CRISPR-Cas9 mediated gene deletions in lager yeast <Emphasis Type="Italic">Saccharomyces pastorianus</Emphasis>

Background

The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae.

Results

This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design.

Conclusions

These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.

     

Biomarker of food intake for assessing the consumption of dairy and egg products

Dairy and egg products constitute an important part of Western diets as they represent an excellent source of high-quality proteins, vitamins, minerals and fats. Dairy and egg products are highly diverse and their associations with a range of nutritional and health outcomes are therefore heterogeneous. Such associations are also often weak or debated due to the difficulty in establishing correct assessments of dietary intake. Therefore, in order to better characterize associations between the consumption of these foods and health outcomes, it is important to identify reliable biomarkers of their intake. Biomarkers of food intake (BFIs) provide an accurate measure of intake, which is independent of the memory and sincerity of the subjects as well as of their knowledge about the consumed foods. We have, therefore, conducted a systematic search of the scientific literature to evaluate the current status of potential BFIs for dairy products and BFIs for egg products commonly consumed in Europe. Strikingly, only a limited number of compounds have been reported as markers for the intake of these products and none of them have been sufficiently validated. A series of challenges hinders the identification and validation of BFI for dairy and egg products, in particular, the heterogeneous composition of these foods and the lack of specificity of the markers identified so far. Further studies are, therefore, necessary to validate these compounds and to discover new candidate BFIs. Untargeted metabolomic strategies may allow the identification of novel biomarkers, which, when taken separately or in combination, could be used to assess the intake of dairy and egg products.     

From agro-industrial wastes to single cell oils: a step towards prospective biorefinery

The reserves of fossil-based fuels, which currently seem sufficient to meet the global demands, is inevitably on the verge of exhaustion. Contemporary raw material for alternate fuel like biodiesel is usually edible plant commodity oils, whose increasing public consumption rate raises the need of finding a non-edible and fungible alternate oil source. In this quest, single cell oils (SCO) from oleaginous yeasts and fungi can provide a sustainable alternate of not only functional but also valuable (polyunsaturated fatty acids (PUFA)-rich) lipids. Researches are been increasingly driven towards increasing the SCO yield in order to realize its commercial importance. However, bulk requirement of expensive synthetic carbon substrate, which inflates the overall SCO production cost, is the major limitation towards complete acceptance of this technology. Even though substrate cost minimization could make the SCO production profitable is uncertain, it is still essential to identify suitable cheap and abundant substrates in an attempt to potentially reduce the overall process economy. One of the most sought-after in-expensive carbon reservoirs, agro-industrial wastes, can be an attractive replacement to expensive synthetic carbon substrates in this regard. The present review assess these possibilities referring to the current experimental investigations on oleaginous yeasts, and fungi reported for conversion of agro-industrial feedstocks into triacylglycerols (TAGs) and PUFA-rich lipids. Multiple associated factors regulating lipid accumulation utilizing such substrates and impeding challenges has been analyzed. The review infers that production of bulk oil in combination to high-value fatty acids, co-production strategies for SCO and different microbial metabolites, and reutilization and value addition to spent wastes could possibly leverage the high operating costs and help in commencing a successful biorefinery. Rigorous research is nevertheless required whether it is PUFA-rich oil production (for competing with algal omega oils) or neutral bulk oil production (for overcoming yield limitations and managing process economy) to establish this potential source as future resource.     

Phytoplankton morphological response to the underwater light conditions in a vegetated wetland

This study analyses the influence of the underwater light climate on the morphometric characteristics of the phytoplankton at the population and community levels. The differences in light conditions across the floodplain were mainly defined by the patchiness of floating macrophytes and humic acids concentration. A morphometric response at the community level to the underwater PAR was registered. Sites with strong light constraints were characterised by non-flagellated organisms or with a small proportion of unicellular flagellates. Short organisms (<10 μm) with a unit volume of less than 1,000 μm³ and a high surface:volume ratio (S/V >2) were the morphotypes related to poorly illuminated environments. Moreover, the organisms showed forms more slender under these limiting conditions. This pattern was different to that registered in well-illuminated sites where longer and larger organisms, with a smaller S/V and frequently flagellated, coexisted with the previously mentioned organisms. The autotrophic picoplankton, the smallest phytoplankton fraction, revealed lower abundances at sites with higher humic substances. Short term morphological changes were additionally studied for the dominant species by means of mesocosm experiments simulating different light climates. Intraspecific morphological plasticity was observed with respect to the filament length and the vacuolization of cells. Electronic Supplementary Material Supplementary material is available for this article at and is available to authorized users.     

Biotechnological production of gluconic acid: future implications

Gluconic acid (GA) is a multifunctional carbonic acid regarded as a bulk chemical in the food, feed, beverage, textile, pharmaceutical, and construction industries. The favored production process is submerged fermentation by Aspergillus niger utilizing glucose as a major carbohydrate source, which accompanied product yield of 98%. However, use of GA and its derivatives is currently restricted because of high prices: about US$ 1.20–8.50/kg. Advancements in biotechnology such as screening of microorganisms, immobilization techniques, and modifications in fermentation process for continuous fermentation, including genetic engineering programmes, could lead to cost-effective production of GA. Among alternative carbohydrate sources, sugarcane molasses, grape must show highest GA yield of 95.8%, and banana must may assist reducing the overall cost of GA production. These methodologies would open new markets and increase applications of GA. Authors’ contributions OVS and RK are the sole contributors of this original review article. This review is based upon the published research in the area of gluconic acid fermentation.     

Mapping quantitative trait loci for root development under hypoxia conditions in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Key message

Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method.

