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The widespread adoption of Poly(3-hydroxybutyrate) (PHB) encounters challenges due to its higher production costs compared to conventional plastics. To overcome this obstacle, this study investigates the use of low-cost raw materials and optimized production methods. Specifically, food processing byproducts such as corn germ and corn bran were utilized as solid substrates through solid-state fermentation, enriched with molasses and cheese whey. Employing the One Factor at a Time technique, we examined the effects of substrate composition, temperature, initial substrate moisture, molasses, and cheese whey on PHB production at the flask scale. Subsequently, experiments were conducted at the bioreactor scale to evaluate the influence of aeration. In flask-scale experiments, the highest PHB yield, reaching 4.1 (g/kg Initial Dry Weight Substrate) (IDWS) after 72 hours, was achieved using a substrate comprising a 1:1 mass ratio of corn germ to corn bran supplemented with 20 % (v/w) cheese whey. Furthermore, PHB production in a 0.5-L packed-bed bioreactor yielded a maximum of 8.4 (g/kg IDWS), indicating a more than 100 % increase in yield after 72 hours, with optimal results achieved at an aeration rate of 0.5 l/(kg IDWS. h)., Competing Interests: Declaration of Competing Interest The 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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metabolic burden is defined by the influence of genetic manipulation and environmental perturbations on the distribution of cellular resources. The rewiring of microbial metabolism for bio-based chemical production often leads to a metabolic burden, followed by adverse physiological effects, such as impaired cell growth and low product yields. Alleviating the burden imposed by undesirable metabolic changes has become an increasingly attractive approach for constructing robust microbial cell factories. In this review, we provide a brief overview of metabolic burden engineering, focusing specifically on recent developments and strategies for diminishing the burden while improving robustness and yield. A variety of examples are presented to showcase the promise of metabolic burden engineering in facilitating the design and construction of robust microbial cell factories. Finally, challenges and limitations encountered in metabolic burden engineering are discussed., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microbial biosurfactant is an emerging vital biomolecule of the 21st century. They are amphiphilic compounds produced by microorganisms and possess unique properties to reduce surface tension activity. The use of microbial surfactants spans most of the industrial fields due to their biodegradability, less toxicity, being environmentally safe, and being synthesized from renewable sources. These would be highly efficient eco-friendly alternatives to petroleum-derived surfactants that would open up new approaches to research on the production of biosurfactants. In the upcoming era, biobased surfactants will become a dominating multifunctional compound in the world market. Research on biosurfactants ranges from the search for novel microorganisms that can produce new molecules, structural and physiochemical characterization of biosurfactants, and fermentation process for enhanced large-scale productivity and green applications. The main goal of this review is to provide an overview of the recent state of knowledge and trends about microbially derived surfactants, various aspects of biosurfactant production, definition, properties, characteristics, diverse advances, and applications. This would lead a long way in the production of biosurfactants as globally successful biomolecules of the current century., (© 2024 Wiley‐VCH GmbH.)

The construction and optimization of microbial cell factories are crucial steps and key technologies in achieving green biomanufacturing. As concern has been aroused regarding the excessive carbon dioxide (CO 2 ) emissions and food security, a new and promising research field, microbial conversion of CO 2 into food compounds, has emerged. The research in this field not only holds significant implications for achieving the carbon peaking and carbon neutrality goals but also plays a role in maintaining food security. This paper provides a comprehensive review and outlook of the research on utilizing CO 2 and its derived low-carbon chemicals for the production of food compounds, focusing on the production of glucose, sugar derivatives, and single-cell proteins and the development of artificial CO 2 fixation pathways.

