Grape and wine analysis: Oenologists to exploit advanced test kits.
Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.
It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.
Megazyme “advanced” wine test kits general characteristics and validation.
Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.
Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.
Interaction of Nectarin 4 with a fungal protein triggers a microbial surveillance and defense mechanism in nectar.
Harper, A. D., Stalnaker, S. H., Wells, L., Darvill, A., Thornburg, R. & York, W. S. (2010). Phytochemistry, 71(17-18), 1963-1969.
Understanding the biochemical mechanisms by which plants respond to microbial infection is a fundamental goal of plant science. Extracellular dermal glycoproteins (EDGPs) are widely expressed in plant tissues and have been implicated in plant defense responses. Although EDGPs are known to interact with fungal proteins, the downstream effects of these interactions are poorly understood. To gain insight into these phenomena, we used tobacco floral nectar as a model system to identify a mechanism by which the EDGP known as Nectarin IV (NEC4) functions as pathogen surveillance molecule. Our data demonstrates that the interaction of NEC4 with a fungal endoglucanase (XEG) promotes the catalytic activity of Nectarin V (NEC5), which catalyzes the conversion of glucose and molecular oxygen to gluconic acid and H2O2. Significantly enhanced NEC5 activity was observed when XEG was added to nectar or nectarin solutions that contain NEC4. This response was also observed when the purified NEC4:XEG complex was added to NEC4-depleted nectarin solutions, which did not respond to XEG alone. These results indicate that formation of the NEC4:XEG complex is a key step leading to induction of NEC5 activity in floral nectar, resulting in an increase in concentrations of reactive oxygen species (ROS), which are known to inhibit microbial growth directly and activate signal transduction pathways that induce innate immunity responses in the plant.
The LysR transcription factor, HexS, is required for glucose inhibition of prodigiosin production by Serratia marcescens.
Stella, N. A., Fender, J. E., Lahr, R. M., Kalivoda, E. J. & Shanks, R. M. (2012). Advances in Microbiology, 2(4).
Generation of many useful microbe-derived secondary metabolites, including the red pigment prodigiosin of the bacterium Serratia marcescens, is inhibited by glucose. In a previous report, a genetic approach was used to determine that glucose dehydrogenase activity (GDH) is required for inhibiting prodigiosin production and transcription of the prodigiosin biosynthetic operon (pigA-N). However, the transcription factor(s) that regulate this process were not characterized. Here we tested the hypothesis that HexS, a LysR-family transcription factor similar to LrhA of Escherichia coli, is required for inhibition of prodigiosin by growth in glucose. We observed that mutation of the hexS gene in S. marcescens allowed the precocious production of prodigiosin in glucose-rich medium conditions that completely inhibited prodigiosin production by the wild type. Unlike previously described mutants able to generate prodigiosin in glucose-rich medium, hexS mutants exhibited GDH activity and medium acidification similar to the wild type. Glucose inhibittion of pigA expression was shown to be dependent upon HexS, suggesting that HexS is a key transcription factor in secondary metabolite regulation in response to medium pH. These data give insight into the prodigiosin regulatory pathway and could be used to enhance the production of secondary metabolites.
Modeling of Continuous Gluconic Acid Production by Fermentation.
Fatmawati, A. & Agustriyanto, R. (2010). Science Journal. 1(1), 82-89.
The batch fermentation kinetic of gluconic acid production has been studied. The continuous fermentation process of glucose by Aspergillus niger to produce gluconic acid under the influence of inlet substrate concentration and hydraulic retention time has also been investigated. The fermentation was modeled to be carried out in a continuous stirred tank reactor. The results showed that at the studied inlet glucose concentration of 150, 200, and 250 g/l, the hydraulic retention time resulted in the increasing amount of cell and gluconic acid concentration but decreasing glucose concentration at the outlet stream of the reactor. The model results also suggested that the possible range of hydraulic retention time for the inlet substrate concentration of 150, 200, and 250 g/l were 3-13, 8-12, and 7-11 h, respectively. Therefore the recommended values of hydraulic retention time were 13, 12 and 11 h for the inlet substrate concentration of 150, 200, and 250 g/l, respectively.
