Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

Selection by UV Mutagenesis and Physiological Characterization of Mutant Strains of the Yeast Saprochaete suaveolens (Former Geotrichum fragrans) with Higher Capacity to Produce Flavor Compounds

Yeast volatile organic compounds (VOCs), i.e. low molecular weight organic acids, alcohols and esters, are considered as potential and sustainable sources of natural aromas that can replace commonly used artificial flavors in food and other industrial sectors. Although research generally focuses on the yeast Saccharomyces cerevisiae, other so-called unconventional yeasts (NCY) are beginning to attract the attention of researchers, particularly for their ability to produce alternative panels of VOCs. With this respect, a Saprochaete suaveolens strain isolated from dragon fruit in Reunion Island was shown to produce α-unsaturated esters from branched-chain amino acids (BCAAs) such as isobutyl, isoamyl or ethyl tiglate, which are rarely found in other yeasts strains. Given that β-oxidation allows the growth of S. suaveolens on BCAAs as sole carbon source, we developped a method based on UV mutagenesis to generate mutants that can no longer grow on BCAAs, while redirecting the carbon flow towards esterification of α-unsaturated esters. Among the 15,000 clones generated through UV irradiation, we identified nine clones unable to grow on BCAAs with one of them able to produce eight times more VOCs as compared to the wild-type strain. This higher production of α-unsaturated esters in this mutant strain coincided with an almost complete loss of enoyl-CoA hydratase activity of the β-oxidation pathways and with a twofold increase of acyl-CoA hydrolase with not significant changes in the enzymes of the Ehrlich pathway. Moreover, from our knowledge, it constituted the first example of VOCs enhancement in a microbial strain by UV mutagenesis.

Selection of Three Indigenous Lebanese Yeast Saccharomyces cerevisiae with Physiological Traits from Grape Varieties in Western Semi-Desert and Pedoclimatic Conditions in the Bekaa Valley

Wine production depends on the fermentation process performed by yeasts, especially (but not solely) strains of the species Saccharomyces cerevisiae, which is a technique that has been practiced from the Middle Ages till modern days. Selecting indigenous starters offers a beneficial technique to manage alcoholic grape juice fermentation, conserving the particular sensory qualities of wine produced from specific regions. This paper investigated yeast biodiversity of four grape varieties (Carignan, Syrah, Grenache, and Aswad Karesh) grown in the pedoclimatic western semi-desert Bekaa Valley. Further research identified, characterized, and selected strains with the most industrial wine interest and economic value to Lebanon. By using molecular methods and by the ITS PCR analysis, the isolates belonging to the Saccharomyces and non-Saccharomyces genus were identified. These isolates taken from four varieties were further characterized by amplification with Interdelta and δ12/δ21 primer pairs, permitting the identification of 96 S. cerevisiae strains. Forty-five genomically homogenous groups were classified through the comparison between their mtDNA RFLP patterns. Based on physiological characterization analysis (H2S and SO2 production, killer phenotype, sugar consumption, malic and acetic acid, etc.), three strains (NL28629, NL28649, and NL28652) showed interesting features, where they were also vigorously fermented in a synthetic medium. These strains can be used as a convenient starter for typical wine production. In particular, Carignan and Syrah had the highest percentage of strains with the most desirable physiological parameters.

Genetic Variability of Tunisian Faba Beans (Vicia faba L.) Based on Seeds’ Morphophysical Properties as Assessed by Statistical Analysis

Faba bean (Vicia faba L.) is a legume crop cultivated for its nutritious seeds that are an important worldwide source of human food and feed. Seeds characterization is a prerequisite step for faba bean quality improvement. The morphophysical characterization of the seeds of twenty-four local faba bean accessions following the UPOV descriptors and the AOAC International standards was carried out and assessed with an approach based on Euclidean statistical model. “205 Bulk” was the unique accession harboring white hilum color seed which is linked to low convicine grain content. Irregular seed shape was the most observed character among the studied accessions except “Badii” displaying an elliptical seed shape; therefore, seed shape did not allow discrimination within our Tunisian germplasm. Interestingly, the physical characters of the seeds showed significant diversity between the accessions for all the measured parameters. A highly significant variability was observed for axial, length, and width dimensions of seeds, with “Memdouh” being the longest and largest seed accession, whereas “01-02” was the shortest and narrowest. Classification of the studied faba bean germplasm accessions based on morphophysical characters using clustering by Euclidean distance revealed three different groups. Moreover, multivariate PCA analysis further classified the faba bean accessions into four main clusters. Correlation study performed by using Spearman’s test established positive correlations within physical parameters of seeds such as between mean length and mean width of seeds. Therefore, using morphophysical parameters screening, valuable phenotypes have been selected for deeper physiological characterization and further breeding programs.

Isolation and characterization of soil actinobacteria as cellulolytic enzyme producer from Aceh Besar, Indonesia

Fitri L, Bessania MA, Septi N, Suhartono S. 2021. Isolation and characterization of soil actinobacteria as cellulolytic enzyme producer from Aceh Besar, Indonesia. Biodiversitas 22: 5169-5180. Cellulolytic actinobacteria are cellulase-producing bacteria capable of degrading cellulose. This study aimed to isolate, characterize, evaluate the cellulolytic ability, and to determine physiological characterization of soil cellulolytic actinobacteria isolated from the Ujung Pancu area, Aceh Besar. Isolation of actinobacteria from soil samples was performed using serial dilution method on Yeast Malt Agar (YMA) medium. Morphological characterization was carried out by growing isolates on YMA, Oatmeal Agar (OA), and Yeast Starch Agar (YSA) media. Cellulolytic ability was determined by calculating the cellulolytic index (IS) on 1% carboxymethyl cellulose (CMC) medium after adding 0.1% congo red solution. Physiological characterization of cellulolytic actinobacteria tested in this study was salinity, pH, and carbon source in liquid Yeast Malt (liquid YM), and the growth was measured at a wavelength of 581nm. The results showed that a total of nine isolates of actinobacteria were isolated, which belonged to the genus Streptomyces. Cellulolytic test results showed that eight isolates had the ability to degrade cellulose. Isolates AUP-04, AUP-03, and AUP-01 had the highest cellulolytic index value. Physiological characterization results revealed that three isolates had different tolerances for salinity levels, pH, and types of carbon sources. AUP-03 isolate grew well at 10% salinity with an OD value of 0.88, isolate AUP-01 grew at 5% salinity with an OD value of 0.49, whereas isolate AUP-04 grew well on media that did not contain salinity. All three isolates grew well at pH 6 with OD values of 0.93, 1.12, and 1.27. AUP-03 and AUP-01 isolates grew well on media containing dextrose as carbon source with OD values of 0.154 and 0.17, respectively, while isolate AUP-04 grew well on glucose-containing media with an OD value of 0.22.

Genetic and physiological characterization of sunflower resistance provided by the wild-derived OrDeb2 gene against highly virulent races of Orobanche cumana Wallr

Key message OrDeb2 confers post-attachment resistance to Orobanche cumana and is located in a 1.38 Mbp genomic interval containing a cluster of receptor-like kinase and receptor-like protein genes with nine high-confidence candidates. Abstract Sunflower broomrape is a holoparasitic angiosperm that parasitizes on sunflower roots, severely constraining crop yield. Breeding for resistance is the most effective method of control. OrDeb2 is a dominant resistance gene introgressed into cultivated sunflower from a wild-related species that confers resistance to highly virulent broomrape races. The objectives of this study were as follows: (i) locate OrDeb2 into the sunflower genome and determine putative candidate genes and (ii) characterize its underlying resistance mechanism. A segregating population from a cross between the sunflower resistant line DEB2, carrying OrDeb2, and a susceptible line was phenotyped for broomrape resistance in four experiments, including different environments and two broomrape races (FGV and GTK). This population was also densely genotyped with microsatellite and SNP markers, which allowed locating OrDeb2 within a 0.9 cM interval in the upper half of Chromosome 4. This interval corresponded to a 1.38 Mbp genomic region of the sunflower reference genome that contained a cluster of genes encoding LRR (leucine-rich repeat) receptor-like proteins lacking a cytoplasmic kinase domain and receptor-like kinases with one or two kinase domains and lacking an extracellular LRR region, which were valuable candidates for OrDeb2. Rhizotron and histological studies showed that OrDeb2 determines a post-attachment resistance response that blocks O. cumana development mainly at the cortex before the establishment of host-parasite vascular connections. This study will contribute to understand the interaction between crops and parasitic weeds, to establish durable breeding strategies based on genetic resistance and provide useful tools for marker-assisted selection and OrDeb2 map-based cloning.

Heightened facial muscle reactivity in preadolescent girls with pathological anxiety

Anticipatory anxiety and heightened responses to uncertainty are central features of anxiety disorders (ADs). We examined facial emotional responding in a sample of preadolescent girls with a range of anxiety symptoms: no/low anxiety (controls) to subthreshold anxiety (subthreshold-AD) to DSM-5 diagnoses of separation, social, and/or generalized ADs. Using a threat anticipation paradigm, we assessed how variations in image valence (negative vs. neutral) and image anticipation (uncertain vs. certain timing) impacted activity of the corrugator supercilii, a forehead muscle implicated in the ‘frown’ response that modulates to emotional stimuli (negative>neutral). Average corrugator magnitude and corrugator time-course were compared between groups. Findings demonstrate greater corrugator activity during anticipation and viewing of negative stimuli, with overall increased corrugator reactivity in subthreshold-AD and AD girls. Time-course analyses revealed anxiety-related sustained corrugator activity during uncertain anticipation of negative images. Results extend the physiological characterization of childhood pathological anxiety, highlighting the impact of subthreshold-AD symptoms.