Transcriptional repression of MdMa1 by MdMYB21 in Ma locus decreases malic acid content in apple fruit.

Malic acid is a major organic acid component of apples and a crucial determinant of fruit organoleptic quality. A candidate gene for malic acid content, designated MdMa1, was previously identified in the Ma locus, which is a major quantitative trait locus (QTL) for apple fruit acidity located on the linkage group 16. Region-based association mapping to detect candidate genes in the Ma locus identified MdMa1 and an additional MdMYB21 gene putatively associated with malic acid. MdMYB21 was significantly associated with fruit malic acid content, accounting for ~7.48% of the observed phenotypic variation in the apple germplasm collection. Analyses of transgenic apple calli, fruits and tomatoes demonstrated that MdMYB21 negatively regulated malic acid accumulation. The apple fruit acidity-related MdMa1 and its tomato ortholog, SlALMT9, exhibited lower expression profiles in apple calli, mature fruits and tomatoes in which MdMYB21 was overexpressed, compared with their corresponding wild-type variety. MdMYB21 directly binds to the MdMa1 promoter and represses its expression. Interestingly, a 2-bp variation in the MdMYB21 promoter region altered its expression and regulation of its target gene, MdMa1, expression. Our findings not only demonstrate the efficiency of integrating QTL and association mapping in the identification of candidate genes controlling complex traits in apples, but also provide insights into the complex regulatory mechanism of fruit malic acid accumulation.

Conditional expression of FumA in Aspergillus niger enhances synthesis of L-malic acid.

Currently, the L-malic acid titer achieved through Aspergillus niger fermentation reaches 201 g/L, meeting industrial demands satisfactorily. However, the co-presence of structurally similar fumaric acid and succinic acid in fermentation products suggests a theoretical potential for further improvement in L-malic acid production. In the tricarboxylic acid cycle, fumarate reductase mediates the conversion of succinic acid to fumaric acid. Subsequently, fumarase catalyzes the conversion of fumaric acid to L-malic acid. Notably, both enzymatic reactions are reversible. Our investigation revealed that A. niger contains only one mitochondria-located fumarase FumA. Employing CRISPR-Cas9 technology, we performed a replacement of the fumA promoter with a doxycycline-induced promoter Tet. Under non-inducing condition, the conditional strain exhibited increased levels of fumaric acid and succinic acid. It strongly suggests that FumA mainly promotes the flow of fumaric acid to L-malic acid. Furthermore, a promoter PmfsA that is exclusively activated in a fermentation medium by calcium carbonate was identified through RNA-sequencing screening. Utilizing PmfsA to regulate fumA expression led to a 9.0% increase in L-malic acid titer, an 8.75% increase in yield (glucose to L-malic acid), and an 8.86% enhancement in productivity. This research serves as a significant step toward expediting the industrialization of L-malic acid synthesis via biological fermentation. Additionally, it offers valuable insights for the biosynthesis of other organic acids.IMPORTANCEThis study focuses on enhancing L-malic acid synthesis by modifying the tricarboxylic acid cycle within the mitochondria of Aspergillus niger. We emphasize the significant role of fumarase in converting fumaric acid into L-malic acid, enhancing our understanding of metabolic pathways in A. niger. The precise regulation of fumA is highlighted as a key factor in enhancing L-malic acid production. Furthermore, this research introduces a stringent conditional promoter (PmfsA), exclusively activated by CaCO3. The utilization of PmfsA for fumA expression resulted in heightened L-malic acid titers. The progress in metabolic engineering and bioprocess optimization holds promise for expediting industrial L-malic acid synthesis via biological fermentation. Moreover, it carries implications for the biosynthesis of various other organic acids.

Targeted 1-H-NMR wine analyses revealed specific metabolomic signatures of yeast populations belonging to the Saccharomyces genus.

