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This review comprehensively examines the advancements in engineering thermostable phytase through genetic modification and immobilization techniques, focusing on developments from the last seven years. Genetic modifications, especially protein engineering, have enhanced enzyme's thermostability and functionality. Immobilization on various supports has further increased thermostability, with 50–60 % activity retention at higher temperature (more than 50 °C). In the food industry, phytase is used in flour processing and bread making, reducing phytate content by around 70 %, thereby improving nutritional value and mineral bioavailability. In the feed industry, it serves as a poultry feed additive, breaking down phytates to enhance nutrient availability and feed efficiency. The enzyme's robustness at high temperatures makes it valuable in feed processing. The integration of microbial production of phytase with genetically engineered strains followed by carrier free immobilization represents a synergistic approach to fortify enzyme structure and improve thermal stability. These advancement in the development of phytase enzyme capable of withstanding high temperatures, thereby pivotal for industrial utilization. [ABSTRACT FROM AUTHOR]

Chlorella vulgaris, a unicellular green microalga, has obtained significant attention due to its high protein content, abundance of bioactive compounds, and broad biotechnological potential. Used in nutraceuticals, pharmaceuticals, and functional foods, it is now gaining traction in cosmetics, biopharmaceuticals, and environmental applications. Recent advancements in fermentation technology, such as the development of high-density fermentation strategies, adaptive evolution of strains, and real-time monitoring systems, have greatly improved the efficiency, scalability, and sustainability of C. vulgaris production, enhancing bioavailability and product quality. This review explores developments in C. vulgaris fermentation, highlighting advancements in strain improvement through genetic engineering, metabolic optimization, mutagenesis, and adaptive evolution, alongside bioprocess engineering and the optimization of fermentation parameters. Key considerations include bioreactor design, downstream processing, and innovative monitoring technologies aimed at maximizing biomass yield and bioactive compound production. Emerging applications of fermented C. vulgaris across industries are also highlighted, along with future perspectives on scaling up production, addressing regulatory challenges, and ensuring biosafety. These insights provide a comprehensive outlook on the future of C. vulgaris fermentation in biotechnological applications. [ABSTRACT FROM AUTHOR]

The cyanobacterium Limnospira platensis, vulgarly Spirulina, has gained significant attention due to its high protein content, rich bioactive compounds, and health benefits, making it a valuable resource in biotechnology, nutraceuticals, food supplements, biopharmaceuticals, and cosmetics. Recent advancements in fermentation technology have considerably improved the efficiency, scalability, and cost-effectiveness of L. platensis production while addressing environmental sustainability and enhancing product quality. Based on well-recognized databases (Google Scholar, PubMed, Scopus, Web of Science), this review explores the latest developments in L. platensis fermentation, emphasizing strain improvement, bioprocess engineering, and optimization of fermentation parameters. It also examines key factors such as bioreactor design, downstream processing, and innovative monitoring technologies aimed at maximizing biomass yield and bioactive compound production. Additionally, emerging applications of fermented L. platensis in various industries and future perspectives, including large-scale production, regulatory barriers, and biosafety considerations, are discussed. These insights provide a comprehensive outlook on the future of L. platensis fermentation in biotechnological applications. [ABSTRACT FROM AUTHOR]

Epigenetics has emerged as a potent field of study for understanding the factors influencing the effectiveness of human disease treatments and for identifying alternations induced by pathogens in host plants. However, there has been a paucity of research on the epigenetic control of the proliferation and pathogenicity of fungal plant pathogens. Fungal plant pathogens such as Magnaporthe oryzae , a significant threat to global rice production, provide an important model for exploring how epigenetic mechanisms govern fungal proliferation and virulence. In M. oryzae , epigenetic alterations, such as DNA methylation, histone modification, and non-coding RNAs, regulate gene expression patterns that influence the pathogen's ability to infect its host. These modifications can enhance fungal adaptability, allowing the pathogen to survive in diverse environments and evade host immune responses. Our primary objective is to provide a comprehensive review of the existing epigenetic research on M. oryzae and shed light on how these changes influence the pathogen's lifecycle, its ability to invade host tissues, and the overall severity of the disease. We begin by examining the epigenetic alterations occurring in M. oryzae and their contributions to the virulence and proliferation of the fungus. To advance our understanding of epigenetic mechanisms in M. oryzae and similar plant diseases, we emphasize the need to address unanswered questions and explore future research directions. This information is crucial for developing new antifungal treatments that target epigenetic pathways, which could lead to improved disease management. [ABSTRACT FROM AUTHOR]

