Adaptive Laboratory Evolution of Antibiotic Resistance Using Different Selection Regimes Lead to Similar Phenotypes and Genotypes

Antibiotic resistance is a global threat to human health, wherefore it is crucial to study the mechanisms of antibiotic resistance as well as its emergence and dissemination. One way to analyze the acquisition of de novo mutations conferring antibiotic resistance is adaptive laboratory evolution. However, various evolution methods exist that utilize different population sizes, selection strengths, and bottlenecks. While evolution in increasing drug gradients guarantees high-level antibiotic resistance promising to identify the most potent resistance conferring mutations, other selection regimes are simpler to implement and therefore allow higher throughput. The specific regimen of adaptive evolution may have a profound impact on the adapted cell state. Indeed, substantial effects of the selection regime on the resulting geno- and phenotypes have been reported in the literature. In this study we compare the geno- and phenotypes of Escherichia coli after evolution to Amikacin, Piperacillin, and Tetracycline under four different selection regimes. Interestingly, key mutations that confer antibiotic resistance as well as phenotypic changes like collateral sensitivity and cross-resistance emerge independently of the selection regime. Yet, lineages that underwent evolution under mild selection displayed a growth advantage independently of the acquired level of antibiotic resistance compared to lineages adapted under maximal selection in a drug gradient. Our data suggests that even though different selection regimens result in subtle genotypic and phenotypic differences key adaptations appear independently of the selection regime

Dependency of Heterochromatin Domains on Replication Factors

Chromatin structure regulates both genome expression and dynamics in eukaryotes, where large heterochromatic regions are epigenetically silenced through the methylation of histone H3K9, histone deacetylation, and the assembly of repressive complexes. Previous genetic screens with the fission yeast Schizosaccharomyces pombe have led to the identification of key enzymatic activities and structural constituents of heterochromatin. We report here on additional factors discovered by screening a library of deletion mutants for silencing defects at the edge of a heterochromatic domain bound by its natural boundary—the IR-R+ element—or by ectopic boundaries. We found that several components of the DNA replication progression complex (RPC), including Mrc1/Claspin, Mcl1/Ctf4, Swi1/Timeless, Swi3/Tipin, and the FACT subunit Pob3, are essential for robust heterochromatic silencing, as are the ubiquitin ligase components Pof3 and Def1, which have been implicated in the removal of stalled DNA and RNA polymerases from chromatin. Moreover, the search identified the cohesin release factor Wpl1 and the forkhead protein Fkh2, both likely to function through genome organization, the Ssz1 chaperone, the Fkbp39 proline cis-trans isomerase, which acts on histone H3P30 and P38 in Saccharomyces cerevisiae, and the chromatin remodeler Fft3. In addition to their effects in the mating-type region, to varying extents, these factors take part in heterochromatic silencing in pericentromeric regions and telomeres, revealing for many a general effect in heterochromatin. This list of factors provides precious new clues with which to study the spatiotemporal organization and dynamics of heterochromatic regions in connection with DNA replication

Milk without cows – can we produce sustainable milk alternatives?

Our food production system is the single most contributor to climate change, and 80% of its impact is caused by animal farming. Increasing animal protein consumption has triggered environmental concerns about sustainability and climate change, which resonate with ethical and public health issues. Therefore, identifying alternatives for animal products is crucial for the planetary health. Even though plant-based drinks can partly mimic bovine milk, most of them have altered taste, functionality or nutritional value, placing them far from the ideal analogue. Advances in biotechnology provide a promising solution by allowing recombinant production of the essential components of milk, nutritionally highly valuable and bioactive proteins. Moreover, it allows the design and tailoring of the final product so it fits our nutritional and health requirements even better than conventional milk. Caseins are a family of key proteins in milk and play a particularly important role in food traits, including the characteristic stretch, melt and mouthfeel of cheese. When produced recombinantly, host-specific post-translational modifications can alter some crucial physico-chemical properties of caseins such as calcium-binding and micelle formation. In my research project I aim to characterize and optimize different microbial hosts as potential milk protein production platforms not only in terms of yield and titer, but also in regard to specific food-related protein functionality. I will also explore the possibility of in situ recombinant casein production coupled to fermentation of various plant substrates for the development of milk-like food products without or minimal waste generation

Engineering Aspergillus oryzae for production of rapeseed protein isoforms and milk proteins

Rapeseed is a commonly grown crop in Northern Europe. However, extraction of oil from rapeseed forms a large side stream of rapeseed press cake, which has a high protein content and therefore a big potential to feed human mouths. Unfortunately, rapeseed proteins do not have desired functionality in food - they are rather bitter and do not provide a nice texture. The SEEDFOOD project aims to create new fundamental knowledge for developing rapeseed storage proteins into a more suitable ingredient for foods. By microbial production and subsequent purification of rapeseed protein isoforms, this PhD project will supply our collaborators with a reliable and customizable supply of proteins, so that they can work on improving palatability. To achieve this, we will engineer filamentous fungi for protein production. Filamentous fungi, such as Aspergillus oryzae, have the potential to secrete several grams per liter of heterologous protein, significantly more than more commonly used hosts for heterologous protein production such as Escherichia coli and Saccharomyces cerevisiae. Additionally, filamentous fungi have more advanced mechanisms for post-translational modifications to the proteins, which helps with folding, secretion, and functionality. To achieve high titers, a high-throughput rational strain construction method, aided by genome-scale metabolic models, will be used, and genetic engineering tools such as CRISPR-Cas will be utilized. After the production host can produce satisfactory levels of rapeseed protein isoforms, it can also be used to produce different types of protein that can help produce more sustainable and healthy foods, such as caseins and sweet proteins

Synthetic gene expression system enhances recombinant protein production in Aspergillus oryzae

The koji mould Aspergillus oryzae has a long history of use for traditional fermentations in East Asia to make foods such as soy sauce, miso and sake. It is naturally capable of secreting high titres of enzymes, which helped establish the species as a favourite for industrial enzyme production in recent decades. This makes A. oryzae an ideal host for producing heterologous proteins for sustainable novel foods such as meat and dairy alternatives. Previous methods for introducing new genes to A. oryzae relied on random multicopy integration, which can cause unpredictable and variable levels of expression, positional effects, and genome instability. Targeted genetic engineering using CRISPR-Cas has the potential to overcome these issues, but it is challenging to reach high levels of recombinant protein due to a lack of suitable promoters. To address this problem, we have developed a modular system where a synthetic transcription factor is expressed which interacts with an activating domain upstream of a core promoter and gene of interest. We screened a library of thirteen core promoters by measuring fluorescence in A. oryzae spores. The library contains promoters with a wide range of expression levels, the strongest of which reaches over sixfold the expression level of the native strong constitutive TEF1 promoter. This represents a significant step forward in improving the production of heterologous proteins in A. oryzae. <br/

Bacterial resistance to CRISPR-Cas antimicrobials

Abstract In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials