the calculated hydraulic conductance. The xylem traits of N. alpina might contribute to its relatively high growth rate and facilitate its spread into forest gaps.     

<Emphasis Type="Italic">Viola woosanensis</Emphasis>, a recurrent spontaneous hybrid between <Emphasis Type="Italic">V. ulleungdoensis</Emphasis> and <Emphasis Type="Italic">V. chaerophylloides</Emphasis> (Violaceae) endemic to Ulleung Island,Korea

Ulleung Island is an oceanic volcanic island in Korea, which has never been connected to the adjacent continent. Previous studies highlighted Ulleung Island as an excellent system to study the pattern and process of early stages of flowering plant evolutions on oceanic island. The predominant mode of speciation in flowering plants on Ulleung Island appears to be anagenesis. However, the potentially important role of hybrid speciation among incompletely reproductively isolated lineages cannot be ruled out. Viola woosanensis (Violaceae) is of purportedly hybrid origin between V. ulleungdoensis (i.e., formerly recognized as V. selkirkii in Ulleung Island) and V. chaerophylloides, based on morphology. To examine the origin of V. woosanensis, we sampled a total of 80 accessions, including V. woosanensis and its putative parental species and sequenced nrDNA ITS, and four highly variable chloroplast noncoding regions (trnL-trnF, rpl16 intron, atpF-atpH, and psbA-trnH). Representative species of Viola from Korea were also included in the phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference). Additive polymorphic sites in the nrDNA ITS regions were confirmed by cloning amplicons from representative species. The molecular data strongly supported the hybrid origin of V. woosanensis, and the maternal and paternal parent were determined to be V. ulleungdoensis and V. chaerophylloides, respectively. The presence of two parental ribotypes in V. woosanensis (with the exception in one population) was confirmed by cloning, suggesting V. woosanensis is primarily the F₁ generation. No trace of backcrossing and introgression with its parents was detected due to low fertility of hybrid species. We found a multiple and unidirectional hybrid origin of V. woosanensis. Additional studies are required to determine which factors contribute to asymmetric gene flow of Viola species in Ulleung Island.