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Polyhydroxyalkanoates (PHAs) are green and sustainable bioplastics that could replace petrochemical synthetic plastics without posing environmental threats to living organisms. In addition, sustainable PHA production could be achieved using marine photosynthetic purple nonsulfur bacteria (PNSBs) that utilize natural seawater, sunlight, carbon dioxide gas, and nitrogen gas for growth. However, PHA production using marine photosynthetic PNSBs has not been economically feasible yet due to its high cost and low productivity. In this work, strain improvement, using genome-wide mutagenesis coupled with high-throughput screening via fluorescence-activated cell sorting, we were able to create Rhodovulum sulfidophilum mutants with enhanced volumetric PHA productivity, with an up to 1.7-fold increase. The best selected mutants (E6 and E6M4) reached the stationary growth phase 1 day faster and accumulated the maximum PHA content 2 days faster than the wild type. Maximizing volumetric PHA productivity before the stationary growth phase is indeed an additional advantage for R. sulfidophilum as a growth-associated PHA producer.

open, Actinomycetes and Fungi are frequently organized in small integrated societies (exemplified by colonies) in which a division of labor occurs. The division of labor has the evolutionary outcome of giving a selective advantage to the growing cells and finally to the fertile progeny. The fertile progeny has the task to preserve the species within the limits of natural evolution and colonization of new niches. A considerable part of the population assembled in a fungus or actinomycete colony (up to 80%) is fated to dye since it cannot embed its genome in spores. We have suggested that, in natural microbial isolates, a common characteristic is to segregate phenotypes in which the production of secondary metabolites and enzymes is maximized in the part of the colony which has lost the capacity to reproduce itself (the sterile caste), while reproduction is delegated to another part of the population (the non-sterile caste, generally represented by spores or other durable cells). Based on the above statements, we were digging in the sterile caste having as goal the identification, characterization and long-term preservation of industrially relevant lineages. In mycelial and polynuclear microorganisms, as fungi and actinomycetes are, simple replication of colonies, or plating by dilution, did not easily allow the separation of genomes. Therefore, to correctly separate the lineages of interest, we have approached the selection of the sterile caste by use of three different techniques: morphological selection of those phenotypes having lost the spore-producing ability (study performed on fungi and actinomycetes), the selection of the sterile caste by use of selective agents (applied to fungi and actinomycetes) and the separation of genomes by massive screening of clones produced with the protoplast technique. More in detail, we used the antibiotic A40926 as selective agent for the identification of high producers of the A40926 glycopeptide (the natural precursor of the clinica, Gli attinomiceti ed i funghi sono spesso organizzati in piccole società integrate (esemplificate da colonie) caratterizzate da una divisione del lavoro. La divisione del lavoro ha il riscontro evolutivo di dare un vantaggio selettivo alle cellule in accrescimento ed in ultima analisi alla progenie fertile. La progenie fertile ha il compito di preservare la specie nei limiti dell'evoluzione naturale e della colonizzazione di nuove nicchie. Una parte considerevole della popolazione riunita in una colonia di funghi o di actinomiceti (fino all'80%) è destinata a soccombere poiché non può incorporare il proprio genoma nelle spore. Abbiamo suggerito che, partendo da isolati microbici naturali, una caratteristica comune di funghi e batteri filamentosi è quella di segregare fenotipi in cui la produzione di metaboliti secondari ed enzimi è massimizzata in una parte della colonia che ha perso la capacità di riprodursi (la casta sterile), mentre la riproduzione è delegata ad un'altra parte della popolazione (la casta non sterile, generalmente rappresentata da spore o altre cellule durevoli). Sulla base di quanto sopra, abbiamo rivolto i nostri sforzi alla casta sterile avendo come obiettivo l'identificazione, la caratterizzazione e la conservazione a lungo termine di popolazioni clonali industrialmente rilevanti. Tuttavia, nei microrganismi miceliari e polinucleati, come i funghi e gli attinomiceti, la semplice replicazione delle colonie o l’isolamento per diluizione, non consentono la efficace separazione dei genomi. Al fine di separare correttamente le popolazioni clonali d’interesse, ci siamo dedicati alla selezione della casta sterile mediante l'utilizzo di tre diverse tecniche: selezione morfologica di quei fenotipi che hanno perso la capacità di produrre spore (studio eseguito su funghi e attinomiceti e su Bacillus subtilis), la selezione della casta sterile mediante l'utilizzo di agenti selettivi (applicato a funghi ed attinomiceti) e la separazione dei genomi mediante, 0, open, Beltrametti, F

