Identification and Construction of Strong Promoters in Yarrowia lipolytica Suitable for Glycerol-Based Bioprocesses.

Yarrowia lipolytica is a non-pathogenic aerobic yeast with numerous industrial biotechnology applications. The organism grows in a wide variety of media, industrial byproducts, and wastes. A need exists for molecular tools to improve heterologous protein expression and pathway reconstitution. In an effort to identify strong native promoters in glycerol-based media, six highly expressed genes were mined from public data, analyzed, and validated. The promoters from the three most highly expressed (H3, ACBP, and TMAL) were cloned upstream of the reporter mCherry in episomal and integrative vectors. Fluorescence was quantified by flow cytometry and promoter strength was benchmarked with known strong promoters (pFBA1in, pEXP1, and pTEF1in) in cells growing in glucose, glycerol, and synthetic glycerol media. The results show that pH3 > pTMAL > pACBP are very strong promoters, with pH3 exceeding all other tested promoters. Hybrid promoters were also constructed, linking the Upstream Activating Sequence 1B (UAS1B8) with H3(260) or TMAL(250) minimal promoters, and compared to the UAS1B8-TEF1(136) promoter. The new hybrid promoters exhibited far superior strength. The novel promoters were utilized to overexpress the lipase LIP2, achieving very high secretion levels. In conclusion, our research identified and characterized several strong Y. lipolytica promoters that expand the capacity to engineer Yarrowia strains and valorize industrial byproducts.

Fish-Derived Protein Hydrolysates Increase Insulin Sensitivity and Alter Intestinal Microbiome in High-Fat-Induced Obese Mice.

Obesity and type 2 diabetes are characterized by low-grade systemic inflammation and glucose intolerance, which can be partially controlled with nutritional interventions. Protein-containing nutritional supplements possess health-promoting benefits. Herein, we examined the effect of dietary supplementation with protein hydrolysates derived from fish sidestreams on obesity and diabetes, utilizing a mouse model of High-Fat Diet-induced obesity and type 2 diabetes. We examined the effect of protein hydrolysates from salmon and mackerel backbone (HSB and HMB, respectively), salmon and mackerel heads (HSH and HMH, respectively), and fish collagen. The results showed that none of the dietary supplements affected weight gain, but HSH partially suppressed glucose intolerance, while HMB and HMH suppressed leptin increase in the adipose tissue. We further analyzed the gut microbiome, which contributes to the metabolic disease implicated in the development of type 2 diabetes, and found that supplementation with selected protein hydrolysates resulted in distinct changes in gut microbiome composition. The most prominent changes occurred when the diet was supplemented with fish collagen since it increased the abundance of beneficial bacteria and restricted the presence of harmful ones. Overall, the results suggest that protein hydrolysates derived from fish sidestreams can be utilized as dietary supplements with significant health benefits in the context of type 2 diabetes and diet-induced changes in the gut microbiome.

Microbial and Biochemical Profile of Different Types of Greek Table Olives.

Analysis of table olives microbiome using next-generation sequencing has enriched the available information about the microbial community composition of this popular fermented food. In this study, 16S and 18S rRNA sequencing was performed on table olives of five Greek popular cultivars, Halkidikis, Thassou, Kalamon, Amfissis, and Konservolia, fermented either by Greek style (in brine or salt-drying) or by Spanish style, in order to evaluate their microbial communities. Moreover, analytical methods were used to evaluate their biochemical properties. The prevailing bacterial species of all olives belonged to Lactobacillaceae, Leuconostocaceae, and Erwiniaceae families, while the most abundant yeasts were of the Pichiaceae family. Principal coordinates analysis showed a clustering of samples cured by salt-drying and of samples stored in brine, regardless of their cultivar. The biochemical evaluation of total phenol content, antioxidant activity, hydroxytyrosol, oleuropein, oleocanthal, and oleacein showed that salt-dried olives had low amounts of hydroxytyrosol, while Spanish-style green olives had the highest amounts of oleocanthal. All the other values exhibited various patterns, implying that more than one factor affects the biochemical identity of the final product. The protocols applied in this study can provide useful insights for the final product, both for the producers and the consumers.

Novel evolved Yarrowia lipolytica strains for enhanced growth and lipid content under high concentrations of crude glycerol.

