Search Results (32)

Yeast genetic improvement is entering a transformative phase, driven by the integration of artificial intelligence (AI), big data analytics, and synthetic microbial communities with conventional methods such as sexual breeding and random mutagenesis. These advancements have substantially expanded the potential for innovative re-engineering of yeast, ranging from single-strain cultures to complex polymicrobial consortia. This review compares traditional genetic manipulation techniques with cutting-edge approaches, highlighting recent breakthroughs in their application to beer and wine fermentation. Among the innovative strategies, adaptive laboratory evolution (ALE) stands out as a non-GMO method capable of rewiring complex fitness-related phenotypes through iterative selection. In contrast, GMO-based synthetic biology approaches, including the most recent developments in CRISPR/Cas9 technologies, enable efficient and scalable genome editing, including multiplexed modifications. These innovations are expected to accelerate product development, reduce costs, and enhance the environmental sustainability of brewing and winemaking. However, despite their technological potential, GMO-based strategies continue to face significant regulatory and market challenges, which limit their widespread adoption in the fermentation industry. Full article

Insect cell lines are a cornerstone of recombinant protein production, providing a versatile platform for biopharmaceutical and research applications. In the early 20th century, scientists first attempted to culture insect cells in vitro, developing continuous cell lines to produce the first insect cell-derived recombinant protein, IFN-β. Initial successes, along with advancements in the use of insect cells for recombinant protein manufacturing, primarily relied on baculovirus expression vector systems (BEVSs), which enable heterologous gene expression in infected cells. Today, growing attention is focused on baculovirus-free systems based on the transfection of insect cells with plasmid DNA. This approach simplifies the final product purification process and facilitates the development of stable monoclonal cell lines that produce recombinant proteins or protein complexes, particularly virus-like particles (VLPs). Thanks to advancements in genetic engineering and the application of adaptive laboratory evolution (ALE) methods, significant strides have been made in overcoming many limitations associated with insect cell BEVSs, ultimately enhancing the reliability, yield, and quality of the biomanufacturing process. Our manuscript discusses the history of developing insect cell lines, presents various recombinant protein production systems utilizing these cells, and summarizes modifications aimed at improving insect cell lines for recombinant protein biomanufacturing. Finally, we explore their implications in pharmaceutical production, particularly on Nuvaxovid^®/Covovax, which is the latest approved vaccine developed using insect cell BEVSs for protection against SARS-CoV-2. Full article

Due to the increasing global demand for feed protein, microbial protein has great potential of being able to feed sustainably. However, the application of microbial protein in the animal cultivation industry is still limited by its high cost and availability on scale. From the viewpoint of industrial production, it is vital to specify the crucial processes and components for further technical exploration and process optimization. This article presents state-of-the-art industrial microbial technologies for non-protein nitrogen (NPN) assimilation in feed protein production. Nitrogen sources are one of the main cost factors in the media used for large-scale microbial protein fermentation. Therefore, the available NPN sources for microbial protein synthesis, NPN utilization mechanisms, and fermentation technologies corresponding to the strain and NPN are reviewed in this paper. Especially, the random mutagenesis and adaptive laboratory evolution (ALE) approach combined with (ultra-) throughput screening provided the main impetus for strain evolution to increase the protein yield. Despite the underlying potential and technological advances in the production of microbial protein, extensive research and development efforts are still required before large-scale commercial application of microbial protein in animal feed. Full article

Propolis is a highly complex, resinous natural product collected by honeybees from tree leaves and buds and mixed with pollen and enzymes. Due to its antimicrobial properties, it has various medical and industrial applications. As a nonconventional strategy, the use of propolis was suggested to control contaminating yeast growth in ethanol fermentations, without significantly affecting the starter yeast of the fermentation, Saccharomyces cerevisiae. In this study, we have developed a highly propolis-resistant S. cerevisiae strain using evolutionary engineering. The evolved strain FD11 had a higher growth rate (µ_max = 0.21 h⁻¹) than the reference strain (µ_max = 0.17 h⁻¹) under propolis stress and showed cross-resistance against caffeine stress. Moreover, it had significantly lower reactive oxygen species levels and higher cell wall integrity than the reference strain. Comparative transcriptomic analysis results revealed that the genes involved in oxidoreductase activity, transmembrane transporter activity, unfolded protein binding and pleiotropic drug resistance were upregulated in FD11. Whole genome re-sequencing analysis revealed mutations in multiple genes including PDR1, encoding a transcription factor regulating pleiotropic drug response. The results imply the importance of pleiotropic drug response and cell wall integrity in propolis resistance and the potential of using propolis-resistant, robust yeast strains in industrial applications. Full article

