A precise and consistent assay for major wall polymer features that distinctively determine biomass saccharification in transgenic rice by near-infrared spectroscopy

Background

The genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling.

Results

Among the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R ²/R ² _cv/R ² _ev and RPD values for a perfect prediction capacity.

Conclusions

Due to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.

     

Stalk Rot Diseases Impact Sweet Sorghum Biofuel Traits

Owing to its sugar-rich stalks and high biomass, sweet sorghum [Sorghum bicolor (L.) Moench] has potential as a source of biofuel feedstock for juice and lignocellulosic-based bioethanol production. However, stalk rot-mediated lodging is an important concern. The potential impacts of disease on sweet sorghum biofuel traits are currently unknown. The objectives of this study were to test the effects of Fusarium stalk rot and charcoal rot on sweet sorghum biofuel traits and to assess the combining ability of the parental genotypes for resistance to the two diseases. Nineteen genotypes including 7 parents and 12 hybrids were tested in the field in 2014 (Ashland, Kansas) and 2015 (Manhattan, Kansas) against Fusarium thapsinum (FT) and Macrophomina phaseolina (MP). Fourteen days after flowering, plants were inoculated with FT and MP. Plants were harvested at 35 days after inoculation and measured for disease severity using stalk lesion length. Grain weight, juice weight, Brix (°Bx), and dried bagasse weight were also determined. Total soluble sugars per plant (TSSP) were determined using juice weight and °Bx. On average, FT and MP resulted in reduced grain weight and dried bagasse weight by 17.4 and 17.6 %, respectively, across genotypes. Depending on the genotype, pathogens reduced juice weight, °Bx, and TSSP in the ranges of 11.3 to 25.9, 0.2 to 16.7, and 21.2 to 33.3 %, respectively. Parental line general and specific combining abilities were found to be statistically insignificant. This study revealed the adverse effects of stalk rot diseases on harvestable biofuel traits and the need to breed sweet sorghum for stalk rot resistance.     

Mechanistic insights into the effect of imidazolium ionic liquid on lipid production by <Emphasis Type="Italic">Geotrichum fermentans</Emphasis>

Background

Ionic liquid (IL) pretreatment has emerged as a promising technique that enables complete utilization of lignocellulosic biomass for biofuel production. However, imidazolium IL has recently been shown to exhibit inhibitory effect on cell growth and product formation of industrial microbes, such as oleaginous microorganisms. To date, the mechanism of this inhibition remains largely unknown.

Results

In this study, the feasibility of [Bmim][OAc]-pretreated rice straw hydrolysate as a substrate for microbial lipid production by Geotrichum fermentans, also known as Trichosporon fermentans, was evaluated. The residual [Bmim][OAc] present in the hydrolysate caused a reduction in biomass and lipid content (43.6 and 28.1%, respectively) of G. fermentans, compared with those of the control (7.8 g/L and 52.6%, respectively). Seven imidazolium ILs, [Emim][DEP], [Emim]Cl, [Amim]Cl, [Bmim]Cl, [Bzmim]Cl, [Emim][OAc], and [Bmim][OAc], capable of efficient pretreatment of lignocellulosic biomass were tested for their effects on the cell growth and lipid accumulation of G. fermentans to better understand the impact of imidazolium IL on the lipid production. All the ILs tested inhibited the cell growth and lipid accumulation. In addition, both the cation and the anion of IL contributed to IL toxicity. The side chain of IL cations showed a clear impact on toxicity. On examining IL anions, [OAc]^? was found to be more toxic than those of [DEP]^? and Cl^?. IL exhibited its toxicity by inhibiting sugar consumption and key enzyme (malic enzyme and ATP-citrate lyase) activities of G. fermentans. Cell membrane permeability was also altered to different extents in the presence of various ILs. Scanning electron microscopy revealed that IL induces fibrous structure on the surface of G. fermentans cell, which might represent an adaptive mechanism of the yeast to IL.

Conclusions

This work gives some mechanistic insights into the impact of imidazolium IL on the cell growth and lipid accumulation of oleaginous yeast, which is important for IL integration in lignocellulosic biofuel production, especially for microbial lipid production.