BACKGROUND: Entomopathogenic fungi, such as Beauveria bassiana, hold promise as biological control agents against insect pests. However, the efficacy of these fungi can be hindered by insect immune responses. One strategy to enhance fungal virulence is to manipulate host immune by targeting key regulatory molecules like 20‐hydroxyecdysone (20E). RESULTS: In this study, we engineered B. bassiana strains to constitutively express the enzyme ecdysteroid UDP‐glucosyltransferase (EGT), which inactivates 20E, a crucial insect molting hormone. The engineered strain Bb::EGT‐1 exhibited robust expression of EGT, leading to a significant reduction in insect 20E levels upon infection. Moreover, infection with Bb::EGT‐1 resulted in accelerated larval mortality. Immune responses analysis revealed repression of insect immune response genes and decreased phenoloxidase (PO) activity in larvae infected with Bb::EGT‐1. Microbiome analysis indicated alterations in bacterial composition within infected insects, with increased abundance observed during infection with Bb::EGT‐1. Additionally, the presence of bacteria hindered hyphal emergence from insect cadavers, suggesting a role for microbial competition in fungal dissemination. CONCLUSIONS: Constitutive expression of EGT in B. bassiana enhances fungal virulence by reducing insect 20E levels, suppressing immune responses, and altering the insect microbiome. These findings highlighted the potential of engineered fungi as effective biocontrol agents against insect pests and provide insights into the complex interactions between entomopathogenic fungi, their hosts, and associated microbes. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

This review explores the production of the fungal polysaccharide pullulan by mutants and natural isolates of Aureobasidium species using strain improvement. Pullulan is a neutral polysaccharide gum whose structure is a maltotriose-containing glucan. This polysaccharide gum has applications in the fields of food, pharmaceuticals, biomedical and wastewater treatment. The strain improvement of Aureobasidium species has focused on the pullulan production process, including the isolation of strains exhibiting reduced pigmentation, polysaccharide overproduction, the production of pullulan with variable molecular weight, and increased osmotolerant strains promoting pullulan production at high carbon source concentrations and pullulan production on hemicellulosic substrates. The majority of studies have emphasized the isolation of reduced pigmentation and pullulan hyperproducer strains since the goal of large-scale commercial pullulan production is to synthesize non-pigmented polysaccharides. A promising area of strain improvement is the isolation of strains that synthesize authentic pullulan from hemicellulosic substrates. If strain improvement in this area is successful, the goal of commercially producing pullulan at a competitive cost will eventually be achieved. [ABSTRACT FROM AUTHOR]

Food security is threatened by biotic stress on crops, e.g., from invasive pests, in the context of climate change. Pest control has traditionally been achieved using synthetic pesticides, yet pollution and the persistence and toxicity of some pesticides are inducing food contamination and, in turn, generating public concern. As a consequence, biopesticides are increasingly used, notably for organic crops. For instance, some microorganisms produce biopesticidal compounds such as secondary metabolites or growth-inhibitory enzymes. Most reviews on this topic mainly focus on describing microbial species and their active compounds. However, there are very few studies and reviews describing various process parameters. Here, we review both microbial biopesticides and factors controlling physicochemical conditions for the scaling up of biopesticide production. We present biopesticides from bacteria (e.g., Bacillus thuringiensis), algae, fungi, and viruses and review the biocontrol mechanisms and applications of commercial biopesticides. The benefits of genetic engineering for enhancing activity and drawbacks such as commercialization are also discussed. [ABSTRACT FROM AUTHOR]