Glycolic acid is an important chemical product widely used in various fields, including cosmetics, detergents, textiles, and more. Currently, microbial production of glycolic acid has disadvantages such as poor genetic stability, low yield, and high cost. Additionally, whole-cell catalytic production of glycolic acid typically requires the addition of relatively expensive sorbitol as a carbon source, which limits its industrial production. To develop an industrially applicable method for glycolic acid production, this study used ethylene glycol as a substrate to screen the glycolic acid-producing strains through whole-cell catalysis, obtaining a Rhodotorula sp. capable of producing glycolic acid. The strain was then subjected to UV mutagenesis and high throughput screening, and the positive mutant strain RMGly-20 was obtained. After optimization in shake flasks, the glycolic acid titer of RMGly-20 reached 17.8 g/L, a 10.1-fold increase compared to the original strain. Using glucose as the carbon source and employing a fed-batch culture in a 5 L fermenter, strain RMGly-20 produced 61.1 g/L of the glycolic acid. This achievement marks the preliminary breeding of a genetically stable glycolic acid-producing strain using a cheap carbon source, providing a new host for the biosynthesis of glycolic acid and promoting further progress toward industrial production.

D-mannitol is a six-carbon sugar alcohol and one of the most abundant polyols in the nature. With antioxidant and osmotic pressure-regulating effects and non-metabolism by the human body, D-mannitol has been widely used in functional food and pharmaceutical industries. At present, a major way for industrial production of D-mannitol is chemical hydrogenation. In addition, D-Mannitol can be produced by microbial metabolism or catalysis. Compared with the chemical hydrogenation, the microbial methods for synthesizing mannitol do not produce sorbitol as a by-product and have the advantages of mild reaction conditions, strong specificity, and high conversion rate. Microbial fermentation is praised for easy access of strains and raw materials and simple separation of the product. Microbial catalysis usually adopts a multi-enzyme coupling strategy, which uses enzymes produced by engineered bacteria for whole-cell catalysis, and the cofactor recycling pathway is introduced to replenish expensive cofactor. This method can achieve high yields with cheap substrates under mild conditions without the formation of by-products. However, the application of microbial methods in the industrial production of D-mannitol is limited by the high costs of fermentation media and substrates and the long reaction time. This article reviews the reported microbial methods for producing D-mannitol, including the use of high-yielding strains and their fermentation processes, the utilization of low-cost substrates, whole-cell catalytic strategies, and the process control for high productivity. The biosynthesis of mannitol is not only of great significance for promoting industrial upgrading and realizing green manufacturing, but also provides strong support for the development of new bio-based products to meet the growing market demand. With the continuous improvement of technological innovation and industrial chain, it is expected to become one of the main ways of mannitol production in the future.

Microbial production of chemicals from renewable biomass has emerged as a crucial route for sustainable bio-manufacturing. Lignocellulose with a renewable property and wide sources is supposed to be a promising feedstock for the second-generation biorefinery. The efficient co-utilization of mixed sugars from lignocellulosic hydrolysates represents one of the key challenges in reducing the production cost. However, most microorganisms prefer glucose over xylose due to carbon catabolite repression, which constrains the efficiency of lignocellulosic conversion. Therefore, developing the microbial platforms capable of simultaneously utilizing glucose and xylose is paramount for economically viable industrial-scale production. This article reviews the key strategies and studies of metabolic engineering for promoting efficient co-utilization of glucose and xylose by microorganisms. The representative strategies include relieving glucose repression, enhancing xylose transport, constructing xylose metabolic pathways, and directed evolution.

Developing more robust and productive industrial yeast is crucial for high-efficiency biomanufacturing. However, the challenges posed by the long time required and the low abundance of mutations generated through genomewide evolutionary engineering hinder the development and optimization of desired hosts for industrial applications. To address these issues, we present a novel solution called the Genomewide Evolution-based CRISPR/Cas with Donor-free (GEbCD) system, in which nonhomologous-end-joining (NHEJ) repair can accelerate the acquisition of highly abundant yeast mutants. Together with modified rad52 of the DNA double-strand break repair in Saccharomyces cerevisiae , a hypermutation host was obtained with a 400-fold enhanced mutation ability. Under multiple environmental stresses the system could rapidly generate millions of mutants in a few rounds of iterative evolution. Using high-throughput screening, an industrial S. cerevisiae SISc-Δrad52-G4-72 (G4-72) was obtained that is strongly robust and has higher productivity. G4-72 grew stably and produced ethanol efficiently in multiple-stress environments, e.g. high temperature and high osmosis. In a pilot-scale fermentation with G4-72, the fermentation temperature was elevated by 8 °C and ethanol production was increased by 6.9% under the multiple stresses posed by the industrial fermentation substrate. Overall, the GEbCD system presents a powerful tool to rapidly generate abundant mutants and desired hosts, and offers a novel strategy for optimizing microbial chassis with regard to demands posed in industrial applications.

