Bioprocess engineering to produce 9-(nonanoyloxy) nonanoic acid by a recombinant <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>-based biocatalyst

Here, Corynebacterium glutamicum ATCC13032 expressing Baeyer–Villiger monooxygenase from Pseudomonas putida KT2440 was designed to produce 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid. Diverse parameters including cultivation and reaction temperatures, type of detergent, and pH were found to improve biotransformation efficiency. The optimal temperature of cultivation for the production of 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid using whole cells of recombinant C. glutamicum was 15 °C, but the reaction temperature was optimal at 30 °C. Enhanced conversion efficiency was obtained by supplying 0.05 g/L of Tween 80 at pH 7.5. Under these optimal conditions, recombinant C. glutamicum produced 0.28 mM of 9-(nonanoyloxy) nonanoic acid with a 75.6% (mol/mol) conversion yield in 2 h. This is the first report on the biotransformation of 10-ketostearic acid to 9-(nonanoyloxy) nonanoic acid with a recombinant whole-cell C. glutamicum-based biocatalyst and the results demonstrate the feasibility of using C. glutamicum as a whole-cell biocatalyst.     

<Emphasis Type="Italic">Synechocystis</Emphasis> PCC 6803 cells heterologously expressing bacterial tyrosine ammonia lyase can use exogenous tyrosine for <Emphasis Type="Italic">p</Emphasis>-coumaric acid production

Phototrophic cyanobacteria may be considered as an alternative host for producing numerous bioactive compounds. We demonstrate that the Synechocystis PCC 6803 expressing tyrosine ammonia-lyase from Rhodobacter sphaeroides under Ptrc1O promoter produce p-coumaric acid at a rate three times higher than that under Ptrc1Ocore promoter, accounting for 18.4 ± 1.5 μg of p-coumaric acid per 10⁸ cells (0.36 mg L^?1). Additionally, our study is the first report to show the biotransformation of tyrosine to p-coumaric acid reaching a maximum 2.4-fold increase when 0.5 mM tyrosine was supplemented to the growth medium. Liquid chromatography-mass spectrometry analysis revealed the occurrence of diverse patterns of metabolites under different concentrations of supplemented tyrosine, suggesting that it is used in additional metabolic pathways.     

Construction of a genome-anchored,high-density genetic map for melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) and identification of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis> race 1 resistance QTL

Key message

Four QTLs and an epistatic interaction were associated with disease severity in response to inoculation with Fusarium oxysporum f. sp. melonis race 1 in a recombinant inbred line population of melon.

Abstract

The USDA Cucumis melo inbred line, MR-1, harbors a wealth of alleles associated with resistance to several major diseases of melon, including powdery mildew, downy mildew, Alternaria leaf blight, and Fusarium wilt. MR-1 was crossed to an Israeli cultivar, Ananas Yok’neam, which is susceptible to all of these diseases, to generate a recombinant inbred line (RIL) population of 172 lines. In this study, the RIL population was genotyped to construct an ultra-dense genetic linkage map with 5663 binned SNPs anchored to the C. melo genome and exhibits the overall high quality of the assembly. The utility of the densely genotyped population was demonstrated through QTL mapping of a well-studied trait, resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (Fom) race 1. A major QTL co-located with the previously validated resistance gene Fom-2. In addition, three minor QTLs and an epistatic interaction contributing to Fom race 1 resistance were identified. The MR-1 × AY RIL population provides a valuable resource for future QTL mapping studies and marker-assisted selection of disease resistance in melon.

     

A saponin isolated from <Emphasis Type="Italic">Agapanthus africanus</Emphasis> differentially induces apoplastic peroxidase activity in wheat and displays fungicidal properties

A spirostane with an attached trisaccharide, (25R)-5α-spirostane-2α,3β,5α-triol 3-O-(O-α-l-rhamnopyranosyl-(1 → 2)-O-(β-d-galactopyranosyl-(1 → 3))-β-d-glucopyranoside), was isolated and identified from the aerial parts of Agapanthus africanus by activity-guided fractionation. Fungicidal properties of the crude extract, semi-purified fractions as well as the purified active saponin from A. africanus were screened in vitro against Fusarium oxysporum. At a concentration of 1 mg mL^?1, the crude extract and semi-purified ethyl acetate and dichloromethane fractions showed significant antifungal activity. The purified saponin inhibited the in vitro mycelial growth of F. oxysporum completely (100 %) at a concentration of 125 µg mL^?1. Furthermore, to verify previously observed induced resistance by crude extracts of A. africanus towards leaf rust, intercellular PR-protein activity was determined in wheat seedlings following foliar application of the purified saponin at 100 µg mL^?1. In vitro peroxidase enzyme activity increased significantly (60 %) in wheat seedlings 48 h after treatment with the purified saponin, demonstrating its role as an elicitor to activate a defence reaction in wheat.     