This review explores the production of the fungal polysaccharide pullulan by mutants and natural isolates of Aureobasidium species using strain improvement. Pullulan is a neutral polysaccharide gum whose structure is a maltotriose-containing glucan. This polysaccharide gum has applications in the fields of food, pharmaceuticals, biomedical and wastewater treatment. The strain improvement of Aureobasidium species has focused on the pullulan production process, including the isolation of strains exhibiting reduced pigmentation, polysaccharide overproduction, the production of pullulan with variable molecular weight, and increased osmotolerant strains promoting pullulan production at high carbon source concentrations and pullulan production on hemicellulosic substrates. The majority of studies have emphasized the isolation of reduced pigmentation and pullulan hyperproducer strains since the goal of large-scale commercial pullulan production is to synthesize non-pigmented polysaccharides. A promising area of strain improvement is the isolation of strains that synthesize authentic pullulan from hemicellulosic substrates. If strain improvement in this area is successful, the goal of commercially producing pullulan at a competitive cost will eventually be achieved.

Abstract Background Oritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production. Results Firstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities. Conclusion Under approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.

Macrofungi commonly referred to as Mushrooms are distributed worldwide and well known for their nutritional, medicinal, and organoleptic properties. Strain improvement in mushrooms is lagging due to paucity of efficient genome modification techniques. Thus, for advanced developments in research and commercial or economical viability and benefit, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) emerged as an efficient genome editing tool. The higher efficiency and precision of the desired genetic modification(s) are the most valuable attributes of this recent technology. The present review comprehensively summarizes various conventional methods utilized for strain improvement in mushrooms including hybridization, protoplast fusion, and di-mon mating. Furthermore, the problems associated with these techniques have been discussed besides providing the potential recluses. The significance of CRISPR/Cas9 strategies employed for improvement in various mushroom genera has been deliberated, as these strategies will paves the way forward for obtaining improved strain and effective cultivation methods for enhancing the yield and quality of the fruit bodies. [ABSTRACT FROM AUTHOR]

Background: Oritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production. Results: Firstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities. Conclusion: Under approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products. [ABSTRACT FROM AUTHOR]

Epigenetics has emerged as a potent field of study for understanding the factors influencing the effectiveness of human disease treatments and for identifying alternations induced by pathogens in host plants. However, there has been a paucity of research on the epigenetic control of the proliferation and pathogenicity of fungal plant pathogens. Fungal plant pathogens such as Magnaporthe oryzae, a significant threat to global rice production, provide an important model for exploring how epigenetic mechanisms govern fungal proliferation and virulence. In M. oryzae, epigenetic alterations, such as DNA methylation, histone modification, and non-coding RNAs, regulate gene expression patterns that influence the pathogen’s ability to infect its host. These modifications can enhance fungal adaptability, allowing the pathogen to survive in diverse environments and evade host immune responses. Our primary objective is to provide a comprehensive review of the existing epigenetic research on M. oryzae and shed light on how these changes influence the pathogen’s lifecycle, its ability to invade host tissues, and the overall severity of the disease. We begin by examining the epigenetic alterations occurring in M. oryzae and their contributions to the virulence and proliferation of the fungus. To advance our understanding of epigenetic mechanisms in M. oryzae and similar plant diseases, we emphasize the need to address unanswered questions and explore future research directions. This information is crucial for developing new antifungal treatments that target epigenetic pathways, which could lead to improved disease management.

These days, bioethanol research is looking at using non-edible plant materials, called lignocellulosic feedstocks, because they are cheap, plentiful, and renewable. However, these materials are complex and require pretreatment to release fermentable sugars. Saccharomyces cerevisiae, the industrial workhorse for bioethanol production, thrives in sugary environments and can handle high levels of ethanol. However, during lignocellulose fermentation, S. cerevisiae faces challenges like high sugar and ethanol concentrations, elevated temperatures, and even some toxic substances present in the pretreated feedstocks. Also, S. cerevisiae struggles to efficiently convert all the sugars (hexose and pentose) present in lignocellulosic hydrolysates. That’s why scientists are exploring the natural variations within Saccharomyces strains and even figuring out ways to improve them. This review highlights why Saccharomyces cerevisiae remains a crucial player for large-scale bioethanol production from lignocellulose and discusses the potential of genome shuffling to create even more efficient yeast strains.