Abstract

Waterlogging is a major environmental stress limiting soybean yield in wet parts of the world. Root development is an important indicator of hypoxia tolerance in soybean. However, little is known about the genetic control of root development under hypoxia. This study was conducted to identify quantitative trait loci (QTLs) responsible for root development under hypoxia. Recombinant inbred lines (RILs) developed from a cross between a hypoxia-sensitive cultivar, Tachinagaha, and a tolerant landrace, Iyodaizu, were used. Seedlings were subjected to hypoxia, and root development was evaluated with the value change in root traits between after and before treatments. We found 230 polymorphic markers spanning 2519.2 cM distributed on all 20 chromosomes (Chrs.). Using these, we found 11 QTLs for root length (RL), root length development (RLD), root surface area (RSA), root surface area development (RSAD), root diameter (RD), and change in average root diameter (CARD) on Chrs. 11, 12, 13 and 14, and 7 QTLs for hypoxia tolerance of these root traits. These included QTLs for RLD and RSAD between markers Satt052 and Satt302 on Chr. 12, which are important markers of hypoxia tolerance in soybean; those QTLs were stable between 2 years. To validate the QTLs, we developed a near-isogenic line with the QTL region derived from Iyodaizu. The line performed well under both hypoxia and waterlogging, suggesting that the region contains one or more genes with large effects on root development. These findings may be useful for fine mapping and positional cloning of gene responsible for root development under hypoxia.

     

Radiological workers sensitivity to 50 Hz pulsed magnetic fields: preliminary results

In the present study the effect of extremely low frequency pulsed magnetic fields (PMF) was evaluated in lymphocyte cultures from 12 subjects occupationally exposed to low doses of ionising radiations. The PMF signal characteristics were repetition frequency 50 Hz, triangular shape, rise time about 1.2 ms and peak intensity 2.5 mT. The cytokinesis-block technique was employed to evaluate genotoxicity and cytotoxicity in terms of micronucleus frequency and cell proliferation, respectively. When PMF-exposed cultures were compared with their respective controls, a slight but statistically significant increase was detected in both the biological parameters investigated ( p<0.05). The results obtained suggest a possible role of specific employments involving exposure to ionising radiation, in the risk associated with electromagnetic field exposure.     

Tumor-directed targeting of liposomes

Tumor-specific targeting is a critical goal in the research area of liposomal drug delivery. Identification of the specific interactions between ligands and target tumor cells is a principle prerequisite in achieving this goal. Generally, tumor cells aberrantly express tumor-associated antigens that can be utilized as appropriate target molecules. Monoclonal antibodies against tumor-associated antigens have been successfully adopted for targeting to various types of cancer cells. The incorporation of humanized monoclonal antibodies or single chain human antibodies, instead of rodent antibodies into immunoliposomes has resulted in better clinical applicability. Tumor-specific ligands other than monoclonal antibodies have also been investigated as in vivo tumor-directing molecules. However, the number of pre-clinical studies of anticancer treatments using tumor-specific liposomal drugs reporting successful targeting and enhanced therapeutic efficacy has been limited. Further refinement of tumor-specific interactions and liposomal formulations will be necessary for the application of the tumor-specific liposomal drug strategy for anticancer chemotherapy or gene therapy.     

Capitalizing on multi-element interactions through balanced nutrition — A pathway to improve nitrogen use efficiency in China,India and North America

A viable option for increasing nitrogen (N) use efficiency and mitigation of negative impacts of N on the environment is to capitalize on multi-element interactions through implementation of nutrient management programs that provide balanced nutrition. Numerous studies have demonstrated the immediate efficacy of this approach in the developing regions like China and India as well as developed countries in North America. Based on 241 site-years of experiments in these countries, the first-year N recovery efficiency (RE) for the conventional or check treatments averaged 21% while the balanced treatments averaged 54% RE, for an average increase of 33% in RE due to balanced nutrition. Effective policies to promote adoption are most likely those that enable site-specific approaches to nutrient management decisions rather than sweeping, nation-wide incentives supporting one nutrient over another. Local farmers, advisers and officials need to be empowered with tools and information to help them define necessary changes in practices to create more balanced nutrient management.     

Differential role of microenvironment in microencapsulation for improved cell tolerance to stress

The effect of the microenvironment in alginate–chitosan–alginate (ACA) microcapsules with liquid core (LCM) and solid core (SCM) on the physiology and stress tolerance of Sacchromyces cerevisiae was studied. The suspended cells were used as control. Cells cultured in liquid core microcapsules showed a nearly twofold increase in the intracellular glycerol content, trehalose content, and the superoxide dismutase (SOD) activity, which are stress tolerance substances, while SCM did not cause the significant physiological variation. In accordance with the physiological modification after being challenged with osmotic stress (NaCl), oxidative stress (H₂O₂), ethanol stress, and heat shock stress, the cell survival in LCM was increased. However, SCM can only protect the cells from damaging under ethanol stress. Cells released from LCM were more resistant to hyperosmotic stress, oxidative stress, and heat shock stress than cells liberated from SCM. Based on reasonable analysis, a method was established to estimate the effect of microenvironment of LCM and SCM on the protection of cells against stress factors. It was found that the resistance of LCM to hyperosmotic stress, oxidative stress, and heat shock stress mainly depend on the domestication effect of LCM’s microenvironment. The physical barrier of LCM constituted by alginate–chitosan membrane and liquid alginate matrix separated the cells from the damage of oxidative stress and ethanol stress. The significant tolerance against ethanol stress of SCM attributed to the physical barrier consists of solid alginate–calcium matrix and alginate–chitosan membrane.