Monoterpene biotransformation by <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Objective

To investigate the biocatalytic potential of Colletotrichum acutatum and Colletotrichum nymphaeae for monoterpene biotransformation.

Results

C. acutatum and C. nymphaeae used limonene, α-pinene, β-pinene, farnesene, citronellol, linalool, geraniol, perillyl alcohol, and carveol as sole carbon and energy sources. Both species biotransformed limonene and linalool, accumulating limonene-1,2-diol and linalool oxides, respectively. α-Pinene was only biotransformed by C. nymphaeae producing campholenic aldehyde, pinanone and verbenone. The biotransformation of limonene by C. nymphaeae yielded 3.34–4.01 g limonene-1,2-diol l^?1, depending on the substrate (R-(+)-limonene, S-(?)-limonene or citrus terpene (an agro-industrial by-product). This is among the highest concentrations already reported for this product.

Conclusions

This is the first report on the biotransformation of these terpenes by Colletotrichum spp. and the biotransformation of limonene to limonene-1,2-diol possibly involves enzymes similar to those found in Grosmannia clavigera.

     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.     

Proteinase inhibitors from <Emphasis Type="Italic">Cajanus platycarpus</Emphasis> accessions active against pod borer <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Cajanus platycarpus, a wild relative of Cajanus cajan, is an important source for various agronomically desirable traits, including resistance towards pod borer, Helicoverpa armigera. In the present study, the inhibitory activity of proteinase inhibitors (PIs) present in crude protein extracted from different accessions of C. platycarpus and cultivars of C. cajan was evaluated against H. armigera under in vitro and in vivo conditions. The PIs active against H. armigera gut trypsin-like proteinases (HGPs), referred to as ‘HGPIs’, were more pronounced in mature dry seeds of C. platycarpus accessions when compared with cultivars, which is also evident through gelatin activity staining studies. Therefore, the inhibitory activity of HGPIs was further evaluated in various plant organs of C. platycarpus accessions, such as leaves, flowers, pods, developing seeds at 8–10 days (DAP-I), 18–20 days (DAP-II), and 28–32 days after pollination (DAP-III). However, the HGPI activity was more pronounced in mature dry seeds > DAP-III > DAP-II > DAP-I > flowers > pods > leaves. The observed quantitative allocation of HGPIs closely resembled “Optimal Defense Theory”. Further, bioassays demonstrated that there was a significant reduction in the body weight of the larvae fed upon crude PI extracts of C. platycarpus accessions with concomitant increase in mortality rate and the formation of larval–pupal intermediates. Nevertheless, such changes were not observed when the larvae were fed on crude PI extracts of C. cajan cultivars. These results suggest that the PI gene(s) from C. platycarpus accessions could be exploited in the management of H. armigera by introgression into C. cajan cultivars.     

Chemical defences of native European coccinellid eggs against intraguild predation by the invasive Asian coccinellid, <Emphasis Type="Italic">Harmonia axyridis</Emphasis> (Pallas)

Harmonia axyridis (Pallas) is a coccinellid of Asian origin that has recently invaded substantial parts of Europe and is suspected to affect native coccinellid populations through intraguild predation and competition for food. Previous work has shown that two species from the Calvia genus appeared to be well protected against H. axyridis predation. To deepen our understanding on chemical protection of Calvia spp. and the predation risk by H. axyridis, we tested for susceptibility and palatability of Calvia spp. and H. axyridis eggs against predation by H. axyridis neonate larvae. Results show that eggs of C. quatuordecimguttata were mostly not eaten by H. axyridis, while eggs of the congeneric C. decemguttata were found to be largely unprotected against predation by the invasive coccinellid. We also observed that H. axyridis first instars successfully cannibalized on conspecific eggs. Removing the surface chemicals from C. quatuordecimguttata eggs resulted in significantly reduced protection from being preyed upon by H. axyridis, while applying these extracts onto C. decemguttata and H. axyridis eggs resulted in increased protection against H. axyridis larvae. The importance of surface chemicals in the interactions between H. axyridis and native coccinellids was confirmed by GC–MS analysis, showing a high diversity of hydrocarbons located on the surface of C. quatuordecimguttata eggs, i.e. more than twice as many when compared to C. decemguttata. Survival of H. axyridis larvae feeding on eggs of C. quatuordecimguttata, C. decemguttata or conspecific eggs, from which surface chemicals were removed by washing them with hexane, was not different from survival on unwashed eggs.     

Cloning and characterization of <Emphasis Type="Italic">Tabas1</Emphasis>-<Emphasis Type="Italic">B1</Emphasis> gene associated with flag leaf chlorophyll content and thousand-grain weight and development of a gene-specific marker in wheat

The chloroplast photosystem of flag leaves contributes the largest proportion of photosynthates to grain in crops and consequently affects grain weight. The plant 2-Cys peroxiredoxin BAS1 is involved in chlorophyll protection against chloroplast damage. In the present study, we cloned a Tabas1 gene in common wheat (Triticum aestivum L.), comprising seven exons and six introns with a complete sequence of 2847 bp and an open reading frame of 789 bp. The gene was located on chromosome 2B, and designated Tabas1-B1. A codominant gene-specific marker TaS1 was developed based on a 1-bp InDel (-/A) in the second intron of Tabas1-B1. Two alleles, Tabas1-B1a and Tabas1-B1b, at the Tabas1-B1 locus were identified by TaS1. Linkage and quantitative trait locus (QTL) mapping indicated that Tabas1-B1 was linked to Xcfa2278 (5.23 cM) and Xbarc167 (10.38 cM) on chromosome 2BL. A stable QTL co-segregating with Tabas1-B1 explained 9.0–19.2 % of phenotypic variations for chlorophyll content (ChlC) and 9.5–15.5 % for thousand-grain weight (TGW), respectively, across three environments. Association analysis further indicated a significant and positive effect of Tabas1-B1a on the ChlC of flag leaf post-anthesis and TGW in two populations across four environments. Geographic distribution analysis suggested a slightly higher frequency of Tabas1-B1a than Tabas1-B1b in the main wheat-growing regions of China. Selection of Tabas1-B1a may increase grain weight in wheat breeding.     

<Emphasis Type="Italic">Lactobacillus futsaii</Emphasis> CS3, a New GABA-Producing Strain Isolated from Thai Fermented Shrimp (<Emphasis Type="Italic">Kung</Emphasis>-<Emphasis Type="Italic">Som</Emphasis>)

Kung-Som is a popular traditional Thai fermented shrimp product. It is rich in glutamic acid, which is the major substrate for the biosynthesis of gamma-aminobutyric acid (GABA) by lactic acid bacteria (LAB). In the present study, LAB from Kung-Som were isolated, screened for GABA formation, and the two isolates that transform glutamic acid most efficiently into GABA were identified. Based on the API-CHL50 fermentation profile and a phylogenetic tree of 16S rDNA sequences, strain CS3 and CS5 were identified as Lactobacillus futsaii, which was for the first time shown to be a promising GABA producer. L. futsaii CS3 was the most efficient microorganism for the conversion of 25 mg/mL monosodium glutamate (MSG) to GABA, with a maximum yield of more than 99% conversion rate within 72 h. The open reading frame (ORF) of the glutamate decarboxylase (gad) gene was identified by PCR. It consists of 1410 bp encoding a polypeptide of 469 amino acids with a predicted molecular weight of 53.64 kDa and an isoelectric point (pI) of 5.56. Moreover, a good quality of the constructed model of L. futsaii CS3 was also estimated. Our results indicate that L. futsaii CS3 could be of interest for the production of GABA-enriched foods by fermentation and for other value-added products.     

The fitness of the cotton mealybug <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis> (Tinsley) is reduced after feeding on virus-infected <Emphasis Type="Italic">Hibiscus rosa</Emphasis>-<Emphasis Type="Italic">sinensis</Emphasis>

The cotton mealybug Phenacoccus solenopsis (Tinsley) and Cotton leaf curl Multan virus (CLCuMV), serious threats to economic crops and garden plants, have invaded southern China and widely infected Hibiscus rosa-sinensis. Whether an inter-species connection has facilitated the invasion process is unclear. In this study the interaction between P. solenopsis and H. rosa-sinensis infected with CLCuMV was investigated in the laboratory. We observed that 1st and 2nd instar nymphs of P. solenopsis preferred to feed on healthy H. rosa-sinensis leaves, whereas 3rd instar nymphs and female adults had no preference between healthy and virus-infected H. rosa-sinensis leaves. The developmental time of each P. solenopsis developmental stage increased significantly after feeding on infected H. rosa-sinensis leaves (p < 0.05). In particular, the development time for 2nd instar female and male nymphs and 3rd instar female nymphs increased by approximately twofold. The generation time of female mealybugs increased from 25.84 d on healthy H. rosa-sinensis to 32.12 d when feeding on CLCuMV-infected H. rosa-sinensis, and the survival rate decreased from 71.04 % on healthy H. rosa-sinensis to 5.80 % on infected plants. Nymph survival was most affected by feeding on infected plants. Additionally, the fecundity of female mealybugs feeding on infected H. rosa-sinensis decreased by 47.8 %. Thus, feeding on CLCuMV-infected H. rosa-sinensis significantly decreased the biological fitness and invading and colonizing abilities of P. solenopsis.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