This study aimed to explore the non-volatile metabolomic variability of a large panel of strains (44) belonging to the Saccharomyces cerevisiae and Saccharomyces uvarum species in the context of the wine alcoholic fermentation. For the S. cerevisiae strains flor, fruit and wine strains isolated from different anthropic niches were compared. This phenotypic survey was achieved with a special focus on acidity management by using natural grape juices showing opposite level of acidity. A 1H NMR based metabolomics approach was developed for quantifying fifteen wine metabolites that showed important quantitative variability within the strains. Thanks to the robustness of the assay and the low amount of sample required, this tool is relevant for the analysis of the metabolomic profile of numerous wines. The S. cerevisiae and S. uvarum species displayed significant differences for malic, succinic, and pyruvic acids, as well as for glycerol and 2,3-butanediol production. As expected, S. uvarum showed weaker fermentation fitness but interesting acidifying properties. The three groups of S. cerevisiae strains showed different metabolic profiles mostly related to their production and consumption of organic acids. More specifically, flor yeast consumed more malic acid and produced more acetic acid than the other S. cerevisiae strains which was never reported before. These features might be linked to the ability of flor yeasts to shift their metabolism during wine oxidation.

Ultrasound-assisted efficient and convenient method of extracting valuable metals (Ni, Co, and Cd) from waste Ni-Cd batteries using DL-malic acid.

Recycling waste Ni-Cd batteries has received much attention recently because of the serious environmental pollution they cause and to avoid the dissipation of valuable metals. Despite significant research, it is still difficult to efficiently recycle valuable and hazardous metals from waste Ni-Cd batteries in an economical and environmentally friendly manner. This study employed a novel process utilizing ultrasound-assisted leaching to recover Ni, Cd, and Co from waste nickel-cadmium (Ni-Cd) batteries. Organic DL-malic acid served as the leaching agent and H2O2 was employed as an oxidizing agent. The effects of various factors on the recovery efficiency of Ni, Cd, and Co, such as leaching temperature, time, DL-malic acid concentration, pulp density, H2O2 concentration, and ultrasound frequency, were also examined. To predict the chemical compounds present before and after the recycling experiments, the solid residues from the metal extraction were analyzed using XRD, XPS, FE-SEM, and EDS element mapping. Concurrently, ICP-OES was utilized to determine the metal content in the leachate. Under optimized conditions of 90 °C, 90 min, 2M DL-malic acid, 160 mL/g pulp density, and 20% ultrasound frequency, over 83% of Ni, 94% of Cd, and 98% of Co were effectively leached from the waste Ni-Cd battery powder. The leaching kinetics of Ni, Cd, and Co followed the surface chemical reaction control model. The activation energies (Ea) for Ni, Cd, and Co leaching were 21.34, 20.47, and 18.38 kJ/mol, respectively. The findings suggest that ultrasound-assisted leaching is an efficient, cost-effective, environmentally friendly, and sustainable alternative for extracting precious and hazardous metals from waste Ni-Cd batteries. Additionally, it reduces industrial chemical usage and enhances waste management sustainability.

Effect of Storage Conditions on the Volatilome, Biochemical Composition and Quality of Golden Delicious and Red Delicious Apple (Malus domestica) Varieties.

The effects of normal (NA) and controlled atmosphere (CA) storage and postharvest treatment with 1-methylcyclopropene (1-MCP) before CA storage for 5 months on the volatilome, biochemical composition and quality of 'Golden Delicious' (GD) and 'Red Delicious' (RD) apples were studied. Apples stored under NA and CA maintained and 1-MCP treatment increased firmness in both cultivars. NA storage resulted in a decrease of glucose, sucrose and fructose levels in both cultivars. When compared to CA storage, 1-MCP treatment caused a more significant decrease in sucrose levels and an increase in glucose levels. Additionally, 1-MCP-treated apples exhibited a significant decrease in malic acid content for both cultivars. All storage conditions led to significant changes in the abundance and composition of the volatilome in both cultivars. GD and RD apples responded differently to 1-MCP treatment compared to CA storage; higher abundance of hexanoate esters and (E,E)-α-farnesene was observed in RD apples treated with 1-MCP. While 1-MCP was effective in reducing (E,E)-α-farnesene abundance in GD apples, its impact on RD apples was more limited. However, for both cultivars, all storage conditions resulted in lower levels of 2-methylbutyl acetate, butyl acetate and hexyl acetate. The effectiveness of 1-MCP is cultivar dependent, with GD showing better results than RD.

Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH.

Currently, the biological production of L-malic acid (L-MA) is mainly based on the fermentation of filamentous fungi at near-neutral pH, but this process requires large amounts of neutralizing agents, resulting in the generation of waste salts when free acid is obtained in the downstream process, and the environmental hazards associated with the waste salts limit the practical application of this process. To produce L-MA in a more environmentally friendly way, we metabolically engineered the acid-tolerant yeast Pichia kudriavzevii and achieved efficient production of L-MA through low pH fermentation. First, an initial L-MA-producing strain that relies on the reductive tricarboxylic acid (rTCA) pathway was constructed. Subsequently, the L-MA titer and yield were further increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. In addition, we found that the insufficient supply of NADH for cytoplasmic malate dehydrogenase (MDH) hindered the L-MA production at low pH, which was resolved by overexpressing the soluble pyridine nucleotide transhydrogenase SthA from E. coli. Transcriptomic and metabolomic data showed that overexpression of EcSthA contributed to the activation of the pentose phosphate pathway and provided additional reducing power for MDH by converting NADPH to NADH. Furthermore, overexpression of EcSthA was found to help reduce the accumulation of the by-product pyruvate but had no effect on the accumulation of succinate. In microaerobic batch fermentation in a 5-L fermenter, the best strain, MA009-10-URA3 produced 199.4 g/L L-MA with a yield of 0.94 g/g glucose (1.27 mol/mol), with a productivity of 1.86 g/L/h. The final pH of the fermentation broth was approximately 3.10, meaning that the amount of neutralizer used was reduced by more than 50% compared to the common fermentation processes using filamentous fungi. To our knowledge, this is the first report of the efficient bioproduction of L-MA at low pH and represents the highest yield of L-MA in yeasts reported to date.

The non-ribosomal peptide synthetase-independent siderophore (NIS) rhizobactin produced by Caballeronia mineralivorans PML1(12) confers the ability to weather minerals.

To mobilize nutrients entrapped into minerals and rocks, heterotrophic bacteria living in nutrient-poor environments have developed different mechanisms based mainly on acidolysis and chelation. However, the genetic bases of these mechanisms remain unidentified. To fill this gap, we considered the model strain Caballeronia mineralivorans PML1(12) known to be effective at weathering. Based on its transcriptomics and proteomics responses in Fe-depleted conditions, we pointed a cluster of genes differentially expressed and putatively involved in the production of siderophores. In this study, we report the characterization of this gene region coding for the production of a non-ribosomal peptide synthetase-independent siderophore (NIS). Targeted mutagenesis associated with functional assays and liquid chromatography coupled to high-resolution tandem mass spectrometry demonstrated the production of a single siderophore, identified as rhizobactin. This siderophore represents the first NIS containing malic acid in its structure. The evidence for the implication of rhizobactin in mineral weathering was demonstrated during a hematite dissolution assay. This study provides the first demonstration of the synthesis of a NIS in the genus Caballeronia and its involvement in mineral weathering. Our conclusions reinforce the idea that strain PML1(12) is particularly well adapted to nutrient-poor environments. IMPORTANCE This work deciphers the molecular and genetic bases used by strain PML1(12) of Caballeronia mineralivorans to mobilize iron and weather minerals. Through the combination of bioinformatics, chemical, and phylogenetic analyses, we characterized the siderophore produced by strain PML1(12) and the related genes. This siderophore was identified as rhizobactin and classified as a non-ribosomal peptide synthetase-independent siderophore (NIS). Contrary to the previously identified NIS synthetases that form siderophores containing citric acid, α-ketoglutarate, or succinic acid, our analyses revealed that rhizobactin contains malic acid in its structure, representing, therefore, the first identified NIS with such an acid and probably a new NIS category. Last, this work demonstrates for the first time the effectiveness at weathering minerals of a siderophore of the NIS family. Our findings offer relevant information for different fields of research, such as environmental genomics, microbiology, chemistry, and soil sciences.

Ethylene inhibits malate accumulation in apple by transcriptional repression of aluminum-activated malate transporter 9 via the WRKY31-ERF72 network.