     

The targeting of starch binding domains from starch synthase III to the cell wall alters cell wall composition and properties

Key message

Starch binding domains of starch synthase III from Arabidopsis thaliana (SBD123) binds preferentially to cell wall polysaccharides rather than to starch in vitro. Transgenic plants overexpressing SBD123 in the cell wall are larger than wild type. Cell wall components are altered in transgenic plants. Transgenic plants are more susceptible to digestion than wild type and present higher released glucose content. Our results suggest that the transgenic plants have an advantage for the production of bioethanol in terms of saccharification of essential substrates.

Abstract

The plant cell wall, which represents a major source of biomass for biofuel production, is composed of cellulose, hemicelluloses, pectins and lignin. A potential biotechnological target for improving the production of biofuels is the modification of plant cell walls. This modification is achieved via several strategies, including, among others, altering biosynthetic pathways and modifying the associations and structures of various cell wall components. In this study, we modified the cell wall of A. thaliana by targeting the starch-binding domains of A. thaliana starch synthase III to this structure. The resulting transgenic plants (E8-SDB123) showed an increased biomass, higher levels of both fermentable sugars and hydrolyzed cellulose and altered cell wall properties such as higher laxity and degradability, which are valuable characteristics for the second-generation biofuels industry. The increased biomass and degradability phenotype of E8-SBD123 plants could be explained by the putative cell-wall loosening effect of the in tandem starch binding domains. Based on these results, our approach represents a promising biotechnological tool for reducing of biomass recalcitrance and therefore, the need for pretreatments.

     

Growth and photosynthetic activity of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> biofilms

Botryococcus braunii is a green microalga capable of producing large amounts of external long-chain hydrocarbons suitable as a source of biofuel. There have been several studies indicating that cultures of B. braunii can reduce the energy and water requirement for mass biofuel production, especially if non-destructive extraction methods for milking hydrocarbons are used. Growing microalgae as a raw material for biofuel using conventional liquid-based cultivation (i.e., raceway ponds) has yet to be shown to be economically successful. An alternative solid growth (biofilm) cultivation method can markedly reduce the energy requirements and costs associated with the harvesting and dewatering processes. We evaluated the growth of biofilms of several strains of B. braunii (from races A, B, L and S) and found that three of the four tested races successfully grew to stationary phase in 10 weeks with no contamination. Among all races, B. braunii BOT22 (race B) reached the highest biomass and lipid yields (3.80 mg dry weight cm^?2 day^?1 and 1.11 mg dry weight cm^?2). Irrespective of the race, almost all photosynthetic parameters (F _V /F ₀ , PI_ABS and the OJIP curve) showed that the biofilm cultures were more stressed during lag and stationary phases than in logarithmic phase. We also studied the Botryococcus biofilm profiles using confocal microscopy and found that this method is suitable for estimating the overall biomass yield when compared with gravimetric measurement. In conclusion, the growth characteristics (biomass and lipid) and photosynthetic performance of all races indicated that B. braunii BOT22 is the most promising strain for biofilm cultivation.     

Salicylic acid promotes plant growth and salt-related gene expression in <Emphasis Type="Italic">Dianthus superbus</Emphasis> L. (Caryophyllaceae) grown under different salt stress conditions

Salt stress is a critical factor that affects the growth and development of plants. Salicylic acid (SA) is an important signal molecule that mitigates the negative effects of salt stress on plants. To elucidate salt tolerance in large pink Dianthus superbus L. (Caryophyllaceae) and the regulatory mechanism of exogenous SA on D. superbus under different salt stresses, we conducted a pot experiment to evaluate leaf biomass, leaf anatomy, soluble protein and sugar content, and the relative expression of salt-induced genes in D. superbus under 0.3, 0.6, and 0.9% NaCl conditions with and without 0.5 mM SA. The result showed that exposure of D. superbus to salt stress lead to a decrease in leaf growth, soluble protein and sugar content, and mesophyll thickness, together with an increase in the expression of MYB and P5CS genes. Foliar application of SA effectively increased leaf biomass, soluble protein and sugar content, and upregulated the expression of MYB and P5CS in the D. superbus, which facilitated in the acclimation of D. superbus to moderate salt stress. However, when the plants were grown under severe salt stress (0.9% NaCl), no significant difference in plant physiological responses and relevant gene expression between plants with and without SA was observed. The findings of this study suggest that exogenous SA can effectively counteract the adverse effects of moderate salt stress on D. superbus growth and development.     