Gli attinomiceti ed i funghi sono spesso organizzati in piccole società integrate (esemplificate da colonie) caratterizzate da una divisione del lavoro. La divisione del lavoro ha il riscontro evolutivo di dare un vantaggio selettivo alle cellule in accrescimento ed in ultima analisi alla progenie fertile. La progenie fertile ha il compito di preservare la specie nei limiti dell'evoluzione naturale e della colonizzazione di nuove nicchie. Una parte considerevole della popolazione riunita in una colonia di funghi o di actinomiceti (fino all'80%) è destinata a soccombere poiché non può incorporare il proprio genoma nelle spore. Abbiamo suggerito che, partendo da isolati microbici naturali, una caratteristica comune di funghi e batteri filamentosi è quella di segregare fenotipi in cui la produzione di metaboliti secondari ed enzimi è massimizzata in una parte della colonia che ha perso la capacità di riprodursi (la casta sterile), mentre la riproduzione è delegata ad un'altra parte della popolazione (la casta non sterile, generalmente rappresentata da spore o altre cellule durevoli). Sulla base di quanto sopra, abbiamo rivolto i nostri sforzi alla casta sterile avendo come obiettivo l'identificazione, la caratterizzazione e la conservazione a lungo termine di popolazioni clonali industrialmente rilevanti. Tuttavia, nei microrganismi miceliari e polinucleati, come i funghi e gli attinomiceti, la semplice replicazione delle colonie o l’isolamento per diluizione, non consentono la efficace separazione dei genomi. Al fine di separare correttamente le popolazioni clonali d’interesse, ci siamo dedicati alla selezione della casta sterile mediante l'utilizzo di tre diverse tecniche: selezione morfologica di quei fenotipi che hanno perso la capacità di produrre spore (studio eseguito su funghi e attinomiceti e su Bacillus subtilis), la selezione della casta sterile mediante l'utilizzo di agenti selettivi (applicato a funghi ed attinomiceti) e la separazione dei genomi, Actinomycetes and Fungi are frequently organized in small integrated societies (exemplified by colonies) in which a division of labor occurs. The division of labor has the evolutionary outcome of giving a selective advantage to the growing cells and finally to the fertile progeny. The fertile progeny has the task to preserve the species within the limits of natural evolution and colonization of new niches. A considerable part of the population assembled in a fungus or actinomycete colony (up to 80%) is fated to dye since it cannot embed its genome in spores. We have suggested that, in natural microbial isolates, a common characteristic is to segregate phenotypes in which the production of secondary metabolites and enzymes is maximized in the part of the colony which has lost the capacity to reproduce itself (the sterile caste), while reproduction is delegated to another part of the population (the non-sterile caste, generally represented by spores or other durable cells). Based on the above statements, we were digging in the sterile caste having as goal the identification, characterization and long-term preservation of industrially relevant lineages. In mycelial and polynuclear microorganisms, as fungi and actinomycetes are, simple replication of colonies, or plating by dilution, did not easily allow the separation of genomes. Therefore, to correctly separate the lineages of interest, we have approached the selection of the sterile caste by use of three different techniques: morphological selection of those phenotypes having lost the spore-producing ability (study performed on fungi and actinomycetes), the selection of the sterile caste by use of selective agents (applied to fungi and actinomycetes) and the separation of genomes by massive screening of clones produced with the protoplast technique. More in detail, we used the antibiotic A40926 as selective agent for the identification of high producers of the A40926 glycopeptide (the natural precursor of the clinica