ε-poly-l-lysine (ε-PL), a natural food preservative, has garnered widespread attention. It is mainly produced by Streptomyces albulus, but the production by wild-type strains fails to meet the demands of industrialization. To address this issue, adaptive laboratory evolution (ALE) was successfully employed in this study, subjecting S. albulus CICC 11022 to environmental stresses such as acidic pH and antibiotics (rifampicin, gentamicin, and streptomycin). As a result of ALE, an evolutionary strain S. albulus C214 was obtained, exhibiting an increase in ε-PL production and cell growth by 153.23% and 234.51%, respectively, as compared with the original strain. Genomic and metabolic analyses revealed that mutations occurred in genes responsible for transcriptional regulation, transporter, cell envelope, energy metabolism, and secondary metabolite synthesis, as well as the enrichment of metabolites involved in the biosynthesis of ε-PL. These findings hold great significance for elucidating the mechanism underlying ε-PL synthesis. Full article

Adaptive laboratory evolution (ALE) is a non-GMO technique utilized for the amelioration of wine yeast strains. Employing two-step ALE strategies, we recently acquired six evolved Saccharomyces cerevisiae populations with improved fermentative abilities compared to their parental strains in synthetic broths. Herein, we evaluated the qualities of the abovementioned evolved populations under real winemaking conditions, using the grape musts Assyrtiko and Roditis. The ethanol-tolerant populations evolved solely with glucose delayed to complete the fermentation due to slow fructose assimilation, albeit showing improved ethanol yields, compared to their parental strains. The volatile compounds of the evolved populations were significantly different from those of parental strains. Statistically significant differences were observed in the organoleptic profiles between the evolved populations’ and parental strains’ wines. Notably, wine from one evolved population (BLR200) was rated higher in overall aroma and quality. This study supports the magnitude of ALE strategies for the generation of novel wine yeasts. Full article

Despite the urgent need for new antibiotics, very few innovative antibiotics have recently entered clinics or clinical trials. To provide a constant supply of new drug candidates optimized in terms of their potential to select for resistance in natural settings, in vitro resistance-predicting studies need to be improved and scaled up. In this review, the following in vitro parameters are presented: frequency of spontaneous mutant selection (FSMS), mutant prevention concentration (MPC), dominant mutant prevention concentration (MPC-D), inferior-mutant prevention concentration (MPC-F), and minimal selective concentration (MSC). The utility of various adaptive laboratory evolution (ALE) approaches (serial transfer, continuous culture, and evolution in spatiotemporal microenvironments) for comparing hits in terms of the level and time required for multistep resistance to emerge is discussed. We also consider how the hit-to-lead stage can benefit from high-throughput ALE setups based on robotic workstations, do-it-yourself (DIY) continuous cultivation systems, microbial evolution and growth arena (MEGA) plates, soft agar gradient evolution (SAGE) plates, microfluidic chips, or microdroplet technology. Finally, approaches for evaluating the fitness of in vitro-generated resistant mutants are presented. This review aims to draw attention to newly emerged ideas on how to improve the in vitro forecasting of the potential of compounds to select for resistance in natural settings. Full article

During adaptive laboratory evolution experiments, any unexpected interruption in data monitoring or control could lead to the loss of valuable experimental data and compromise the integrity of the entire experiment. Most homemade mini-bioreactors are built employing microcontrollers such as Arduino. Although affordable, these platforms lack the robustness of the programmable logic controller (PLC), which enhances the safety and robustness of the control process. Here, we describe the design and validation of a PLC-controlled morbidostat, an innovative automated continuous-culture mini-bioreactor specifically created to study the evolutionary pathways to drug resistance in microorganisms. This morbidostat includes several improvements, both at the hardware and software level, for better online monitoring and a more robust operation. The device was validated employing Escherichia coli, exploring its adaptive evolution in the presence of didecyldimethylammonium chloride (DDAC), a quaternary ammonium compound widely used for its antimicrobial properties. E. coli was subjected to increasing concentrations of DDAC over 3 days. Our results demonstrated a significant increase in DDAC susceptibility, with evolved populations exhibiting substantial changes in their growth after exposure. Full article