Recalcitrance Assessment of the Agro-industrial Residues from Five <Emphasis Type="Italic">Agave</Emphasis> Species: Ionic Liquid Pretreatment,Saccharification and Structural Characterization

Agave has recently shown its potential as a bioenergy feedstock with promising features such as higher biomass productivity than leading bioenergy feedstock while at the same time being drought-resistant with low water requirements and high sugar to ethanol conversion using ionic liquid (IL) pretreatment. IL pretreatment was studied to develop the first direct side-by-side comparative recalcitrance assessment of the agro-industrial residues from five Agave species [Agave americana (AME), A. angustifolia (ANG), A. fourcroydes (FOU), A. salmiana (SAL), and A. tequilana (TEQ)] using compositional analysis, X-ray diffraction, and the lignin syringyl/guaiacyl subunit ratio (S/G) by pyrolysis molecular beam mass spectrometry (PyMBMS). Prominent calcium oxalate peaks were found only in unpretreated AME, SAL, and TEQ. The S/G ratios of all five unpretreated Agave species were between 1.27 and 1.57 while the IL-pretreated samples were from 1.39 to 1.72. The highest overall sugar production was obtained with IL-pretreated FOU with 492 mg glucose/g biomass and 157 mg xylose/g biomass at 120 °C and 3 h using 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc]). An estimated theoretical ethanol yield from the studied agro-industrial residues from the five Agave species was in the range of 1060 to 5800 L ethanol/ha/year. These comparison results demonstrate the potential of the Agave spp. as a suitable biofuel feedstock which can be employed within a biorefinery scheme.     

Volatile compounds from beneficial or pathogenic bacteria differentially regulate root exudation,transcription of iron transporters,and defense signaling pathways in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Key message

Our results show that Sorghum bicolor is able to recognize bacteria through its volatile compounds and differentially respond to beneficial or pathogens via eliciting nutritional or defense adaptive traits.

Abstract

Plants establish beneficial, harmful, or neutral relationships with bacteria. Plant growth promoting rhizobacteria (PGPR) emit volatile compounds (VCs), which may act as molecular cues influencing plant development, nutrition, and/or defense. In this study, we compared the effects of VCs produced by bacteria with different lifestyles, including Arthrobacter agilis UMCV2, Bacillus methylotrophicus M4-96, Sinorhizobium meliloti 1021, the plant pathogen Pseudomonas aeruginosa PAO1, and the commensal rhizobacterium Bacillus sp. L2-64, on S. bicolor. We show that VCs from all tested bacteria, except Bacillus sp. L2-64, increased biomass and chlorophyll content, and improved root architecture, but notheworthy A. agilis induced the release of attractant molecules, whereas P. aeruginosa activated the exudation of growth inhibitory compounds by roots. An analysis of the expression of iron-transporters SbIRT1, SbIRT2, SbYS1, and SbYS2 and genes related to plant defense pathways COI1 and PR-1 indicated that beneficial, pathogenic, and commensal bacteria could up-regulate iron transporters, whereas only beneficial and pathogenic species could induce a defense response. These results show how S. bicolor could recognize bacteria through their volatiles profiles and highlight that PGPR or pathogens can elicit nutritional or defensive traits in plants.

     

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.     

The eurythermal adaptivity and temperature tolerance of a newly isolated psychrotolerant Arctic <Emphasis Type="Italic">Chlorella</Emphasis> sp.

A new strain of Chlorella sp. (Chlorella-Arc), isolated from Arctic glacier melt water, was found to have high specific growth rates (μ) between 3 and 27 °C, with a maximum specific growth rate of 0.85 day^?1 at 15 °C, indicating that this strain was a eurythermal strain with a broad temperature tolerance range. To understand its acclimation strategies to low and high temperatures, the physiological and biochemical responses of the Chlorella-Arc to temperature were studied and compared with those of a temperate Chlorella pyrenoidosa strain (Chlorella-Temp). As indicated by declining F _v/F _m, photoinhibition occurred in Chlorella-Arc at low temperature. However, Chlorella-Arc reduced the size of the light-harvesting complex (LHC) to alleviate photoinhibition, as indicated by an increasing Chl a/b ratio with decreasing temperatures. Interestingly, Chlorella-Arc tended to secrete soluble sugar into the culture medium with increasing temperature, while its intracellular soluble sugar content did not vary with temperature changes, indicating that the algal cells might suffer from osmotic stress at high temperature, which could be adjusted by excretion of soluble sugar. Chlorella-Arc accumulated protein and lipids under lower temperatures (<15 °C), and its metabolism switched to synthesis of soluble sugar as temperatures rose. This reflects a flexible ability of Chlorella-Arc to regulate carbon and energy distribution when exposed to wide temperature shifts. More saturated fatty acids (SFA) in Chlorella-Arc than Chlorella-Temp also might serve as the energy source for growth in the cold and contribute to its cold tolerance.     