Malic acid accumulation in the vacuole largely determines acidity and perception of sweetness of apple. It has long been observed that reduction in malate level is associated with increase in ethylene production during the ripening process of climacteric fruits, but the molecular mechanism linking ethylene to malate reduction is unclear. Here, we show that ethylene-modulated WRKY transcription factor 31 (WRKY31)-Ethylene Response Factor 72 (ERF72)-ALUMINUM ACTIVATED MALATE TRANSPORTER 9 (Ma1) network regulates malate accumulation in apple fruit. ERF72 binds to the promoter of ALMT9, a key tonoplast transporter for malate accumulation of apple, transcriptionally repressing ALMT9 expression in response to ethylene. WRKY31 interacts with ERF72, suppressing its transcriptional inhibition activity on ALMT9. In addition, WRKY31 directly binds to the promoters of ERF72 and ALMT9, transcriptionally repressing and activating ERF72 and ALMT9, respectively. The expression of WRKY31 decreases in response to ethylene, lowering the transcription of ALMT9 directly and via its interactions with ERF72. These findings reveal that the regulatory complex WRKY31 forms with ERF72 responds to ethylene, linking the ethylene signal to ALMT9 expression in reducing malate transport into the vacuole during fruit ripening.

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor.

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.

Co-Crystalline Solid Solution Affords a High-Soluble and Fast-Absorbing Form of Praziquantel.

Praziquantel (PZQ) is a chiral class-II drug, and it is used as a racemate for the treatment of schistosomiasis. The knowledge of several cocrystals with dicarboxylic acids has prompted the realization of solid solutions of PZQ with both enantiomers of malic acid and tartaric acid. Here, the solid form landscape of such a six-component system has been investigated. In the process, two new cocrystals were structural-characterized and three non-stoichiometric, mixed crystal forms identified and isolated. Thermal and solubility analysis indicates a fourfold solubility advantage for the newly prepared solid solutions over the pure drug. In addition, a pharmacokinetic study was conducted in rats, which involved innovative mini-capsules for the oral administration of the solid samples. The available data indicate that the faster dissolution rate of the solid solutions translates in faster absorption of the drug and helps maintain a constant steady-state concentration.

Continuous Synthesis of Cinnarizine Salt with Malic Acid by Applying Green Chemistry Using Water-Assisted Twin Screw Extrusion.

Organic solvent-free process or green chemistry is needed for manufacturing pharmaceutical salts to avoid various environmental, safety, and manufacturing cost issues involved. In this study, a cinnarizine (CNZ) salt with malic acid at a 1:1 molar ratio was successfully prepared by twin screw extrusion (TSE) with water assistance. The feasibility of salt formation was first evaluated by screening several carboxylic acids by neat grinding (NG) and liquid-assisted grinding (LAG) using a mortar and pestle, which indicated that malic acid and succinic acid could form salts with CNZ. Further studies on salt formation were conducted using malic acid. The examination by hot-stage microscopy revealed that the addition of water could facilitate the formation and crystallization of CNZ-malic acid salt even though CNZ is poorly water-soluble. The feasibility of salt formation was confirmed by determining the pH-solubility relationship between CNZ and malic acid, where a pHmax of 2.7 and a salt solubility of 2.47 mg/mL were observed. Authentic salt crystals were prepared by solution crystallization from organic solvents for examining crystal properties and structure by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Fourier transform infrared (FTIR) spectroscopy, solid-state 13C and 15N nuclear magnetic resonance (NMR), and single-crystal X-ray diffraction (SXD). These techniques also established that a salt, and not a cocrystal, was indeed formed. The CNZ salt crystals were then prepared by TSE of a 1:1 CNZ-malic acid mixture, where the addition of small amounts of water resulted in a complete conversion of the mixture into the salt form. The salts prepared by solvent crystallization and water-assisted TSE had identical properties, and their moisture sorption profiles were also similar, indicating that TSE is a viable method for salt preparation by green chemistry. Since TSE can be conducted in a continuous manner, the results of the present investigation, if combined with other continuous processes, suggest the possibility of continuous manufacturing of drug products from the synthesis of active pharmaceutical ingredients (APIs) to the production of final dosage forms.

Preparation of Poly(ß-malic acid) via Direct Carbonylative Polymerization of Benzyl Glycidate.

Poly(malic acid) (PMLA) is a water-soluble, biodegradable, biocompatible, and nontoxic polyester in the poly(hydroxyalkanoate) (PHA) family. it features various applications in pharmaceutical field. Herein, NaCo(CO)4 and pyridine derivatives are employed for direct carbonylative polymerization of benzyl glycidate (BG) for poly(ß-malic acid) production. Further investigation on reaction mechanism reveals that this polymerization undergoes a direct chain growth, rather than a sequential process involving ß-lactone intermediate. The low cost and facile preparation of epoxide substrate render this methodology extremely appealing that avoids the rather tedious procedures for ß-malolactonate synthesis required toward ring opening polymerization. This study also represents an alternative strategy over traditional methods for poly(ß-malic acid) production using step growth polycondensation of malic acid.

Efficacy of a 1% malic acid spray for xerostomia treatment: A systematic review and meta-analysis.

OBJECTIVES: To assess the efficacy of topical sialogogue spray containing malic acid 1% for treating xerostomia. METHODS: We searched PubMed, Cochrane Library, Embase, ClinicalTrials.gov and Web of Science databases. Literature search, screening, study selection, data collection, data extraction and assessment of bias risk were independently conducted by two reviewers. The study appraisal was performed by Cochrane Collaboration's tool for assessing bias risk. The systematic review registration number was PROSPERO-CRD42021241322. All statistical analyses were performed using Review Manager version 5.4. RESULTS: Five original articles involving 244 patients with xerostomia who received topical sialogogue spray (malic acid 1%) or placebo for two weeks were included in this review. Based on the questionnaire survey, the topical sialogogue spray (malic acid 1%) improved the symptoms of dry mouth significantly better than the placebo, which was reflected in the Dry Mouth Questionnaire (DMQ), Xerostomia Inventory (XI) and Visual Analogue Scale (VAS) scores. Regarding the increase in unstimulated and stimulated saliva flow rates, the intervention group was also better than the placebo group after a two-week course of treatment. CONCLUSIONS: Although the included studies are limited, our results show that topical sialogogue spray (malic acid 1%) is an effective method for the treatment of xerostomia. Additional randomised controlled trials in the future are needed to provide high-quality evidence of this therapy and to improve clinical practice guidelines.

New malic acid producer strains of Saccharomyces cerevisiae for preserving wine acidity during alcoholic fermentation.

In the context of climate change, the chemical composition of wines is characterized by a massive drop of malic acid concentration in grape berries. Then wine professionals have to find out physical and/or microbiological solutions to manage wine acidity. The aim of this study is to develop wine Saccharomyces cerevisiae strains able to produce significant amount of malic acid during the alcoholic fermentation. By applying a large phenotypic survey in small scale fermentations, the production level of malic acid in seven grape juices confirmed the importance of the grape juice in the production of malic acid during the alcoholic fermentation. Beside the grape juice effect, our results demonstrated that extreme individuals able to produce up to 3 g/L of malic acid can be selected by crossing together appropriate parental strains. A multivariate analysis of the dataset generated illustrate that the initial the amount of malic acid produced by yeast is a determining exogenous factor for controlling the final pH of wine. Interestingly most of the acidifying strains selected are particularly enriched in alleles that have been previously reported for increasing the level of malic acid at the end of the alcoholic fermentation. A small set of acidifying strains were compared with strains able to consume a large amount of malic acid previously selected. The total acidity of resulting wines was statistically different and a panelist of 28 judges was able to discriminate the two groups of strains during a free sorting task analysis.

Addition of Organic Acids to Base Wines: Impacts on the Technological Characteristics and the Foam Quality of Sparkling Wines.