The cyanobacterium Limnospira platensis, vulgarly Spirulina, has gained significant attention due to its high protein content, rich bioactive compounds, and health benefits, making it a valuable resource in biotechnology, nutraceuticals, food supplements, biopharmaceuticals, and cosmetics. Recent advancements in fermentation technology have considerably improved the efficiency, scalability, and cost-effectiveness of L. platensis production while addressing environmental sustainability and enhancing product quality. Based on well-recognized databases (Google Scholar, PubMed, Scopus, Web of Science), this review explores the latest developments in L. platensis fermentation, emphasizing strain improvement, bioprocess engineering, and optimization of fermentation parameters. It also examines key factors such as bioreactor design, downstream processing, and innovative monitoring technologies aimed at maximizing biomass yield and bioactive compound production. Additionally, emerging applications of fermented L. platensis in various industries and future perspectives, including large-scale production, regulatory barriers, and biosafety considerations, are discussed. These insights provide a comprehensive outlook on the future of L. platensis fermentation in biotechnological applications.

Chlorella vulgaris, a unicellular green microalga, has obtained significant attention due to its high protein content, abundance of bioactive compounds, and broad biotechnological potential. Used in nutraceuticals, pharmaceuticals, and functional foods, it is now gaining traction in cosmetics, biopharmaceuticals, and environmental applications. Recent advancements in fermentation technology, such as the development of high-density fermentation strategies, adaptive evolution of strains, and real-time monitoring systems, have greatly improved the efficiency, scalability, and sustainability of C. vulgaris production, enhancing bioavailability and product quality. This review explores developments in C. vulgaris fermentation, highlighting advancements in strain improvement through genetic engineering, metabolic optimization, mutagenesis, and adaptive evolution, alongside bioprocess engineering and the optimization of fermentation parameters. Key considerations include bioreactor design, downstream processing, and innovative monitoring technologies aimed at maximizing biomass yield and bioactive compound production. Emerging applications of fermented C. vulgaris across industries are also highlighted, along with future perspectives on scaling up production, addressing regulatory challenges, and ensuring biosafety. These insights provide a comprehensive outlook on the future of C. vulgaris fermentation in biotechnological applications.

Strain improvement via chemical mutagen could impart traits with better enzyme production or improved characteristics. The present study sought to investigate the physicochemical properties of pullulanase produced from the wild Bacillus sp and the mutant. The pullulanases produced from the wild and the mutant Bacillus sp. (obtained via induction with ethyl methyl sulfonate) were purified in a-three step purification procedure and were also characterized. The wild and mutant pullulanases, which have molecular masses of 40 and 43.23 kDa, showed yields of 2.3% with 6.0-fold purification and 2.0% with 5.0-fold purification, respectively, and were most active at 50 and 40 °C and pH 7 and 8, respectively. The highest stability of the wild and mutant was between 40 and 50 °C after 1 h, although the mutant retained greater enzymatic activity between pH 6 and 9 than the wild. The mutant had a decreased Km of 0.03 mM as opposed to the wild type of 1.6 mM. In comparison to the wild, the mutant demonstrated a better capacity for tolerating metal ions and chelating agents. These exceptional characteristics of the mutant pullulanase may have been caused by a single mutation, which could improve its utility in industrial and commercial applications. [ABSTRACT FROM AUTHOR]