D-glucaric acid is an important organic acid with numerous applications in therapy, food, and materials, contributing significantly to its substantial market value. The biosynthesis of D-glucaric acid (GA) from renewable sources such as glucose has garnered significant attention due to its potential for sustainable and cost-effective production. This review summarizes the current understanding of the cell factories for GA production in different chassis strains, from static to dynamic control strategies for regulating their metabolic networks. We highlight recent advances in the optimization of D-glucaric acid biosynthesis, including metabolic dynamic control, alternative feedstocks, metabolic compartments, and so on. Additionally, we compare the differences between different chassis strains and discuss the challenges that each chassis strain must overcome to achieve highly efficient GA productions. In this review, the processes of engineering a desirable cell factory for highly efficient GA production are just like an epitome of metabolic engineering of strains for chemical biosynthesis, inferring general trends for industrial chassis strain developments., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

We here explore the potential of the fungal genus Aureobasidium as a prototype for a microbial chassis for industrial biotechnology in the context of a developing circular bioeconomy. The study emphasizes the physiological advantages of Aureobasidium, including its polyextremotolerance, broad substrate spectrum, and diverse product range, making it a promising candidate for cost-effective and sustainable industrial processes. In the second part, recent advances in genetic tool development, as well as approaches for up-scaled fermentation, are described. This review adds to the growing body of scientific literature on this remarkable fungus and reveals its potential for future use in the biotechnological industry., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Starch is an attractive feedstock in biorefinery processes, while the low natural conversion rate of most microorganisms limits its applications. Herein, starch metabolic pathway was systematically investigated using Bacillus licheniformis DW2 as the host organism. Initially, the effects of overexpressing amylolytic enzymes on starch hydrolysis were evaluated. Subsequently, the transmembrane transport system and intracellular degradation module were modified to accelerate the uptake of hydrolysates and their further conversion to glucose-6-phosphate. The DW2-derived strains exhibited robust growth in starch medium, and productivity of bacitracin and subtilisin were improved by 38.5% and 32.6%, with an 32.3% and 22.9% increase of starch conversion rate, respectively. Lastly, the employment of engineering strategies enabled another B. licheniformis WX-02 to produce poly-γ-glutamic acid from starch with a 2.1-fold increase of starch conversion rate. This study not only provided excellent B. licheniformis chassis for sustainable bioproduction from starch, but shed light on researches of substrate utilization., Competing Interests: Declaration of competing interest The 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., (Copyright © 2024. Published by Elsevier Ltd.)

Microbes that use the single-carbon substrates methanol and methane offer great promise to bioindustry along with substantial environmental benefits. Methanotrophs and other methylotrophs can be engineered and optimized to produce a wide range of products, from biopolymers to biofuels and beyond. While significant limitations remain, including delivery of single-carbon feedstock to bioreactors, efficient growth, and scale-up, these challenges are being addressed and notable improvements have been rapid. Development of expression chassis, use of genome-scale and regulatory models based on omics data, improvements in bioreactor design and operation, and development of green product recovery schemes are enabling the rapid development of single-carbon bioconversion in the industrial space., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recent SARS-CoV-2 pandemic elevated research interest in microorganism-related diseases, and protective health application importance such as vaccination and immune promoter agents emerged. Among the production methods for proteins, recombinant technology is an efficient alternative and frequently preferred method. However, since the production and purification processes vary due to the protein nature, the effect of these differences on the cost remains ambiguous. In this study, brucellosis and its two important vaccine candidate proteins (rOmp25 and rEipB) with different properties were selected as models, and industrial-scale production processes were compared with the SuperPro Designer ® for estimating the unit production cost. Simulation study showed raw material cost by roughly 60% was one of the barriers to lower-cost production and 52.5 and 559.8 $/g were estimated for rEipB and rOmp25, respectively.