The yeast Saccharomyces pastorianus is responsible for the annual worldwide production of almost 200 billion liters of lager-type beer. S. pastorianus is a hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been studied for well over a century. Scientific interest in S. pastorianus intensified upon the discovery, in 2011, of its S. eubayanus ancestor. Moreover, advances in whole-genome sequencing and genome editing now enable deeper exploration of the complex hybrid and aneuploid genome architectures of S. pastorianus strains. These developments not only provide novel insights into the emergence and domestication of S. pastorianus but also generate new opportunities for its industrial application. This review paper combines historical, technical and socioeconomic perspectives to analyze the evolutionary origin and genetics of S. pastorianus. In addition, it provides an overview of available methods for industrial strain improvement and an outlook on future industrial application of lager-brewing yeasts. Particular attention is given to the ongoing debate on whether current S. pastorianus originates from a single or multiple hybridization events and to the potential role of genome editing in developing industrial brewing yeast strains., BT/Industrial Microbiology, BT/Biotechnology

Protoplasts are microbial or vegetable cells lacking a cell wall. These can be obtained from microalgae by an enzymatic hydrolysis process in the presence of an osmotic stabilizer. In general, protoplasts are experimentally useful in physiological, geneticand bio-chemical studies, so their acquisition and fusion will continue to be an active research area in modern biotechnology. The fusion of protoplasts in microalgae constitutes a tool for strain improvement because it allows both intra and interspecific genetic recombina-tion, resulting in organisms with new or improved characteristics of industrial interest. In this review we briefly describe themethod-ology for obtaining protoplasts, as well as fusion methods and the main applications of microalgal platforms., Los protoplastos son células microbianas o vegetales que carecen de pared celular. Estos pueden obtenerse a partir de microalgaspor un proceso de hidrólisis enzimática en presencia de un estabilizador osmótico. En general, los protoplastos son experimental-mente útiles en estudios fisiológicos, genéticos y bioquímicos, por lo que su obtención y fusión continuarán siendo un área de in-vestigación activa en la biotecnología moderna. La fusión de protoplastos en microalgas constituye una herramienta para el mejora-miento de cepas pues permite la recombinación genética intra e interespecífica, logrando así organismos con nuevas características de interés industrial. En esta revisión, describimos brevemente la metodología para obtener protoplastos, métodos de fusión ylas principales aplicaciones de las plataformas basadas en microalgas.

Protoplasts are microbial or vegetable cells lacking a cell wall. These can be obtained from microalgae by an enzymatic hydrolysis process in the presence of an osmotic stabilizer. In general, protoplasts are experimentally useful in physiological, genetic and biochemical studies, so their acquisition and fusion will continue to be an active research area in modern biotechnology. The fusion of protoplasts in microalgae constitutes a tool for strain improvement because it allows both intra and interspecific genetic recombination, resulting in organisms with new or improved characteristics of industrial interest. In this review we briefly describe the methodology for obtaining protoplasts, as well as fusion methods and the main applications of microalgal platforms., Los protoplastos son células microbianas o vegetales que carecen de pared celular. Estos pueden obtenerse a partir de microalgas por un proceso de hidrólisis enzimática en presencia de un estabilizador osmótico. En general, los protoplastos son experimentalmente útiles en estudios fisiológicos, genéticos y bioquímicos, por lo que su obtención y fusión continuarán siendo un área de investigación activa en la biotecnología moderna. La fusión de protoplastos en microalgas constituye una herramienta para el mejoramiento de cepas pues permite la recombinación genética intra e interespecífica, logrando así organismos con nuevas características de interés industrial. En esta revisión, describimos brevemente la metodología para obtener protoplastos, métodos de fusión y las principales aplicaciones de las plataformas basadas en microalgas.