Secondary metabolites are produced by microbes in minimal quantities in the natural environment out of necessity. However, in the pharmaceutical industry, their overproduction becomes essential. To achieve higher yields, genetic modifications are employed to create strains that surpass the productivity of the initially isolated strains. While rational screening and genetic engineering have emerged as valuable practices in recent years, the cost-effective technique of mutagenesis and selection, known as "random screening," remains a preferred method for efficient short-term strain development. This review aims to comprehensively explore all aspects of strain improvement, focusing on why random mutagenesis continues to be widely adopted. [ABSTRACT FROM AUTHOR]

The biosynthesis of citric acid (CA) and its derivatives is of great interest due to its wide range of applications in various manufacturing sectors. The fungus Aspergillus niger is mainly used for the commercial production of CA, using sucrose and molasses as the primary carbon sources. Since the 1960s, intensive research has been underway to introduce Yarrowia lipolytica yeast as an alternative to traditional fungal technology. This review discusses the practical uses of CA and its derivatives. Also, the challenges and developments that have led to efficient and green CA synthesis technologies using Y. lipolytica are outlined. The nutrient medium requirements and the use of various carbon sources, encompassing pure substrates and industry, agriculture, and food waste are considered. Additionally, the choice and improvement of strain producers, including efficient mutagenesis, genetic modification, and screening methods, are discussed. [ABSTRACT FROM AUTHOR]

Long-chain polyunsaturated fatty acids (LC-PUFA) are nutritionally and pharmacologically well-recognized beneficial compounds widely used as nutrient supplements for various applications. As the human body cannot accomplish de novo synthesis of PUFA, we depend on exogenous bioresources for the supply of these essential PUFA for day-to-day activities. The PUFA global market is expected to reach about US$ 9.7 billion by 2032. The recent drop in natural fish catch and global climate change severely hamper the global PUFA supply chain. As a sustainable, promising resource, marine zoosporic microorganisms are considered to be safe and alternative primary producers of beneficial PUFAs for direct and indirect human consumption. However, by considering the limitations associated with strain efficiency, final product yield and production cost, more viable low-cost production strategies and establishment of highly efficient, stable, high production cell lines specific for marine zoosporic microbes are essential to meet global PUFA market demand. The exploration of the latest techniques for research and development could shape the dynamics better for the global market for marine zoosporic organism-based PUFA products. Therefore, this review first summarizes low-cost production and strain improvement strategies, and later highlights the advantages and commercial prospects for marine zoosporic microbe-derived PUFA. [ABSTRACT FROM AUTHOR]

Brevibacillus sp. SPR20 produced potentially antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). The synthesis of these substances is controlled by their biosynthetic gene clusters. Several mutagenesis methods are used to overcome the restriction of gene regulations when genetic information is absent. Atmospheric and room temperature plasma (ARTP) is a powerful technique to initiate random mutagenesis for microbial strain improvement. This study utilized an argon-based ARTP to conduct the mutations on SPR20. The positive mutants of 40% occurred. The M27 mutant exhibited an increase in anti-MRSA activity when compared to the wild-type strain, with the MIC values of 250–500 and 500 μg/mL, respectively. M27 had genetic stability because it exhibited constant activity throughout fifteen generations. This mutant had similar morphology and antibiotic susceptibility to the wild type. Comparative proteomic analysis identified some specific proteins that were upregulated in M27. These proteins were involved in the metabolism of amino acids, cell structure and movement, and catalytic enzymes. These might result in the enhancement of the anti-MRSA activity of the ARTP-treated SPR20 mutant. This study supports the ARTP technology designed to increase the production of valuable antibacterial agents. [ABSTRACT FROM AUTHOR]

In the present study, Aspergillus uvarum was subjected to random mutagenesis, by treatment with chemical mutagen i.e., Ethyl methanesulfonate (EMS). Among the various mutant strains of A. uvarum, the strain that was obtained after treatment with EMS at 12% v/v concentration and 60 min of exposure time showed 1.8-fold and 1.4-fold increased cellulase activity using submerged (SmF) and solid state fermentation (SSF), respectively, as compared to the parent wild strain. Partial purification of the cellulase by ammonium sulphate fractionation and dialysis showed 1.46 and 2.02 fold purification, respectively. Among the several concentrations of sodium alginate and calcium chloride used for immobilization, partially purified cellulase demonstrated maximum activity with 2% w/v concentration of sodium alginate and 0.2 M concentration of calcium chloride. The optimal temperature and pH for immobilized cellulase were observed to be 60 °C and 7, respectively. Furthermore, the immobilized enzyme presented increased reusability, having 18.5% relative activity after five consecutive uses, and was more stable, maintaining 64.5 and 48.7% of its initial activity at 4 and 30 °C, respectively, after 30 days of storage. [ABSTRACT FROM AUTHOR]