The growing biotechnology industry has focused a lot of attention on biosurfactants because of several advantages over synthetic surfactants. These benefits include worldwide public health, environmental sustainability, and the increasing demand from sectors for environmentally friendly products. Replacement with biosurfactants can reduce upto 8% lifetime CO 2 emissions avoiding about 1.5 million tons of greenhouse gas released into the atmosphere. Therefore, the demand for biosurfactants has risen sharply occupying about 10% (∼10 million tons/year) of the world production of surfactants. Biosurfactants' distinct amphipathic structure, which is made up of both hydrophilic and hydrophobic components, enables these molecules to perform essential functions in emulsification, foam formation, detergency, and oil dispersion-all of which are highly valued characteristic in a variety of sectors. Today, a variety of biosurfactants are manufactured on a commercial scale for use in the food, petroleum, and agricultural industries, as well as the pharmaceutical and cosmetic industries. We provide a thorough analysis of the body of knowledge on microbial biosurfactants that has been gained over time in this research. We also discuss the benefits and obstacles that need to be overcome for the effective development and use of biosurfactants, as well as their present and future industrial uses., Competing Interests: Declaration of Competing Interest Authors have no conflicts of interest., (Copyright © 2024. Published by Elsevier GmbH.)

Astaxanthin is a red xanthophyll with high economic and industrial value in the pharmaceutical, nutraceutical, cosmetic and food industries. In recent years, the biotechnological production of astaxanthin has attracted much attention as a sustainable alternative to the predominating petrochemical-dependent chemical synthesis. In this regard, Xanthophyllomyces dendrorhous is regarded as a promising microorganism for industrial production of astaxanthin. Unfortunately, biotechnological production of the carotenoid is currently expensive. The present study investigated soy molasses (SM) and residual brewers' yeast as cheap fermentation feedstocks for the cultivation of X. dendrorhous and astaxanthin production. Yeast extract was obtained from residual brewers' yeast using various techniques and then combined with SM to formulate a two-component growth medium which was subsequently used to cultivate X. dendrorhous. Generally, the yeast extract produced from residual brewers' yeast supported X. dendrorhous growth and astaxanthin production at levels comparable to those seen with commercial yeast extract. Overall, cultivating X. dendrorhous in an SM-based medium containing 5% SM and 0.2% yeast extract obtained from residual brewers' yeast resulted in significantly higher (> 20% more) biomass accumulation compared to the control media (YPD). A similar slightly higher astaxanthin output (up to 14% more) was recorded in the SM-based medium compared to YPD. The formulated cultivation medium in this study provides an opportunity to reduce the production cost of astaxanthin from X. dendrorhous while simultaneously reducing the environmental impact related to the disposal of the industrial waste used as feedstock. KEY POINTS: • Cheap culture media were formulated from soy molasses and brewers' spent yeast • The formulated medium resulted in at least 20% more biomass than the control • Up to 14% more astaxanthin was produced in molasses-based medium., (© 2024. The Author(s).)

Extremophilic microorganisms play a key role in understanding how life on Earth originated and evolved over centuries. Their ability to thrive in harsh environments relies on a plethora of mechanisms developed to survive at extreme temperatures, pressures, salinity, and pH values. From a biotechnological point of view, thermophiles are considered a robust tool for synthetic biology as well as a reliable starting material for the development of sustainable bioprocesses. This review discusses the current progress in the biomanufacturing of high-added bioproducts from thermophilic microorganisms and their industrial applications.

Traditional Chinese food therapies often motivate the development of modern medicines, and learning from them will bring bright prospects. Monascus, a conventional Chinese fungus with centuries of use in the food industry, produces various metabolites, including natural pigments, lipid-lowering substances, and other bioactive ingredients. Recent Monascus studies focused on the metabolite biosynthesis mechanisms, strain modifications, and fermentation process optimizations, significantly advancing Monascus development on a lab scale. However, the advanced manufacture for Monascus is lacking, restricting its scale production. Here, the synthetic biology techniques and their challenges for engineering filamentous fungi were summarized, especially for Monascus. With further in-depth discussions of automatic solid-state fermentation manufacturing and prospects for combining synthetic biology and process intensification, the industrial scale production of Monascus will succeed with the help of Monascus improvement and intelligent fermentation control, promoting Monascus applications in food, cosmetic, agriculture, medicine, and environmental protection industries., (© 2024 Wiley‐VCH GmbH.)