Genetic diversity of phosphate-solubilizing peanut (Arachis hypogaea L.) associated bacteria and mechanisms involved in this ability.
Anzuay, M. S., Frola, O., Angelini, J. G., Ludueña, L. M., Fabra, A. & Taurian, T. (2013). Symbiosis , 60(3), 143-154.
In this study, attempts were made to analyze mechanisms involved in the bacterial phosphate-solubilizing ability of peanut isolates. Bacteria were taxonomically identified by analysis of 16S rDNA sequence. Levels of soluble P released by the isolates in unbuffered or buffered with Tris–HCl or MES NBRIP-BPB medium as well as the production of D-gluconic acid were determined in their culture. Presence of two of the genes encoding the cofactor PQQ of GDH enzyme was analyzed in the genome of this bacterial collection. 16S rDNA sequence analysis indicated that isolates belong to genera Serratia, Enterobacter, Pantoea, Acinetobacter, Bacillus and Enterococcus. All bacteria showed ability to solubilize tricalcium phosphate either in unbuffered or buffered medium. Nevertheless, addition of buffer solutions reduced levels of Pi liberated by the isolates. Although almost all isolates produced detectable amounts of D-gluconic acid, no correlation with levels of P soluble released were observed. The presence of pqqE and pqqC genes was detected only in Gram negative bacteria. It was concluded from this study that the mechanism involved in phosphate solubilization is organic acids production and, presence of pqq genes in all Gram negative bacteria analyzed encourages to confirm their role in bacterial phosphate solubilizing ability as well to identify genes involved in this PGP trait in Gram positive bacteria.
Aerobic deconstruction of cellulosic biomass by an insect-associated Streptomyces.
Takasuka, T. E., Book, A. J., Lewin, G. R., Currie, C. R. & Fox, B. G. (2013). Scientific Reports, 3.
Streptomyces are best known for producing antimicrobial secondary metabolites, but they are also recognized for their contributions to biomass utilization. Despite their importance to carbon cycling in terrestrial ecosystems, our understanding of the cellulolytic ability of Streptomyces is currently limited to a few soil-isolates. Here, we demonstrate the biomass-deconstructing capability of Streptomyces sp. SirexAA-E (ActE), an aerobic bacterium associated with the invasive pine-boring woodwasp Sirex noctilio. When grown on plant biomass, ActE secretes a suite of enzymes including endo- and exo-cellulases, CBM33 polysaccharide-monooxygenases, and hemicellulases. Genome-wide transcriptomic and proteomic analyses, and biochemical assays have revealed the key enzymes used to deconstruct crystalline cellulose, other pure polysaccharides, and biomass. The mixture of enzymes obtained from growth on biomass has biomass-degrading activity comparable to a cellulolytic enzyme cocktail from the fungus Trichoderma reesei, and thus provides a compelling example of high cellulolytic capacity in an aerobic bacterium.
Serratia marcescens quinoprotein glucose dehydrogenase activity mediates medium acidification and inhibition of prodigiosin production by glucose.
Fender, J. E., Bender, C. M., Stella, N. A., Lahr, R. M., Kalivoda, E. J. & Shanks, R. M. (2012). Applied and Environmental Microbiology, 78(17), 6225-6235.
Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that D-glucono-1,5-lactone and D-gluconic acid, but not D-gluconate, were able to inhibit prodigiosin production. These data support a model in which the oxidation of D-glucose by quinoprotein GDH initiates a reduction in pH that inhibits prodigiosin production through transcriptional control of the prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites.
Applying systems biology tools to study n‐butanol degradation in Pseudomonas putida KT2440.
Vallon, T., Simon, O., Rendgen‐Heugle, B., Frana, S., Mückschel, B., Broicher, A., Siemann-Herzberg, M., Pfannenstiel, J., Hauer, B., Huber, A., Breuer, M. & Breuer, M. (2015). Engineering in Life Sciences, 15(8), 760-771.