Strains' improvement technology plays an essential role in enhancing the quality of industrial strains. Several traditional methods and modern techniques have been used to further improve strain engineering programs. The advances stated in strain engineering and the increasing demand for microbial metabolites leads to the invention of the genome shuffling technique, which ensures a specific phenotype improvement through inducing mutation and recursive protoplast fusion. In such technique, the selection of multi-parental strains with distinct phenotypic traits is crucial. In addition, as this evolutionary strain improvement technique involves combinative approaches, it does not require any gene sequence data for genome alteration and, therefore, strains developed by this elite technique will not be considered as genetically modified organisms. In this review, the different stages involved in the genome shuffling technique and its wide applications in various phenotype improvements will be addressed. Taken together, data discussed here highlight that the use of genome shuffling for strain improvement will be a plus for solving complex phenotypic traits and in promoting the rapid development of other industrially important strains. [ABSTRACT FROM AUTHOR]

Propionic acid (PA) is a 3-carbon carboxylic acid widely used in the chemical, food and pharmaceutical industries. Current production of PA is primarily via petrochemical routes and alternative sources using renewable feedstocks are desirable. The goal of the present study was to develop bacterial strains for efficient PA production by fermentation of sugarcane-derived saccharides in inverted sugarcane syrup. Adaptive evolution and random chemical mutagenesis of Propionibacterium acidipropionici were individually evaluated. Although evolutionary engineering proved to be a suitable strategy for producing strains tolerant to acid stress, chemical mutagenesis coupled with a novel screening methodology proved to be a superior strategy for strain improvement. The selected strain presented high PA productivity (0.55 g L−1 h−1) and titer (40 g L−1) in relation to the parental strain (0.38 g L−1 h−1 and 27 g L−1, respectively), in fermentations using inverted sugarcane syrup. [Display omitted] • P. acidipropionici is more tolerant to propionate than other propionibacteria. • Acid tolerance did not correlate with higher propionate production. • Random mutagenesis coupled to rounds of selection results in improved strains. • The M8 strain ferments high test molasses with a 60% production rate improvement. [ABSTRACT FROM AUTHOR]

Key points Hydrogen can be generated by microorganisms. Dark fermentation is efficient for biological hydrogen production. Strain improvement is critical to enhancing hydrogen-producing ability.

In the control of plant diseases, biocontrol has the advantages of being efficient and safe for human health and the environment. The filamentous fungus Trichoderma harzianum and its closely related species can inhibit the growth of many phytopathogenic fungi, and have been developed as commercial biocontrol agents for decades. In this review, we summarize studies on T. harzianum species complex from the perspective of strain improvement. To elevate the biocontrol ability, the production of extracellular proteins and compounds with antimicrobial or plant immunity-eliciting activities need to be enhanced. In addition, resistance to various environmental stressors should be strengthened. Engineering the gene regulatory system has the potential to modulate a variety of biological processes related to biocontrol. With the rapidly developing technologies for fungal genetic engineering, T. harzianum strains with increased biocontrol activities are expected to be constructed to promote the sustainable development of agriculture. [ABSTRACT FROM AUTHOR]