Protoplasts are microbial or vegetable cells lacking a cell wall. These can be obtained from microalgae by an enzymatic hydrolysis process in the presence of an osmotic stabilizer. In general, protoplasts are experimentally useful in physiological, genetic and biochemical studies, so their acquisition and fusion will continue to be an active research area in modern biotechnology. The fusion of protoplasts in microalgae constitutes a tool for strain improvement because it allows both intra and interspecific genetic recombination, resulting in organisms with new or improved characteristics of industrial interest. In this review we briefly describe the methodology for obtaining protoplasts, as well as fusion methods and the main applications of microalgal platforms., Los protoplastos son células microbianas o vegetales que carecen de pared celular. Estos pueden obtenerse a partir de microalgas por un proceso de hidrólisis enzimática en presencia de un estabilizador osmótico. En general, los protoplastos son experimentalmente útiles en estudios fisiológicos, genéticos y bioquímicos, por lo que su obtención y fusión continuarán siendo un área de investigación activa en la biotecnología moderna. La fusión de protoplastos en microalgas constituye una herramienta para el mejoramiento de cepas pues permite la recombinación genética intra e interespecífica, logrando así organismos con nuevas características de interés industrial. En esta revisión, describimos brevemente la metodología para obtener protoplastos, métodos de fusión y las principales aplicaciones de las plataformas basadas en microalgas.

Protoplasts are microbial or vegetable cells lacking a cell wall. These can be obtained from microalgae by an enzymatic hydrolysis process in the presence of an osmotic stabilizer. In general, protoplasts are experimentally useful in physiological, genetic and biochemical studies, so their acquisition and fusion will continue to be an active research area in modern biotechnology. The fusion of protoplasts in microalgae constitutes a tool for strain improvement because it allows both intra and interspecific genetic recombination, resulting in organisms with new or improved characteristics of industrial interest. In this review we briefly describe the methodology for obtaining protoplasts, as well as fusion methods and the main applications of microalgal platforms., Los protoplastos son células microbianas o vegetales que carecen de pared celular. Estos pueden obtenerse a partir de microalgas por un proceso de hidrólisis enzimática en presencia de un estabilizador osmótico. En general, los protoplastos son experimentalmente útiles en estudios fisiológicos, genéticos y bioquímicos, por lo que su obtención y fusión continuarán siendo un área de investigación activa en la biotecnología moderna. La fusión de protoplastos en microalgas constituye una herramienta para el mejoramiento de cepas pues permite la recombinación genética intra e interespecífica, logrando así organismos con nuevas características de interés industrial. En esta revisión, describimos brevemente la metodología para obtener protoplastos, métodos de fusión y las principales aplicaciones de las plataformas basadas en microalgas.

The yeast Saccharomyces pastorianus is responsible for the annual worldwide production of almost 200 billion liters of lager-type beer. S. pastorianus is a hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been studied for well over a century. Scientific interest in S. pastorianus intensified upon the discovery, in 2011, of its S. eubayanus ancestor. Moreover, advances in whole-genome sequencing and genome editing now enable deeper exploration of the complex hybrid and aneuploid genome architectures of S. pastorianus strains. These developments not only provide novel insights into the emergence and domestication of S. pastorianus but also generate new opportunities for its industrial application. This review paper combines historical, technical and socioeconomic perspectives to analyze the evolutionary origin and genetics of S. pastorianus. In addition, it provides an overview of available methods for industrial strain improvement and an outlook on future industrial application of lager-brewing yeasts. Particular attention is given to the ongoing debate on whether current S. pastorianus originates from a single or multiple hybridization events and to the potential role of genome editing in developing industrial brewing yeast strains., BT/Industriele Microbiologie, BT/Biotechnologie

Strain development is frequently used to improve the performance and functionality of industrially important microbes. As traditional mutagenesis screen is especially utilized by the food industry to improve strains used in food fermentation, high-throughput and cost-effective screening tools are important in mutant selection. The emerging droplet-based microfluidics technology miniaturizes the volume for cell cultivation and phenotype interrogation down to the pico-liter scales, which facilitates screening of microbes for improved phenotypical properties tremendously. In this mini-review, we present recent application of the droplet-based microfluidics in microbial strain improvement with a focus on its potential use in the screening of lactic acid bacteria.

Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes., BT/Industrial Microbiology

A key issue for lowering the cost of farming microalgae was explored herein, which is the requirement to utilise a strain possessing the host of desirable farming traits. Wild strains are not fully geared for farming which translates into higher costs. This may be avoidable if microalgae were more amenable to improvement. It was demonstrated herein that evolutionary engineering of Chlamydomonas reinhardtii can bring amenability and domestication whereby novel methods were used to develop salinity tolerance and low light absorbance. C. reinhardtii was selected as the subject species due to several reasons, although most notably because of its superior sexual capabilities that place it as a clear front runner for domestication. The traits of focus in this thesis was salinity tolerance and low chlorophyll content; the former being chosen, for among other things, the clear need to use seawater for a cheap economical media for mass culture, and it being a simple trait to demonstrate the effectiveness of breeding. The latter trait being chosen due to the requirement for better solar conversions efficiencies in mass culture, and the current lack of an effective selection strategy for this important trait. With salinity tolerance, the breeding strategy of genome shuffling was successfully employed to increase NaCl growth tolerance in liquid culture from a maximum of 300 mM to a maximum of 700 mM. Prior to executing the breeding program, investigations into method development was required due to a lack of information available in the literature. These investigations focused on the optimisation of sexual efficiency in populations of C. reinhardtii, which are already sexually mixed. Further to this, investigations into the inherent salt tolerance and mutagenicity of C. reinhardtii were conducted. The methods derived from these investigations culminated into a simple and effective published genome shuffling program with very successful outcomes. With low chlorophyll content, a

Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes., BT/Industriele Microbiologie

Our goal in the present study was to evaluate the potential for lipid production of two cell populations of the marine microalgae Chlorococcum littorale under different climate conditions. We selected, in a previous study and via fluorescence activated cell sorting (FACS), a new cell population of Chlorococcum littorale, namely S5. S5 showed a stable doubled triacylglycerol (TAG) productivity in comparison with the original population. A previously developed model was expanded to include day:night cycles and validated to predict biomass and outdoor TAG productivities at different locations. Four different locations were chosen to simulate the response of C. littorale to different day lengths and light intensities (the Netherlands, Norway, Brazil and Spain). Indoor experiments (simulated summer) were carried out with Original and S5, showing that S5 had a doubled TAG productivity under N-starvation. Finally, simulations of biomass and TAG productivities of Original and S5 at different locations were performed. At locations with lower light intensities, Norway and the Netherland s, biomass productivities were higher than at locations with higher light intensities, Brazil/Spain. Such results might be associated with light-saturation effects. TAG productivities, however, showed no effect of local light intensity. Locations at higher latitudes, Norway/Netherlands, cannot sustain phototrophic year-round production, hence, the yearly average TAG productivities were doubled in Brazil/Spain (from 1.4–1.6 to 3.0–3.2 g m − 2 d − 1 ). Likewise, C. littorale S5 was simulated with doubled TAG productivities when compared with Original, at all locations (2.5–2.7 (low light) to 4.7–5.2 g m − 2 d − 1 (high light)). The present results confirm the industrial potential of Chlorococcum littorale, both Original and S5, as a source of TAG. Furthermore, our results can be used for comparison and to estimate future production scenarios.

Despite extensive research in the last decades, microalgae are still only economically feasible for high valued markets. Strain improvement is a strategy to increase productivities, hence reducing costs. In this work, we focus on microalgae selection: taking advantage of the natural biological variability of species to select variations based on desired characteristics. We focused on triacylglycerol (TAG), which have applications ranging from biodiesel to high-value omega-3 fatty-acids. Hence, we demonstrated a strategy to sort microalgae cells with increased TAG productivity.

Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to take up and phosphorylate glucose, fructose, and sucrose, the major sugars from agricultural crops that are used as the primary feedstocks for industrial amino acid fermentation. This means that worldwide amino acid production using this organism has depended exclusively on the PTS. Recently, a better understanding not only of PTS-mediated sugar uptake but also of global regulation associated with the PTS has permitted the correction of certain negative aspects of this sugar transport system for amino acid production. In addition, the recent identification of different glucose uptake systems in this organism has led to a strategy for the generation of C. glutamicum strains that express non-PTS routes instead of the original PTS. The potential practical advantages of the development of such strains are discussed.