BACKGROUND: Yarrowia lipolytica is a well-studied oleaginous yeast known for its ability to accumulate and store intracellular lipids, while growing on diverse, non-conventional substrates. Amongst them, crude glycerol, a low-cost by-product of the biodiesel industry, appears to be an interesting option for scaling up a sustainable single-cell oil production process. Adaptive laboratory evolution (ALE) is a powerful tool to force metabolic adaptations endowing tolerance to stressful environmental conditions, generating superior phenotypes with industrial relevance. RESULTS: Y. lipolytica MUCL 28849 underwent ALE in a synthetic medium with increasing concentration of pure or crude glycerol as a stressing factor (9-20% v/v) for 520 generations. In one case of pure glycerol, chemical mutagenesis with ethyl methanesulfonate (EMS) was applied prior to ALE. Growth profile, biomass production and lipid content of 660 evolved strains (EVS), revealed 5 superior isolates; exhibiting from 1.9 to 3.6-fold increase of dry biomass and from 1.1 to 1.6-fold increase of lipid concentration compared to the parental strain, when grown in 15% v/v crude glycerol. NGS for differential gene expression analysis, showed induced expression in all EVS affecting nucleosomal structure and regulation of transcription. As strains differentiated, further changes accumulated in membrane transport and protein transport processes. Genes involved in glycerol catabolism and triacylglycerol biosynthesis were overexpressed in two EVS. Mismatches and gaps in the expressed sequences identified altered splicing and mutations in the EVS, with most of them, affecting different components of septin ring formation in the budding process. The selected YLE155 EVS, used for scale-up cultivation in a 3L benchtop bioreactor with 20% v/v crude glycerol, achieved extended exponential phase, twofold increase of dry biomass and lipid yields at 48 h, while citric acid secretion and glycerol consumption rates were 40% and 50% lower, respectively, compared to the parental strain, after 24 h of cultivation. CONCLUSION: ALE and EMS-ALE under increasing concentrations of pure or crude glycerol generated novel Y. lipolytica strains with enhanced biomass and lipid content. Differential gene expression analysis and scale-up of YLE155, illustrated the potential of the evolved strains to serve as suitable "chassis" for rational engineering approaches towards both increased lipid accumulation, and production of high-added value compounds, through efficient utilization of crude glycerol.

Genomic data of two Greek Vitis varieties.

The genetic material of Vitis varieties is crucial for the wine sector. In addition, genomic technologies applied in vitis germplasm characterization are important for the conservation of indigenous genetic reservoirs. Until recently the most common method to genetically identify vitis varieties was the use of Simple Sequence Repeats (SSR) along with SNP chips. Yet, with the progress in Next Generation Sequencing (NGS) technologies and the reduced sequencing cost per base, a twist in plant species genetic identification methods has occurred. Among them, the low coverage Whole-Genome Sequencing (lcWGS) method with downstream bioinformatic analysis for variant discovery and phylogenetic characterization is gaining scientific attention. In this dataset, shotgun sequencing data of two different Greek Vitis varieties, 'Razaki' and 'Vlachiko' are presented. Vitis cultivars were collected from the Aristotle University of Thessaloniki's (AUTH) ampelographic collection and have been previously phenotypically and genetically characterized. WGS libraries were sequenced on an IlluminaⓇ NovaSeq 6000 platform with the IlluminaⓇ NovaSeq 6000 S2 Reagent Kit (300 cycles). Raw sequence data used for analysis are available in NCBI under the Sequence Read Archive (SRA), with BioProject ID PRJNA805368. Reads were aligned to the reference genome of Vitis vinifera available from the EnsemblPlants database and formal analysis was conducted with the Genome Analysis Toolkit 4 (GATK4) pipeline. Data can be used to enrich our knowledge related to the genetic background of vitis cultivars and can also serve as a threshold in the scientific community towards the construction of a genomic database of vitis cultivars.

Effects of Magnesium Oxide and Magnesium Hydroxide Microparticle Foliar Treatment on Tomato PR Gene Expression and Leaf Microbiome.

Recently, metal oxides and magnesium hydroxide nanoparticles (NPs) with high surface-to-volume ratios were shown to possess antibacterial properties with applications in biomedicine and agriculture. To assess recent observations from field trials on tomatoes showing resistance to pathogen attacks, porous micron-scale particles composed of nano-grains of MgO were hydrated and sprayed on the leaves of healthy tomato (Solanum lycopersicum) plants in a 20-day program. The results showed that the spray induced (a) a modest and selective stress gene response that was consistent with the absence of phytotoxicity and the production of salicylic acid as a signalling response to pathogens; (b) a shift of the phylloplane microbiota from near 100% dominance by Gram (-) bacteria, leaving extremophiles and cyanobacteria to cover the void; and (c) a response of the fungal leaf phylloplane that showed that the leaf epiphytome was unchanged but the fungal load was reduced by about 70%. The direct microbiome changes together with the low level priming of the plant's immune system may explain the previously observed resistance to pathogen assaults in field tomato plants sprayed with the same hydrated porous micron-scale particles.

Diet Supplementation with Fish-Derived Extracts Suppresses Diabetes and Modulates Intestinal Microbiome in a Murine Model of Diet-Induced Obesity.

Metabolic syndrome-related diseases affect millions of people worldwide. It is well established that changes in nutritional habits and lifestyle can improve or prevent metabolic-related pathologies such as type-2 diabetes and obesity. Previous reports have shown that nutritional supplements have the capacity to limit glucose intolerance and suppress diabetes development. In this study, we investigated the effect of dietary supplementation with fish-derived extracts on obesity and type 2 diabetes and their impact on gut microbial composition. We showed that nutritional supplements containing Fish Complex (FC), Fish Complex combined with Cod Powder (FC + CP), or Cod Powder combined with Collagen (CP + C) improved glucose intolerance, independent of abdominal fat accumulation, in a mouse model of diet-induced obesity and type 2 diabetes. In addition, collagen-containing supplements distinctly modulate the gut microbiome in high-fat induced obesity in mice. Our results suggest that fish-derived supplements suppress diet-induced type 2 diabetes, which may be partly mediated through changes in the gut microbiome. Thus, fish-derived supplements and particularly the ones containing fish collagen have potential beneficial properties as dietary supplements in managing type 2 diabetes and metabolic syndrome via modulation of the gut microbiome.