Biochemicals are widely used in the medicine and food industries and are more efficient and safer than synthetic chemicals. The amphipathic surfactants can interact with the microorganisms and embed the extracellular metabolites, which induce microbial metabolites secretion and biosynthesis, performing an attractive prospect of promoting the biochemical production. However, the commonness and differences of surfactant-mediated bio-manufacture in various fields are largely unexplored. Accordingly, this review comprehensively summarized the properties of surfactants, different application scenarios of surfactant-meditated bio-manufacture, and the mechanism of surfactants increasing metabolites production. Various biochemical productions such as pigments, amino acids, and alcohols could be enhanced using the cloud point and the micelles of surfactants. Besides, the amphiphilicity of surfactants also promoted the utilization of fermentation substrates, especially lignocellulose and waste sludge, by microorganisms, indirectly increasing the metabolites production. The increase in target metabolites production was attributed to the surfactants changing the permeability and composition of the cell membrane, hence improving the secretion ability of microorganisms. Moreover, surfactants could regulate the energy metabolism, the redox state and metabolic flow in microorganisms, which induced target metabolites synthesis. This review aimed to broaden the application fields of surfactants and provide novel insights into the production of microbial biochemicals., Competing Interests: Declaration of competing interest The 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The global food system is shifting towards cellular agriculture, a second domestication marked by cultivating microorganisms and tissues for sustainable food production. This involves tissue engineering, precision fermentation, and microbial biomass fermentation to establish food value chains independent of traditional agriculture. However, these techniques rely on growth media sourced from agricultural, chemical (fossil fuels), and mining supply chains, raising concerns about land use competition, emissions, and resource depletion. Fermentable sugars, nitrogen, and phosphates are key ingredients derived from starch crops, energy-intensive fossil fuel based processes, and finite phosphorus resources, respectively. This review explores sustainable alternatives to reduce land use and emissions associated with cellular agriculture media ingredients. Sustainable alternatives to first generation sugars (lignocellulosic substrates, sidestreams, and gaseous feedstocks), sustainable nitrogen sources (sidestreams, green ammonia, biological nitrogen fixation), and efficient use of phosphates are reviewed. Especially cellulosic sugars, gaseous chemoautotrophic feedstocks, green ammonia, and phosphate recycling are the most promising technologies but economic constraints hinder large-scale adoption, necessitating more efficient processes and cost reduction. Collaborative efforts are vital for a biotechnological future grounded in sustainable feedstocks, mitigating competition with agricultural land and emissions., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bioethanol is a sustainable energy alternative and can contribute to global greenhouse-gas emission reductions by over 60%. Its industrial production faces various bottlenecks, including sub-optimal efficiency resulting from bacteria. Broad-spectrum removal of these contaminants results in negligible gains, suggesting that the process is shaped by ecological interactions within the microbial community. Here, we survey the microbiome across all process steps at two biorefineries, over three timepoints in a production season. Leveraging shotgun metagenomics and cultivation-based approaches, we identify beneficial bacteria and find improved outcome when yeast-to-bacteria ratios increase during fermentation. We provide a microbial gene catalogue which reveals bacteria-specific pathways associated with performance. We also show that Limosilactobacillus fermentum overgrowth lowers production, with one strain reducing yield by ~5% in laboratory fermentations, potentially due to its metabolite profile. Temperature is found to be a major driver for strain-level dynamics. Improved microbial management strategies could unlock environmental and economic gains in this US $ 60 billion industry enabling its wider adoption., (© 2024. The Author(s).)