Overexpression of cytochrome p450 125 in <Emphasis Type="Italic">Mycobacterium</Emphasis>: a rational strategy in the promotion of phytosterol biotransformation

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD⁺/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD⁺ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD⁺ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD⁺/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.     

Enhancement of NAD(H) pool for formation of oxidized biochemicals in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The NAD⁺/NADH ratio and the total NAD(H) play important roles for whole-cell biochemical redox transformations. After the carbon source is exhausted, the degradation of NAD(H) could contribute to a decline in the rate of a desired conversion. In this study, methods to slow the native rate of NAD(H) degradation were examined using whole-cell Escherichia coli with two model oxidative NAD⁺-dependent biotransformations. A high phosphate concentration (50 mM) was observed to slow NAD(H) degradation. We also constructed E. coli strains with deletions in genes coding several enzymes involved in NAD⁺ degradation. In shake-flask experiments, the total NAD(H) concentration positively correlated with conversion of xylitol to l-xylulose by xylitol 4-dehydrogenase, and the greatest conversion (80%) was observed using MG1655 nadR nudC mazG/pZE12-xdh/pCS27-nox. Controlled 1-L batch processes comparing E. coli nadR nudC mazG with a wild-type background strain demonstrated a 30% increase in final l-xylulose concentration (5.6 vs. 7.9 g/L) and a 25% increase in conversion (0.53 vs. 0.66 g/g). MG1655 nadR nudC mazG was also examined for the conversion of galactitol to l-tagatose by galactitol 2-dehydrogenase. A batch process using 15 g/L glycerol and 10 g/L galactitol generated over 9.4 g/L l-tagatose, corresponding to 90% conversion and a yield of 0.95 g l-tagatose/g galactitol consumed. The results demonstrate the value of minimizing NAD(H) degradation as a means to improve NAD⁺-dependent biotransformations.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

Associations of MMP-2 −1306 C/T and MMP-9 −1562 C/T polymorphisms with breast cancer risk among different populations: a meta-analysis

The meta-analysis aims to investigate association between two matrix metalloproteinases (MMPs) polymorphisms (MMP-2 ?1306 C/T and MMP-9 ?1562 C/T) and breast cancer risk. Eligible studies were retrieved from relevant databases, based on predefined criteria. Quality assessment was evaluated by Newcastle–Ottawa Scale. Odds ratio (OR) with its 95% confidence interval (CI) was selected as the effect size for the meta-analysis. As a result, 13 studies were included. MMP-2 ?1306 C/T polymorphism was not significantly associated with breast cancer risk under all genetic models (P > 0.05). However, subgroup analysis stratified by ethnicity showed a significant association between MMP-2 ?1306 C/T polymorphism and reduced breast cancer risk in Asian populations under allelic model (OR 0.60, 95% CI 0.39–0.90, P = 0.02) and dominant model (OR 0.55, 95% CI 0.34–0.89, P = 0.02). MMP-9 ?1562 C/T polymorphism was significantly related to increased breast cancer risk under allelic model (OR 1.50, 95% CI 1.06–2.12, P = 0.02), additive model (OR 1.45, 95% CI 1.02–2.05, P = 0.04) and recessive model (OR 1.54, 95% CI 1.13–2.12, OR 0.007). A significant association between MMP-9 ?1562 C/T polymorphism and increased breast cancer risk in Caucasian was detected under most of the genetic models (P < 0.05). MMP-2 ?1306 C/T polymorphism might be significantly associated with reduced breast cancer risk in Asian, while MMP-9 ?1562 C/T might be closely related to increased breast cancer risk, especially in Caucasian.     

