Interspecific hybridization is a valuable tool for developing and improving brewing yeast in a number of industry-relevant aspects. However, the genomes of newly formed hybrids can be unstable. Here, we exploited this trait by adapting four brewing yeast strains, three of which were interspecific lager hybrids with different ploidy levels, to high ethanol concentrations in an attempt to generate variant strains with improved fermentation performance in high-gravity wort. Through a batch fermentation-based adaptation process and selection based on a two-step screening process, we obtained eight variant strains which we compared to the wild-type strains in 2-liter-scale wort fermentations replicating industrial conditions. The results revealed that the adapted variants outperformed the strains from which they were derived, and the majority also possessed several desirable brewing-relevant traits, such as increased ester formation and ethanol tolerance, as well as decreased diacetyl formation. The variants obtained from the polyploid hybrids appeared to show greater improvements in fermentation performance than those derived from diploid strains. Interestingly, it was not only the hybrid strains, but also the parent strain, that appeared to adapt and showed considerable changes in genome size. Genome sequencing and ploidy analysis revealed that changes had occurred at both the chromosome and single nucleotide levels in all variants. Our study demonstrates the possibility of improving lager yeast hybrids through adaptive evolution by generating stable and superior variants that possess traits relevant to industrial lager beer fermentation. Recent studies have shown that hybridization is a valuable tool for creating new and diverse strains of lager yeast. Adaptive evolution is another strain development tool that can be applied in order to improve upon desirable traits. Here, we apply adaptive evolution to newly created lager yeast hybrids by subjecting them to environments containing high ethanol levels. We isolated and characterized a number of adapted variants which possess improved fermentation properties and ethanol tolerance. Genome analysis revealed substantial changes in the variants compared to the original strains. These improved variant strains were produced without any genetic modification and are suitable for industrial lager beer fermentations.

BackgroundThe CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.ResultsIn this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger strain with improved capacity to produce galactarate, a potential chemical building block, was generated.ConclusionsThe developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.Electronic supplementary materialThe online version of this article (10.1186/s40694-019-0066-9) contains supplementary material, which is available to authorized users.

There are no earlier reports with successful isolation of plasma membranes from lignin-forming tissues of conifers. A method to isolate cellular membranes from extracellular lignin-producing tissue-cultured cells and developing xylem of Norway spruce was optimized. Modifications to the homogenization buffer were needed to obtain membranes from these phenolics-rich tissues. Membranes were separated by aqueous polymer two-phase partitioning. Chlorophyll a determination, marker enzyme assays and western blot analyses using antibodies for each membrane type showed that mitochondrial, chloroplastic and to a certain extent also ER and Golgi membranes were efficiently diminished from the upper phase, but tonoplast and plasma membranes distributed evenly between the upper and lower phases. Redox enzymes present in the partially purified membrane fractions were assayed in order to reveal the origin of H(2)O(2) needed for lignification. The membranes of spruce contained enzymes able to generate superoxide in the presence of NAD(P)H. Besides members of the flavodoxin and flavodoxin-like family proteins, cytochrome b5, cytochrome P450 and several stress responsive proteins were identified by nitroblue tetrazolium staining of isoelectric focusing gels and by mass spectrometry. Naphthoquinones juglone and menadione increased superoxide production in activity-stained gels. Some juglone-activated enzymes were preferentially using NADH. With NADH, menadione activated only some of the enzymes that juglone did, whereas with NADPH the activation patterns were identical. Duroquinone, a benzoquinone, did not affect superoxide production. Superoxide dismutase, ascorbate peroxidase, catalase and an acidic class III peroxidase isoenzyme were detected in partially purified spruce membranes. The possible locations and functions of these enzymes are discussed.

Due to the rapidly increasing sequence information on gene variants generated by evolution and our improved abilities to engineer novel biological activities, microbial cells can be evolved for the production of a growing spectrum of compounds. For high productivity, efficient carbon channeling towards the end product is a key element. In large scale production systems the genetic modifications that ensure optimal performance cannot be dependent on plasmid-based regulators, but need to be engineered stably into the host genome. Here we describe a CRISPR/Cas9 mediated high-throughput workflow for combinatorial and multiplexed replacement of native promoters with synthetic promoters and the following high-throughput phenotype characterization in the yeast Saccharomyces cerevisiae. The workflow is demonstrated with three central metabolic genes, ZWF1, PGI1 and TKL1 encoding a glucose-6-phosphate dehydrogenase, phosphoglucose isomerase and transketolase, respectively. The synthetic promoter donor DNA libraries were generated by PCR and transformed to yeast cells. A 50% efficiency was achieved for simultaneous replacement at three individual loci using short 60-bp flanking homology sequences in the donor promoters. Phenotypic strain characterization was validated and demonstrated using liquid handling automation and 150 µl cultivation volume in 96-well plate format. The established workflow offers a robust platform for automated engineering and improvement of yeast strains.

Bioprocess monitoring can improve the understanding and control of biotechnological processes. When analyses are carried out as automated online measurements, manual steps of the analysis procedures are avoided, thus decreasing both the time required for analyses and systematic errors. In this study, an online capillary electrophoresis (CE) system with flow-through sample vial made in-house and action control programming was assembled to monitor carboxylic acid production by Kluyveromyces lactis and Saccharomyces cerevisiae during two different bioreactor cultivations. The relative standard deviations were less than 0.6% for intraday migration times and the total analysis time was less than 20 min. The system operated continuously and automatically up to 6 days and produced data concerning carboxylic acid production during the cultivations. The successful test runs demonstrated that this system has potential for the monitoring of biotechnological processes.