In silico tools such as genome-scale metabolic models have shown to be powerful for metabolic engineering of microorganisms. Saccharomyces pastorianus is a complex aneuploid hybrid between the mesophilic Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus . This species is of biotechnological importance because it is the primary yeast used in lager beer fermentation and is also a key model for studying the evolution of hybrid genomes, including expression pattern of ortholog genes, composition of protein complexes, and phenotypic plasticity. Here, we created the iSP_1513 GSMM for S. pastorianus CBS1513 to allow top-down computational approaches to predict the evolution of metabolic pathways and to aid strain optimization in production processes. The iSP_1513 comprises 4,062 reactions, 1,808 alleles, and 2,747 metabolites, and takes into account the functional redundancy in the gene-protein-reaction rule caused by the presence of orthologous genes. Moreover, a universal algorithm to constrain GSMM reactions using transcriptome data was developed as a python library and enabled the integration of temperature as parameter. Essentiality data sets, growth data on various carbohydrates and volatile metabolites secretion were used to validate the model and showed the potential of media engineering to improve specific flavor compounds. The iSP_1513 also highlighted the different contributions of the parental sub-genomes to the oxidative and non-oxidative parts of the pentose phosphate pathway. Overall, the iSP_1513 GSMM represent an important step toward understanding the metabolic capabilities, evolutionary trajectories, and adaptation potential of S. pastorianus in different industrial settings., Importance: Genome-scale metabolic models (GSMM) have been successfully applied to predict cellular behavior and design cell factories in several model organisms, but no models to date are currently available for hybrid species due to their more complex genetics and general lack of molecular data. In this study, we generated a bespoke GSMM, iSP_1513, for this industrial aneuploid hybrid Saccharomyces pastorianus , which takes into account the aneuploidy and functional redundancy from orthologous parental alleles. This model will (i) help understand the metabolic capabilities and adaptive potential of S. pastorianus (domestication processes), (ii) aid top-down predictions for strain development (industrial biotechnology), and (iii) allow predictions of evolutionary trajectories of metabolic pathways in aneuploid hybrids (evolutionary genetics)., Competing Interests: The authors declare no conflict of interest.

Butyl butyrate is a short-chain fatty acid ester (C8) with a fruity aroma. It has broad prospects in the fields of foods, cosmetics and biofuels. At present, butyl butyrate is produced by chemical synthesis in the industry, but it is highly dependent on petroleum-based products. The growing concerns regarding the future scarcity of fossil fuels have been strongly promoted the transition from traditional fossil fuels and products to renewable bioenergy and biochemicals. Therefore, it is necessary to develop a green biochemical technology to replace traditional petroleum-based materials. In recent years, microorganisms such as Escherichia coli and Clostridium have been engineered to serve as cell factories for the sustainable one-pot production of short-chain fatty acid esters, including butyl butyrate. This opinion highlights the recent development in the use of lipases and alcohol acyltransferases (AATs) for butyl butyrate production in microbial fermentation, as well as future perspectives., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Levan, a β-(2,6)-linked fructose polymer, exhibits diverse properties that impart versatility, rendering it a highly sought-after biopolymer with various industrial applications. Levan can be produced by various microorganisms using sucrose, food industry byproducts and agricultural wastes. Microbial levan represents the most potent cost-effective process for commercial-scale levan production. This study reviews the optimization of levan production by understanding its biosynthesis, physicochemical properties and the fermentation process. In addition, genetic and protein engineering for its increased production and emerging methods for its detection are introduced and discussed. All of these comprehensive studies could serve as powerful tools to optimize levan production and broaden its applications across various industries., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