Characterization of an IAA-glucose hydrolase gene <Emphasis Type="Italic">TaTGW6</Emphasis> associated with grain weight in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In rice, the TGW6 gene determines grain weight and encodes a protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. Its homolog in wheat, TaTGW6, is considered as a candidate gene related to grain development. To amplify this gene, we designed primers based on a homologous conserved domain of the rice TGW6 gene. Sequence analysis indicated that TaTGW6 comprises only one exon, with 1656 bp in total and an open reading frame of 1035 bp. Three alleles at TaTGW6 locus detected by the primer pair TG23 were designated as TaTGW6-a, TaTGW6-b and TaTGW6-c, respectively. Compared with TaTGW6-a, TaTGW6-b had a 6-bp InDel at the position 170 downstream of initiation codon, and TaTGW6-c was a null mutant. Both TaTGW6-b and TaTGW6-c could significantly increase grain size and weight other than TaTGW6-a; however, the former two alleles showed a low frequency distribution in modern varieties. TaTGW6 was located on chromosome 4AL using a recombinant inbred line population and a set of Chinese Spring nullisomic-tetrasomic lines. It was linked to the SSR locus Xbarc1047 with a genetic distance of 6.62 cM and explained 15.8–21.0 % of phenotypic variation of grain weight in four environments. Association analysis using a natural population and Chinese wheat mini-core collections additionally validated the relationship of TaTGW6 with grain weight; the gene could explain 7.7–12.4 % of phenotypic variation in three environments. Quantitative real-time PCR revealed that TaTGW6-b showed relatively lower expression than TaTGW6-a in immature grain at 20 and 30 days post-anthesis and in mature grain. The low expression of TaTGW6 generally associated with low IAA content, but with high grain weight. The novel functional marker, designated as TG23, can be used for marker-assisted selection to improve grain weight in wheat and also provides insights into the regulatory mechanism underlying grain weight.     

An NADPH-dependent <Emphasis Type="Italic">Lactobacillus composti</Emphasis> short-chain dehydrogenase/reductase: characterization and application to (<Emphasis Type="Italic">R</Emphasis>)-1-phenylethanol synthesis

A new anti-Prelog short-chain dehydrogenase/reductase (SDR) encoding gene lcsdr was cloned from Lactobacillus composti DSM 18527, and heterologously expressed in Escherichia coli. LcSDR is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and has a molecular weight of approximately 30 kDa. The optimal pH and temperature were 6.5 and 30?°C, respectively. The maximal reaction rate V_max was 133.9 U mg^?1; the Michaelis–Menten constant K _m of LcSDR were 0.345 mM for acetophenone (1a), and 0.085 mM for NADPH. Through introducing an EsGDH-catalyzed NADPH regeneration system, a biocatalytic process for (R)-1-phenylethanol ((R)-1b) was developed with outstanding time–space yield. Under the optimized conditions, 50 g l^?1 1a was converted to (R)-1b in 2 h with a yield of 93.8%, enantiomeric excess of product (e.e._p) above 99% and space–time yield of 562.8 g l^?1 d^?1.     

Toxicity and Detoxification Mechanism of Black Pepper and Its Major Constituent in Controlling <Emphasis Type="Italic">Rhynchophorus ferrugineus</Emphasis> Olivier (Curculionidae: Coleoptera)

The survival, feeding response, and detoxification mechanism of Rhynchophorus ferrugineus Olivier, a key pest responsible for destruction of date palm, was examined with different extracts of Piper nigrum and its major constituent (piperine) identified by GC-MS. In the present study, toxicity of different extracts of black pepper was evaluated by incorporating diffferent doses of extracts into the artificial diet of red palm weevil larvae. All extracts showed dose-dependent insecticidal activity to the tested eighth-instar red palm weevil larvae. Among all the extracts, maximum larvicidal activity was exhibited by chloroform (LD₅₀ = 342.62 mg/l), followed by dichloromethane (LD₅₀ = 357.78 mg/l), acetone (LD₅₀ = 372.57 mg/l), and ethanol (LD₅₀ = 408.88 mg/l). However, piperine, a major constituent of all black pepper extracts identified by GC-MS in the present work, was found to be the most potent treatment exhibiting the least LD₅₀ (219.88 mg/l). In addition, nutritional indices evaluated by calculating the efficiency of the conversion of ingested food (ECI) and digested food (ECD) at the same dose (219.88 mg/l) showed that there was maximum reduction in the ECI (49.90%) and ECD (62.21%) index of larvae fed diets incorporated with piperine. Larvae that were fed diets incorporated with different black pepper extracts experienced increases in the expression of detoxification genes (glutathione S-transferase and cytochrome P450), and this upregulation in detoxification genes (glutathione S-transferase, cytochrome P450 and esterase) was tremendously high in larvae fed diets incorporated with piperine. Results suggest that piperine is a promising bio-pesticide agent for the control of R. ferrugineus Olivier.