Accumulation and tolerance characteristics of lead in <Emphasis Type="Italic">Althaea rosea</Emphasis> Cav. and <Emphasis Type="Italic">Malva crispa</Emphasis> L.

Two ornamental plants of Althaea rosea Cav. and Malva crispa L. were exposed to various concentrations of lead (Pb) (0, 50, 100, 200 and 500 mg·kg^?1) for 70 days to evaluate the accumulating potential and the tolerance characteristics. The results showed that both plant species grown normally under Pb stress, and A. rosea had a higher tolerance than M. crispa, while M. crispa had a higher ability in Pb accumulation than A. rosea. Besides, lower Pb concentration (50 mg·kg^?1) stimulated the shoot biomass in both plant species. Pb accumulation in plants was consistent with the increase of Pb levels, and the main accumulation sites were the roots and the older leaves. In addition, the photosynthetic pigments content and chlorophyll fluorescence parameters were influenced by Pb stress. In such case, both of the plants could improve the activities of antioxidant enzymes of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), and the contents of the total soluble sugar and soluble protein, which reached the highest value at Pb 100 mg·kg^?1, as well as the accumulation of the total thiols (T-SH) and non-protein thiols (NP-SH) to adapt to Pb stress. Thus, it provides the theoretical basis and possibility for ornamental plants of A. rosea and M. crispa in phytoremediation of Pb contaminated areas.     

Overexpression of <Emphasis Type="Italic">flv3</Emphasis> improves photosynthesis in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803 by enhancement of alternative electron flow

Background

To ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability.

Results

In the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O₂ photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O₂ evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo¹³C-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain.

Conclusions

Overexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO₂ incorporation is enhanced by the flv3 overexpression. Increase in O₂ evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

     

Comparative evaluation of <Emphasis Type="Italic">Populus</Emphasis> variants total sugar release and structural features following pretreatment and digestion by two distinct biological systems

Background

Populus natural variants have been shown to realize a broad range of sugar yields during saccharification, however, the structural features responsible for higher sugar release from natural variants are not clear. In addition, the sugar release patterns resulting from digestion with two distinct biological systems, fungal enzymes and Clostridium thermocellum, have yet to be evaluated and compared. This study evaluates the effect of structural features of three natural variant Populus lines, which includes the line BESC standard, with respect to the overall process of sugar release for two different biological systems.

Results

Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar release from hydrothermal pretreatment combined with either enzymatic hydrolysis or Clostridium thermocellum fermentation compared to the Populus natural variant, BESC standard. However, C. thermocellum outperformed the fungal cellulases yielding 96.0, 95.5, and 85.9% glucan plus xylan release from SKWE 24-2, BESC 876, and BESC standard, respectively. Among the feedstock properties evaluated, cellulose accessibility and glycome profiling provided insights into factors that govern differences in sugar release between the low recalcitrant lines and the BESC standard line. However, because this distinction was more apparent in the solids after pretreatment than in the untreated biomass, pretreatment was necessary to differentiate recalcitrance among Populus lines. Glycome profiling analysis showed that SKWE 24-2 contained the most loosely bound cell wall glycans, followed by BESC 876, and BESC standard. Additionally, lower molecular weight lignin may be favorable for effective hydrolysis, since C. thermocellum reduced lignin molecular weight more than fungal enzymes across all Populus lines.

Conclusions

Low recalcitrant Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar yields than BESC standard when hydrothermal pretreatment was combined with biological digestion. However, C. thermocellum was determined to be a more robust and effective biological catalyst than a commercial fungal cellulase cocktail. As anticipated, recalcitrance was not readily predicted through analytical methods that determined structural properties alone. However, combining structural analysis with pretreatment enabled the identification of attributes that govern recalcitrance, namely cellulose accessibility, xylan content in the pretreated solids, and non-cellulosic glycan extractability.