During the epoch of sustainable development, leveraging cellular systems for production of diverse chemicals via fermentation has garnered attention. Industrial fermentation, extending beyond strain efficiency and optimal conditions, necessitates a profound understanding of microorganism growth characteristics. Specific growth rate (SGR) is designated as a key variable due to its influence on cellular physiology, product synthesis rates and end-product quality. Despite its significance, the lack of real-time measurements and robust control systems hampers SGR control strategy implementation. The narrative in this contribution delves into the challenges associated with the SGR control and presents perspectives on various control strategies, integration of soft-sensors for real-time measurement and control of SGR. The discussion highlights practical and simple SGR control schemes, suggesting their seamless integration into industrial fermenters. Recommendations provided aim to propose new algorithms accommodating mechanistic and data-driven modelling for enhanced progress in industrial fermentation in the context of sustainable bioprocessing., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The hyphal surface of cells of filamentous fungi is covered with cell wall, which is mainly composed of polysaccharides. Since the cell wall is the first structure to come in contact with the infection host, the environment, and the fungus itself, the elucidation of the cell wall structure and biogenesis is essential for understanding fungal ecology. Among filamentous fungi, the genus Aspergillus is an important group in the industrial, food, and medical fields. It is known that Aspergillus species form hyphal pellets in shake liquid culture. The authors previously found the role of α-1,3-glucan in hyphal aggregation in Aspergillus species. In addition, extracellular polysaccharide galactosaminogalactan contributed to hyphal aggregation as well, and dual disruption of biosynthesis genes of α-1,3-glucan and galactosaminogalactan resulted in complete hyphal dispersion in shake liquid culture. The characteristic of mycelia to form pellets under liquid culture conditions was the main reason why the growth measurement methods used for unicellular organisms could not be applied. We reported that hyphal growth of the dual disruption mutant could be measured by optical density. A real-time plate reader could be used to determine the growth curve of the mycelial growth of the dual disruption mutant. This measurement approach not only provides basic microbiological insights in filamentous fungi, but also has the potential to be applied to high-throughput screening of anti-Aspergillus drugs.

This work explores astaxanthin (AXT), a valuable xanthophyll ketocarotenoid pigment with significant health benefits and diverse applications across various industries. It discusses the prevalence of synthetic AXT, and the development of natural-based alternatives derived from microorganisms such as microalgae, bacteria, and yeast. The chapter examines the potential of microbial AXT production, highlighting the advantages and challenges associated with natural AXT. Key microorganisms like Haematococcus pluvialis, Paracoccus carotinifaciens, and Phaffia rhodozyma are emphasized for their role in commercially producing this valuable ketocarotenoid. The narrative covers the complexities and opportunities in microbial AXT production, from cell structure implications to downstream processing strategies. Additionally, the chapter addresses current applications, commercialization trends, and market dynamics of natural microbial AXT, emphasizing the importance of cost-effective production, regulatory compliance, and technological advancements to reduce the market cost of the final product. As demand for natural microbial-based AXT rises, this chapter envisions a future where research, innovation, and collaboration drive sustainable and competitive microbial AXT production, fostering growth in this dynamic market., Competing Interests: Conflicts of interest There are no conflicts to declare., (Copyright © 2024. Published by Elsevier Inc.)

Background: Stress-tolerant yeasts are highly desirable for cost-effective bioprocessing. Several strategies have been documented to develop robust yeasts, such as genetic and metabolic engineering, artificial selection, and natural selection strategies, among others. However, the significant drawbacks of such techniques have motivated the exploration of naturally occurring stress-tolerant yeasts. We previously explored the biodiversity of non-conventional dung beetle-associated yeasts from extremophilic and pristine environments in Botswana (Nwaefuna AE et.al., Yeast, 2023). Here, we assessed their tolerance to industrially relevant stressors individually, such as elevated concentrations of osmolytes, organic acids, ethanol, and oxidizing agents, as well as elevated temperatures., Results: Our findings suggest that these dung beetle-associated yeasts tolerate various stresses comparable to those of the robust bioethanol yeast strain, Saccharomyces cerevisiae (Ethanol Red™). Fifty-six percent of the yeast isolates were tolerant of temperatures up to 42 °C, 12.4% of them could tolerate ethanol concentrations up to 9% (v/v), 43.2% of them were tolerant to formic acid concentrations up to 20 mM, 22.7% were tolerant to acetic acid concentrations up to 45 mM, 34.0% of them could tolerate hydrogen peroxide up to 7 mM, and 44.3% of the yeasts could tolerate osmotic stress up to 1.5 M., Conclusion: The ability to tolerate multiple stresses is a desirable trait in the selection of novel production strains for diverse biotechnological applications, such as bioethanol production. Our study shows that the exploration of natural diversity in the search for stress-tolerant yeasts is an appealing approach for the development of robust yeasts., (© 2023. The Author(s).)

Background: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and β-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and β-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production., Results: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system., Conclusions: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future., (© 2023. The Author(s).)