The presence of toxic compounds in lignocellulosic hydrolysates (LCH) is among the main barriers affecting the efficiency of lignocellulose-based fermentation processes, in particular, to produce biofuels, hindering the production of intracellular lipids by oleaginous yeasts. These microbial oils are promising sustainable alternatives to vegetable oils for biodiesel production. In this study, we explored adaptive laboratory evolution (ALE), under methanol- and high glycerol concentration-induced selective pressures, to improve the robustness of a Rhodotorula toruloides strain, previously selected to produce lipids from sugar beet hydrolysates by completely using the major C (carbon) sources present. An evolved strain, multi-tolerant not only to methanol but to four major inhibitors present in LCH (acetic acid, formic acid, hydroxymethylfurfural, and furfural) was isolated and the mechanisms underlying such multi-tolerance were examined, at the cellular envelope level. Results indicate that the evolved multi-tolerant strain has a cell wall that is less susceptible to zymolyase and a decreased permeability, based on the propidium iodide fluorescent probe, in the absence or presence of those inhibitors. The improved performance of this multi-tolerant strain for lipid production from a synthetic lignocellulosic hydrolysate medium, supplemented with those inhibitors, was confirmed. Full article

Bioethanol is an important biofuel which can be produced from the abundant low-value lignocelluloses. However, the highly toxic inhibitory compounds formed in the hydrolysate and the ineffective utilization of xylose as a co-substrate are the primarily bottlenecks that hinder the commercialization of lignocellulosic bioethanol. In this study, aiming to properly solve the above obstacles, an engineered Saccharomyces cerevisiae strain was constructed by introducing the xylose reductase (XR)–xylitol dehydrogenase (XDH) pathway, overexpressing the non-oxidized pentose phosphate pathway, and deleting aldose reductase GRE3 and alkaline phosphatase PHO13 using a GTR-CRISPR system, followed by adaptive laboratory evolution (ALE). After screening, the isolated S. cerevisiae YL13-2 mutant was capable of robust xylose-utilizing, and exhibited high tolerance to the inhibitors in undetoxified steam-exploded corn stover hydrolysate (SECSH). An ethanol concentration of 22.96 g/L with a yield of 0.454 g/g can be obtained at the end of batch fermentation when using SECSH as substrate without nutrient supplementation. Moreover, aiming to simplify the downstream process and reduce the energy required in bioethanol production, fermentation using fed-batch hydrolyzed SECSH containing higher titer sugars with a YL13-2 strain was also investigated. As expect, a higher concentration of ethanol (51.12 g/L) was received, with an average productivity and yield of 0.71 g/L h and 0.436 g/g, respectively. The findings of this research provide an effective method for the production of bioethanol from lignocellulose, and could be used in large-scale applications in future works. Full article

Increased human population and the rapid decline of fossil fuels resulted in a global tendency to look for alternative fuel sources. Environmental concerns about fossil fuel combustion led to a sharp move towards renewable and environmentally friendly biofuels. Ethanol has been the primary fossil fuel alternative due to its low carbon emission rates, high octane content and comparatively facile microbial production processes. In parallel to the increased use of bioethanol in various fields such as transportation, heating and power generation, improvements in ethanol production processes turned out to be a global hot topic. Ethanol is by far the leading yeast output amongst a broad spectrum of bio-based industries. Thus, as a well-known platform microorganism and native ethanol producer, baker’s yeast Saccharomyces cerevisiae has been the primary subject of interest for both academic and industrial perspectives in terms of enhanced ethanol production processes. Metabolic engineering strategies have been primarily adopted for direct manipulation of genes of interest responsible in mainstreams of ethanol metabolism. To overcome limitations of rational metabolic engineering, an alternative bottom-up strategy called inverse metabolic engineering has been widely used. In this context, evolutionary engineering, also known as adaptive laboratory evolution (ALE), which is based on random mutagenesis and systematic selection, is a powerful strategy to improve bioethanol production of S. cerevisiae. In this review, we focus on key examples of metabolic and evolutionary engineering for improved first- and second-generation S. cerevisiae bioethanol production processes. We delve into the current state of the field and show that metabolic and evolutionary engineering strategies are intertwined and many metabolically engineered strains for bioethanol production can be further improved by powerful evolutionary engineering strategies. We also discuss potential future directions that involve recent advancements in directed genome evolution, including CRISPR-Cas9 technology. Full article