To smoothen the process of n-butanol formation in Pseudomonas putida KT2440, detailed knowledge of the impact of this organic solvent on cell physiology and regulation is of outmost importance. Here, we conducted a detailed systems biology study to elucidate cellular responses at the metabolic, proteomic, and transcriptional level. Pseudomonas putida KT2440 was cultivated in multiple chemostat fermentations using n-butanol either as sole carbon source or together with glucose. Pseudomonas putida KT2440 revealed maximum growth rates (µ) of 0.3 h-1 with n-butanol as sole carbon source and of 0.4 h-1 using equal C-molar amounts of glucose and n-butanol. While C-mole specific substrate consumption and biomass/substrate yields appeared equal at these growth conditions, the cellular physiology was found to be substantially different: adenylate energy charge levels of 0.85 were found when n-butanol served as sole carbon source (similar to glucose as sole carbon source), but were reduced to 0.4 when n-butanol was coconsumed at stable growth conditions. Furthermore, characteristic maintenance parameters changed with increasing n-butanol consumption. 13C flux analysis revealed that central metabolism was split into a glucose-fueled Entner–Doudoroff/pentose-phosphate pathway and an n-butanol-fueled tricarboxylic acid cycle when both substrates were coconsumed. With the help of transcriptome and proteome analysis, the degradation pathway of n-butanol could be unraveled, thus representing an important basis for rendering P. putida KT2440 from an n-butanol consumer to a producer in future metabolic engineering studies.
Rapid Assessment of Gray Mold (Botrytis cinerea) Infection in Grapes Using Biosensors System.
Cinquanta, L., Albanese, D., De Curtis, F., Malvano, F., Crescitelli, A. & Di Matteo, M. (2015). American Journal of Enology and Viticulture, 66(4).
Botrytis cinerea is responsible for the gray mold disease, which causes considerable economic losses for winemakers. Its evaluation in wine grapes is commonly performed through visual estimation, which was demonstrated to be prone to assessor bias. Rapid and simple enzymatic carbon screen printed amperometric biosensors were here used to evaluate gluconic acid and glycerol content on wine grapes at different B. cinerea infection degrees. The lower concentrations measurable by screen-printed amperometric biosensors were 3 mg/L for gluconic acid (corresponding to an infection degree lower than 1%) and 35 mg/L for glycerol; the response times with a flow rate of 0.5 mL/min were in a range of 0.5 to 2 min in the linear ranges. This study demonstrates the effectiveness of the biosensors for rapid analysis of gluconic acid and glycerol in grapes, confirming their high correlation with B. cinerea degree of infection (R2 = 0.98). Thus, the biosensor developed to measure gluconic acid in grapes (or must), was more precise, and gave a faster response than methods that currently exist allowing the percentage of infection of grape berries by B. cinerea to be evaluated.
Revalorization of strawberry surpluses by bio-transforming its glucose content into gluconic acid.
Cañete-Rodríguez, A. M., Santos-Dueñas, I. M., Jiménez-Hornero, J. E., Torija-Martínez, M. J., Mas, A. & García-García, I. (2016). Food and Bioproducts Processing, 99, 188-196.
Modern societies produce massive surpluses of food, by-products and wastes that increase the interest for their revalorization. This work examines the use of a culture of Gluconobacter japonicus CECT 8443, without pH control, to convert selectively the glucose content of industrially pasteurized strawberry purée into gluconic acid for the development of new beverages. However, depending on the initial concentration of glucose, the microorganism could transform the acid formed into other compounds; for this reason, in this work the effect of initial sugar concentration on the preservation of the acid was investigated. The results show that the gluconic acid formed in strawberry purée containing no added sugars started to disappear after glucose depletion, but the acid concentration remained constant if sugar-enriched purée was used. The use of this industrial substrate resulted in the presence of yeasts and hence in some fructose uptake; however, the fructose consumption was negligible until after 20–30 h. The use of food by-products is an excellent opportunity not only to recover valuable compounds but for the development of new chemical and biotechnological approaches for their revalorization. This strategy should improve regional economies and contribute to a sustainable management of these underexploited resources.