Climate change is causing a significant decrease in the total acidity of grapes and related wines. This represents a serious issue for sparkling wine production. Consequently, before the second fermentation, the acidification of base wines is often necessary. However, the impacts of the most important organic acids on the foam properties of sparkling wines are not yet well known. The impacts of the addition of tartaric, malic, citric, and lactic acid on the quality of Falanghina and Bombino sparkling wines were evaluated. Analyses were performed soon after the second fermentation and one year after aging sur lees. The addition of each different organic acid to the two base wines resulted in significant changes in the sparkling wines not only in terms of pH, titratable acidity, and buffering capacity but also in the content of total amino acids and, in some cases, in the height of the foam and its stability over time. For both grape varieties, acidified wines showed a lower content of total amino acids in comparison with the control wines. The addition of lactic acid determined a higher persistency of the foam even after one year of aging sur lees only in Falanghina wines. The results obtained herein highlight the importance of organic acids and the pH of the base wines for the content of amino acids in sparkling wines. No strict correlation between organic acid addition and the foamability of wines was observed.

Analysis of organic acid metabolism reveals citric acid and malic acid play major roles in determining acid quality during the development of kiwifruit (Actinidia eriantha).

BACKGROUND: Actinidia eriantha is one of the most important kiwifruit species in Actinidia. The relative high accumulation of organic acids in fruit of A. eriantha is an unfavorable factor for organoleptic quality. To identify key metabolic enzymes and genes involved in organic acids accumulation during fruit development, physiological, biochemical, and molecular experiments were conducted for the dynamic fruit samples of a new kiwifruit cultivar, A. eriantha 'Ganlv 1'. RESULTS: The contents of citric acid and malic acid increased greatly during fruit development, while quinic acid content decreased obviously. Significant positive correlations were observed between fruit titratable acidity and the contents of both citric acid and malic acid, and a significant negative correlation was found between fruit titratable acidity and the quinic acid content. The high accumulation of citric acid was found to be caused by the increased activity of citrate synthase (CS), and the decreased activities of two degradation-related enzymes, mitochondrial aconitase and nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase. In addition, the accumulation of malic acid depended mainly on the increased synthesis catalyzed by NAD-dependent malate dehydrogenase (NAD-MDH) and phosphoenolpyruvate carboxylase. Further analysis suggested that AeCS2 and AeMDH2 played pivotal roles in controlling the activities of CS and NAD-MDH respectively. CONCLUSION: The high accumulation level of citric acid relied on both the strong synthesis ability and the weak degradation ability. The accumulation level of malic acid was mainly affected by the synthesis. The novel information would be helpful for our understanding of the formation of fruit acidity quality. © 2023 Society of Chemical Industry.

Recent advances in the production of malic acid by native fungi and engineered microbes.

Malic acid is mainly produced by chemical methods which lead to various environmental sustainability concerns associated with CO2 emissions and resulting global warming. Since malic acid is naturally synthesized, microorganisms offer an eco-friendly and cost-effective alternative for its production. An additional advantage of microbial production is the synthesis of pure L-form of malic acid. Due to its numerous applications, biotechnologically- produced L-malic acid is a much sought-after platform chemical. Malic acid can be produced by microbial fermentation via oxidative/reductive TCA and glyoxylate pathways. This article elaborates the potential and limitations of high malic acid producing native fungi belonging to Aspergillus, Penicillium, Ustilago and Aureobasidium spp. The utilization of industrial side streams and low value renewable substrates such as crude glycerol and lignocellulosic biomass is also discussed with a view to develop a competitive bio-based production process. The major impediments present in the form of toxic compounds from lignocellulosic residues or synthesized during fermentation along with their remedial measures are also described. The article also focuses on production of polymalic acid from renewable substrates which opens up a cost-cutting dimension in production of this biodegradable polymer. Finally, the recent strategies being employed for its production in recombinant organisms have also been covered.

CAM photosynthesis: the acid test.

There is currently considerable interest in the prospects for bioengineering crassulacean acid metabolism (CAM) photosynthesis - or key elements associated with it, such as increased water-use efficiency - into C3 plants. Resolving how CAM photosynthesis evolved from the ancestral C3 pathway could provide valuable insights into the targets for such bioengineering efforts. It has been proposed that the ability to accumulate organic acids at night may be common among C3 plants, and that the transition to CAM might simply require enhancement of pre-existing fluxes, without the need for changes in circadian or diurnal regulation. We show, in a survey encompassing 40 families of vascular plants, that nocturnal acidification is a feature entirely restricted to CAM species. Although many C3 species can synthesize malate during the light period, we argue that the switch to night-time malic acid accumulation requires a fundamental metabolic reprogramming that couples glycolytic breakdown of storage carbohydrate to the process of net dark CO2 fixation. This central element of the CAM pathway, even when expressed at a low level, represents a biochemical capability not seen in C3 plants, and so is better regarded as a discrete evolutionary innovation than as part of a metabolic continuum between C3 and CAM.