Bacillus subtilis strain 168 is commonly used as a host to produce recombinant proteins and as a chassis for bio-based chemicals production. However, its preferred nitrogen source is organic nitrogen, which greatly increases production costs. In this study, adaptive laboratory evolution (ALE) was used to improve B. subtilis 168 growth using NH₄Cl as the sole nitrogen source. The cell density (OD₆₀₀) of a mutant strain LJ-3 was 208.7% higher than that of the original strain. We also optimized the metal ions in the medium and this resulted in a further increase in growth rate by 151.3%. Reintroduction of the sfp+ gene into strain LJ-3 led to the LJ-31 clone, which restored LJ-3’s ability to synthesize surfactin. The fermentation system was optimized (C/N, aeration, pH) in a 5 L bioreactor. Dry cell weight of 7.4 g/L and surfactin concentration of 4.1 g/L were achieved using the optimized mineral salt medium after 22 h of batch fermentation with a Y_P/S value of 0.082 g/g and a Y_P/X of 0.55 g/g. HPLC analysis identified the surfactin isoforms produced by strain LJ-31 in the synthetic medium as C₁₃-surfactin 13.3%, C₁₄-surfactin 44.02%, and C₁₅-surfactin 32.79%. Hence, the variant LJ-3 isolated by ALE is a promising engineering chassis for efficient and cost-effective production of a variety of metabolites. Full article

Changes in biological properties over several generations, induced by controlling short-term evolutionary processes in the laboratory through selective pressure, and whole-genome re-sequencing, help determine the genetic basis of microorganism’s adaptive laboratory evolution (ALE). Due to the versatility of this technique and the imminent urgency for alternatives to petroleum-based strategies, ALE has been actively conducted for several yeasts, primarily using the conventional species Saccharomyces cerevisiae, but also non-conventional yeasts. As a hot topic at the moment since genetically modified organisms are a debatable subject and a global consensus on their employment has not yet been attained, a panoply of new studies employing ALE approaches have emerged and many different applications have been exploited in this context. In the present review, we gathered, for the first time, relevant studies showing the ALE of non-conventional yeast species towards their biotechnological improvement, cataloging them according to the aim of the study, and comparing them considering the species used, the outcome of the experiment, and the employed methodology. This review sheds light on the applicability of ALE as a powerful tool to enhance species features and improve their performance in biotechnology, with emphasis on the non-conventional yeast species, as an alternative or in combination with genome editing approaches. Full article

Adaptive laboratory evolution (ALE) is a valuable complementary tool for modern strain development. Insights from ALE experiments enable the improvement of microbial cell factories regarding the growth rate and substrate utilization, among others. Most ALE experiments are conducted by serial passaging, a method that involves large amounts of repetitive manual labor and comes with inherent experimental design flaws. The acquisition of meaningful and reliable process data is a burdensome task and is often undervalued and neglected, but also unfeasible in shake flask experiments due to technical limitations. Some of these limitations are alleviated by emerging automated ALE methods on the μL and mL scale. A novel approach to conducting ALE experiments is described that is faster and more efficient than previously used methods. The conventional shake flask approach was translated to a parallelized, L scale stirred-tank bioreactor system that runs controlled, automated, repeated batch processes. The method was validated with a growth optimization experiment of E. coli K-12 MG1655 grown with glycerol minimal media as a benchmark. Off-gas analysis enables the continuous estimation of the biomass concentration and growth rate using a black-box model based on first principles (soft sensor). The proposed method led to the same stable growth rates of E. coli with the non-native carbon source glycerol 9.4 times faster than the traditional manual approach with serial passaging in uncontrolled shake flasks and 3.6 times faster than an automated approach on the mL scale. Furthermore, it is shown that the cumulative number of cell divisions (CCD) alone is not a suitable timescale for measuring and comparing evolutionary progress. Full article

Adaptive laboratory evolution (ALE) is a useful experimental methodology for fundamental scientific research and industrial applications to create microbial cell factories. By using ALE, cells are adapted to the environment that researchers set based on their objectives through the serial transfer of cell populations in batch cultivations or continuous cultures and the fitness of the cells (i.e., cell growth) under such an environment increases. Then, omics analyses of the evolved mutants, including genome sequencing, transcriptome, proteome and metabolome analyses, are performed. It is expected that researchers can understand the evolutionary adaptation processes, and for industrial applications, researchers can create useful microorganisms that exhibit increased carbon source availability, stress tolerance, and production of target compounds based on omics analysis data. In this review article, the methodologies for ALE in microorganisms are introduced. Moreover, the application of ALE for the creation of useful microorganisms as cell factories has also been introduced. Full article