     

Overexpression of UDP-glucose dehydrogenase from <Emphasis Type="Italic">Larix gmelinii</Emphasis> enhances growth and cold tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine diphosphate glucose dehydrogenase (UGDH) plays an important role in biosynthesis of hemicellulose by catalyzing oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in biosynthesis of the plant cell wall. In this study, a UGDH ortholog referred to as LgUGDH was isolated from Larix gmelinii using PCR and rapid amplification of cDNA ends techniques. Real-time PCR shows that the LgUGDH gene was expressed primarily in larch stems in addition to its roots and leaves, and Southern blot analysis indicates that UGDH is encoded by two paralogous genes in L. gmelinii. Overexpression of LgUGDH increased the content of soluble sugars and hemicelluloses and enhanced vegetative growth and cold tolerance in transgenic Arabidopsis thaliana. These results reveal that L. gmelinii UGDH participates in sucrose/polysaccharide metabolism and cell wall biosynthesis and may be a good candidate gene for enhancing plant growth, cold tolerance, and hemicellulose content.     

Fecundity of the tropical catadromous eels <Emphasis Type="Italic">Anguilla bicolor bicolor</Emphasis>, <Emphasis Type="Italic">A. bengalensis bengalensis</Emphasis> and <Emphasis Type="Italic">A. marmorata</Emphasis>

Maturation is one of the most important ontogenetic transitions in an individual’s life. However, the reproductive ecology of the tropical anguillid eel genus Anguilla at the onset of oceanic spawning migration is poorly understood. To understand the reproductive ecology, the fecundity of the tropical eels Anguilla bicolor bicolor, A. bengalensis bengalensis and A. marmorata was examined using advanced migrating silver eels (Stage IV and V). A close linear relationship was found between total length and fecundity in A. bengalensis bengalensis. The fecundities of A. bicolor bicolor (0.55 to 4.96 × 10⁶), A. bengalensis bengalensis (0.33–1.72 × 10⁶) and A. marmorata (0.99 × 10⁶) were within the range of those observed in temperate eels.     

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Objective

To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential.

Results

Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain.

Conclusion

A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

     

<Emphasis Type="Italic">qRfg3</Emphasis>, a novel quantitative resistance locus against <Emphasis Type="Italic">Gibberella</Emphasis> stalk rot in maize

Key message

A quantitative trait locus  qRfg3 imparts recessive resistance to maize Gibberella stalk rot. qRfg3 has been mapped into a 350-kb interval and could reduce the disease severity index by ~26.6%.

Abstract

Gibberella stalk rot, caused by the fungal pathogen Fusarium graminearum, severely affects maize yield and grain quality worldwide. To identify more resistance quantitative trait loci (QTLs) against this disease, we analyzed a recombinant inbred line (RIL) population derived from a cross between resistant H127R and susceptible C7-2 inbred lines. Within this population, maize resistance to Gibberella stalk rot had high broad-sense heritability. A major QTL, qRfg3, on chromosome 3 was consistently detected across three field trials, accounting for 10.7–19.4% of the total phenotypic variation. Using a progeny-based sequential fine-mapping strategy, we narrowed qRfg3 down to an interval of ~350 kb. We further demonstrated that qRfg3 is a recessive resistance locus to Gibberella stalk rot that reduced the disease severity index by ~26.6%. Both the gene location and recessive genetic mode distinguish qRfg3 from other stalk rot resistance loci. Hence, qRfg3 is valuable as a complement to existing resistance QTLs to improve maize resistance to Gibberella stalk rot.

     

Development of a novel engineered antibody targeting <Emphasis Type="Italic">Neisseria</Emphasis> species

Objectives

A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).

Results

Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.

Conclusions

The recombinant scFv could detect Neisseria strains at 10⁶ CFU/ml.

     

Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose.

Results

The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the P_R promoter and kanamycin resistance gene (P_R-kan ^R ) at 30 °C, but have no effect on P_R-kan ^R gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~10⁷ cfu per µg of genomic DNA, with no empty vectors detected.

Conclusions

Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.