An approach for estimating the maximum specific growth rate of Gluconobacter japonicus in strawberry purée without cell concentration data.
Cañete-Rodríguez, A. M., Santos-Dueñas, I. M., Jiménez-Hornero, J. E., Torija-Martínez, M. J., Mas, A. & García-García, I. (2016). Biochemical Engineering Journal, 105, 314-320.
The estimation of the maximum specific growth rate (µmax) for non-readily culturable bacteria, growing on complex media containing suspended solids, is a difficult task considering the important problems in obtaining reliable measures of cell concentration. An example of this situation can be a culture of Gluconobacter japonicus growing in strawberry purée for producing gluconic acid. Based on the dependency between energy requirements of the genus Gluconobacter and substrate uptake as well as its constant relationship between gluconic acid production and total substrate uptake, the total substrate concentration profile during the exponential growth phase could be used for estimating µmax without cell concentration measures. In this case, the high selectivity of the strain for glucose in comparison to fructose resulted in no fructose consumption during the batch; so, just using the glucose concentrations data during the exponential phase allow us to obtain an estimation of µmax. Additionally, a rough estimation of the apparent and stoichiometric yields of cell on glucose is also possible.
An accurate description of Aspergillus niger organic acid batch fermentation through dynamic metabolic modelling.
Upton, D. J., McQueen-Mason, S. J. & Wood, A. J. (2017). Biotechnology for Biofuels, 10(1), 258.
Background: Aspergillus niger fermentation has provided the chief source of industrial citric acid for over 50 years. Traditional strain development of this organism was achieved through random mutagenesis, but advances in genomics have enabled the development of genome-scale metabolic modelling that can be used to make predictive improvements in fermentation performance. The parent citric acid-producing strain of A. niger, ATCC 1015, has been described previously by a genome-scale metabolic model that encapsulates its response to ambient pH. Here, we report the development of a novel double optimisation modelling approach that generates time-dependent citric acid fermentation using dynamic flux balance analysis. Results: The output from this model shows a good match with empirical fermentation data. Our studies suggest that citric acid production commences upon a switch to phosphate-limited growth and this is validated by fitting to empirical data, which confirms the diauxic growth behaviour and the role of phosphate storage as polyphosphate. Conclusions: The calibrated time-course model reflects observed metabolic events and generates reliable in silico data for industrially relevant fermentative time series, and for the behaviour of engineered strains suggesting that our approach can be used as a powerful tool for predictive metabolic engineering.
Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose.
Guo, Z. P., Duquesne, S., Bozonnet, S., Nicaud, J. M., Marty, A. & O’Donohue, M. J. (2017). Biotechnology for Biofuels, 10(1), 298.
Background: A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate. Results: The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively. Conclusions: This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.
Strain Serratia sp. S119: A potential biofertilizer for peanut and maize and a model bacterium to study phosphate solubilization mechanisms.
Ludueña, L. M., Anzuay, M. S., Angelini, J. G., McIntosh, M., Becker, A., Rupp, O., Goesmann, A., Blom, J., Fabra, A. & Taurian, T. (2018).
Applied Soil Ecology, In Press.
Strain Serratia sp. S119 is a peanut native bacterium with high phosphate solubilizing activity that promotes the growth of peanut and maize in the cultivation area of Córdoba in Argentina. The aims of this study were to obtain and analyze the genome sequence of Serratia sp. S119 to understand the genetic basis of its beneficial properties on plant growth, and to demonstrate phosphate solubilizing ability in early stages of bacterial growth. Results obtained indicated that soluble P and gluconic acid were detected during exponential growth phase in bacterial supernatant. Analysis of the genome sequence of Serratia sp. S119 obtained from this study showed the presence of genes related to several plant growth promoting traits. The genome sequence of this strain is a valuable source of information to study bacterial response to phosphate starvation and to investigate interaction between this bacterium with host plants under nutritional deficient environments.