Photosynthesis and leaf structure of F1 hybrids between Cymbidium ensifolium (C3) and C. bicolor subsp. pubescens (CAM).

BACKGROUND AND AIMS: The introduction of crassulacean acid metabolism (CAM) into C3 crops has been considered as a means of improving water-use efficiency. In this study, we investigated photosynthetic and leaf structural traits in F1 hybrids between Cymbidium ensifolium (female C3 parent) and C. bicolor subsp. pubescens (male CAM parent) of the Orchidaceae. METHODS: Seven F1 hybrids produced through artificial pollination and in vitro culture were grown in a greenhouse with the parent plants. Structural, biochemical, and physiological traits involved in CAM in their leaves were investigated. KEY RESULTS: Cymbidium ensifolium accumulated very low levels of malate without diel fluctuation, whereas C. bicolor subsp. pubescens showed nocturnal accumulation and diurnal consumption of malate. The F1s also accumulated malate at night, but much less than C. bicolor subsp. pubescens. This feature was consistent with low nocturnal fixation of atmospheric CO2 in the F1s. δ 13C values of the F1s were intermediate between those of the parents. The leaf thickness was thicker in C. bicolor subsp. pubescens than in C. ensifolium, and those of the F1s were more similar to that of C. ensifolium. This was due to the difference in mesophyll cell size. The chloroplast coverage of mesophyll cell perimeter adjacent to intercellular air spaces of C. bicolor subsp. pubescens was lower than that of C. ensifolium, and those of the F1s were intermediate between them. Interestingly, one F1 had structural and physiological traits more similar to those of C. bicolor subsp. pubescens than the other F1s. Nevertheless, all F1s contained intermediate levels of phosphoenolpyruvate carboxylase but as much pyruvate,Pi dikinase as C. bicolor subsp. pubescens. CONCLUSIONS: CAM traits were intricately inherited in the F1 hybrids, the level of CAM expression varied widely among F1 plants, and the CAM traits examined were not necessarily co-ordinately transmitted to the F1s.

"Multidimensional correlation analysis of temperature and contact time on eradication of biofilms of Cronobacter sakazakii on abiotic surfaces by combination of hypochlorite and malic acid".

AIM: In the present study, malic acid in combination with sodium hypochlorite is evaluated for eradication of biofilms formed by Cronobacter sakazakii strains individually and in a cocktail on different abiotic surfaces. METHOD AND RESULTS: The biofilm formation by five strains of C. sakazakii and their cocktail culture on different substrates was studied in Tryptone Soy Broth (TSB) and reconstituted Powdered Infant Formula (PIF). Further, the effect of temperature (4, 27, 37 and 50°C) and contact time (10, 20, 30, 40, 50 and 60 min) on antibiofilm potential of test solution (0.0625 mol l-1 malic acid and 0.00004 mol l-1 sodium hypochlorite) against biofilm formed by C. sakazakii cocktail culture was investigated on these surfaces. The effect was evaluated in terms of viable cell count and biofilm texture using scanning electron microscopy (SEM). Principal Component Analysis (PCA) revealed that the maximum biofilm reduction was observed for stainless steel at 4°C after 60 min of contact whereas at 25, 37 and 50°C, maximum biofilm reduction was observed for polycarbonate. For glass and polyurethane, maximum log reductions were observed at 50°C. The SEM images revealed cell surface deformation and disruption in biofilms after treatment with the test solution. CONCLUSIONS: The antibiofilm potential was observed to be greatly affected by contact time and temperature. These results indicated that the combination of malic acid NaOCl can effectively kill and remove C. sakazakii biofilms from food contact surfaces and enteral feeding tubes.