Studies on the microorganisms used in food production are of interest because microbial genotypes are reflected in food qualities such as taste, flavor, and yield. However, several microbes are nonmodel organisms, and their analysis is often limited by the lack of genetic tools. Tetragenococcus halophilus, a halophilic lactic acid bacterium used in soy sauce fermentation starter culture, is one such microorganism. The lack of DNA transformation techniques for T. halophilus makes gene complementation and disruption assays difficult. Here, we report that the endogenous insertion sequence ISTeha4, belonging to the IS4 family, is translocated at an extremely high frequency in T. halophilus and causes insertional mutations at various loci. We developed a method named targeting spontaneous insertional mutations in genomes (TIMING), which combines high-frequency insertional mutations and efficient PCR screening, enabling the isolation of gene mutants of interest from a library. The method provides a reverse genetics and strain improvement tool, does not require the introduction of exogenous DNA constructs, and enables the analysis of nonmodel microorganisms lacking DNA transformation techniques. Our results highlight the important role of insertion sequences as a source of spontaneous mutagenesis and genetic diversity in bacteria. IMPORTANCE Genetic and strain improvement tools to manipulate a gene of interest are required for the nontransformable lactic acid bacterium Tetragenococcus halophilus. Here, we demonstrate that an endogenous transposable element, ISTeha4, is transposed into the host genome at an extremely high frequency. A genotype-based and non-genetically engineered screening system was constructed to isolate knockout mutants using this transposable element. The method described enables a better understanding of the genotype-phenotype relationship and serves as a tool to develop food-grade-appropriate mutants of T. halophilus. [ABSTRACT FROM AUTHOR]

Streptomyces albus J1074 is one of the most popular heterologous expression platforms among streptomycetes. Identification of new genes and mutations that influence specialized metabolism in this species is therefore of great applied interest. Here, we describe S. albus KO-1304 that was isolated as a spontaneous lincomycin-resistant variant of double rpsLR94GrsmGR15SG40E mutant KO-1295. Besides altered antibiotic resistance profile, KO-1304 exhibited increased antibiotic activity as compared to its parental strains. KO-1304 genome sequencing revealed mutations within gene XNR_2147 encoding putative TetR-like protein. Gene XNR_2146 for efflux protein is the most likely target of repressing action of Xnr_2147. Our data agree with the scenario where lincomycin resistance phenotype of KO-1304 arose from inability of mutated Xnr_2147 protein to repress XNR_2146. Introduction of additional copy of XNR_2146 into wild type strain increased antibiotic activity of the latter, attesting to the practical value of transporter genes for strain improvement. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 system is a cutting-edge gene-editing tool that has recently been introduced for the strain improvement of fungi to enhance their cellulolytic activity. Cellulase is an enzyme complex that hydrolyzes cellulosic fraction of lignocellulosic biomass into glucose which can be converted subsequently into biofuels and other value-added products. Among diverse microbial communities, fungi are considered the most potent cellulolytic candidates but the naturally low cellulase titers have been the major bottleneck in using them for industrial and biotechnological applications. However, CRISPR/Cas9 mediated fungal strain engineering has opened up new avenues to overcome this challenge by enabling precise and efficient genome editing which induces the overexpression of cellulase genes, deletion of cellulase repressors, and alterations in the regulatory elements governing the overall cellulase gene expression. The effective CRISPR/Cas9 systems have already been designed for the functional genome editing of industrially competent cellulolytic fungal strains such as Aspergillus strains, Trichoderma reesei , and Penicillium chrysogenum , however, the technological advancements to establish it across the fungal kingdom are still desired. The employment of CRISPR/Cas9 in fungal strain improvement poses various limitations such as off-target effects, the need for designing suitable delivery methods, and the appropriate selection markers, therefore, future research should focus on addressing these issues and further refining the CRISPR/Cas9 system for fungal strain improvement. Conclusively, this RNA-directed DNA endonuclease system is a promising future tool for improving cellulase production in fungi leading to the development of numerous industrially competent strains, thus, supporting the sustainable development goal (SDG 7) of affordable and clean energy through efficient bioconversion of lignocellulosic biomass into biofuels coupled with other value-added products. [ABSTRACT FROM AUTHOR]

2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76 ± 0.02 g/L after 72 h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75 ± 0.08 g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36 ± 0.06 g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28 ± 0.20 g/L of 2-PE with a yield of 0.076 g/g glucose and productivity of 0.048 g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15 g/L after 32 h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization. [ABSTRACT FROM AUTHOR]

This review comprehensively examines the advancements in engineering thermostable phytase through genetic modification and immobilization techniques, focusing on developments from the last seven years. Genetic modifications, especially protein engineering, have enhanced enzyme's thermostability and functionality. Immobilization on various supports has further increased thermostability, with 50-60 % activity retention at higher temperature (more than 50 °C). In the food industry, phytase is used in flour processing and bread making, reducing phytate content by around 70 %, thereby improving nutritional value and mineral bioavailability. In the feed industry, it serves as a poultry feed additive, breaking down phytates to enhance nutrient availability and feed efficiency. The enzyme's robustness at high temperatures makes it valuable in feed processing. The integration of microbial production of phytase with genetically engineered strains followed by carrier free immobilization represents a synergistic approach to fortify enzyme structure and improve thermal stability. These advancement in the development of phytase enzyme capable of withstanding high temperatures, thereby pivotal for industrial utilization., Competing Interests: Competing interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© The Korean Society of Food Science and Technology 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

In recent years, mass spectrometry-based metabolomics has been established as a powerful and versatile technique for studying cellular metabolism by comprehensive analysis of metabolites in the cell. Although there are many scientific reports on the use of metabolomics for the elucidation of mechanism and physiological changes occurring in the cell, there are surprisingly very few reports on its use for the identification of rate-limiting steps in a synthetic biological system that can lead to the actual improvement of the host organism. In this mini review, we discuss different strategies for improving strain performance using metabolomics data and compare the application of metabolomics-driven strain improvement techniques in different host microorganisms. Finally, we highlight several success stories on the use of metabolomics-driven strain improvement strategies, which led to significant bioproductivity improvements.

Transposons are mobile genetic elements that can give rise to gene mutation and genome rearrangement. Due to their mobility, transposons have been exploited as genetic tools for modification of plants, animals, and microbes. Although a plethora of reviews have summarized families of transposons, the transposons from fermentation bacteria have not been systematically documented, which thereby constrain the exploitation for metabolic engineering and synthetic biology purposes. In this review, we summarize the transposons from the most used fermentation bacteria including Escherichia coli, Bacillus subtilis, Lactococcus lactis, Corynebacterium glutamicum, Klebsiella pneumoniae, and Zymomonas mobilis by literature retrieval and data mining from GenBank and KEGG. We also outline the state-of-the-art advances in basic research and industrial applications especially when allied with other genetic tools. Overall, this review aims to provide valuable insights for transposon-mediated strain improvement. Key points: • The transposons from the most-used fermentation bacteria are systematically summarized. • The applications of transposons in strain improvement are comprehensively reviewed. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a major health concern worldwide. In our previous study, some bacterial isolates exhibited antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). However, the production of antibacterial substances by native microorganisms is limited by biosynthetic genes. This study aimed to improve the antibacterial activity of SPR19 using atmospheric and room temperature plasma mutagenesis (ARTP). The results showed that SPR19 belonged to the Brevibacillus genus. The growth curves and production kinetics of antibacterial substances were investigated. Argon-based ARTP was applied to SPR19, and the 469 mutants were preliminarily screened using agar overlay method. The remaining 25 mutants were confirmed by agar well diffusion assay against S. aureus TISTR 517 and MRSA isolates 142, 1096, and 2468. M285 exhibited the highest activity compared to the wild-type strain (10.34–13.59%) and this mutant was stable to produce the active substances throughout 15 generations consistently. The antibacterial substances from M285 were tolerant to various conditions (heat, enzyme, surfactant, and pH) while retaining more than 90% of their activities. Therefore, Brevibacillus sp. SPR19 is a potential source of antibacterial substances. ARTP mutagenesis is a powerful method for strain improvement that can be utilized to treat MRSA infection in the future. [ABSTRACT FROM AUTHOR]