The development of synthetic biology has greatly promoted the construction of microbial cell factories, providing an important strategy for green and efficient chemical production. However, the bottleneck of poor tolerance to harsh industrial environments has become the key factor hampering the productivity of microbial cells. Adaptive evolution is an important method to domesticate microorganisms for a certain period by applying targeted selection pressure to obtain desired phenotypic or physiological properties that are adapted to a specific environment. Recently, with the development of technologies such as microfluidics, biosensors, and omics analysis, adaptive evolution has laid the foundation for efficient productivity of microbial cell factories. Herein, we discuss the key technologies of adaptive evolution and their important applications in improvement of environmental tolerance and production efficiency of microbial cell factories. Moreover, we looked forward to the prospects of adaptive evolution to realize industrial production by microbial cell factories.

Production of amylases by fungi under solid-state fermentation is considered the best methodology for commercial scaling that addresses the ever-escalating needs of the worldwide enzyme market. Here response surface methodology (RSM) was used for the optimization of process variables for α-amylase enzyme production from Trichoderma virens using watermelon rinds (WMR) under solid-state fermentation (SSF). The statistical model included four variables, each detected at two levels, followed by model development with partial purification and characterization of α-amylase. The partially purified α-amylase was characterized with regard to optimum pH, temperature, kinetic constant, and substrate specificity. The results indicated that both pH and moisture content had a significant effect (P < 0.05) on α-amylase production (880 U/g) under optimized process conditions at a 3-day incubation time, moisture content of 50%, 30 °C, and pH 6.98. Statistical optimization using RSM showed R 2 values of 0.9934, demonstrating the validity of the model. Five α-amylases were separated by using DEAE-Sepharose and characterized with a wide range of optimized pH values (pH 4.5-9.0), temperature optima (40-60 °C), low Km values (2.27-3.3 mg/mL), and high substrate specificity toward large substrates. In conclusion, this study presents an efficient and green approach for utilization of agro-waste for production of the valuable α-amylase enzyme using RSM under SSF. RSM was particularly beneficial for the optimization and analysis of the effective process parameters., (© 2021. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Evolutionary engineering of microbes provides a powerful tool for untargeted optimization of (engineered) cell factories and identification of genetic targets for further research. Directed evolution is an intrinsically time-intensive effort, and automated methods can significantly reduce manual labor. Here, design considerations for various evolutionary engineering methods are described, and generic workflows for batch-, chemostat-, and accelerostat-based evolution in automated bioreactors are provided. These methods can be used to evolve yeast cultures for >1000 generations and are designed to require minimal manual intervention., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Metabolic engineering can have a pivotal role in increasing the environmental sustainability of the transportation and chemical manufacturing sectors. The field has already developed engineered microorganisms that are currently being used in industrial-scale processes. However, it is often challenging to achieve the titres, yields and productivities required for commercial viability. The efficiency of microbial chemical production is usually dependent on the physiological traits of the host organism, which may either impose limitations on engineered biosynthetic pathways or, conversely, boost their performance. In this Review, we discuss different aspects of microbial physiology that often create obstacles for metabolic engineering, and present solutions to overcome them. We also describe various instances in which natural or engineered physiological traits in host organisms have been harnessed to benefit engineered metabolic pathways for chemical production., (© 2021. Springer Nature Limited.)

Marine microorganisms represent virtually unlimited sources of novel biological compounds and can survive extreme conditions. Cellulases, a group of enzymes that are able to degrade cellulosic materials, are in high demand in various industrial and biotechnological applications, such as in the medical and pharmaceutical industries, food, fuel, agriculture, and single-cell protein, and as probiotics in aquaculture. The cellulosic biopolymer is a renewable resource and is a linearly arranged polysaccharide of glucose, with repeating units of disaccharide connected via β-1,4-glycosidic bonds, which are broken down by cellulase. A great deal of biodiversity resides in the ocean, and marine systems produce a wide range of distinct, new bioactive compounds that remain available but dormant for many years. The marine environment is filled with biomass from known and unknown vertebrates and invertebrate microorganisms, with much potential for use in medicine and biotechnology. Hence, complex polysaccharides derived from marine sources are a rich resource of microorganisms equipped with enzymes for polysaccharides degradation. Marine cellulases' extracts from the isolates are tested for their functional role in degrading seaweed and modifying wastes to low molecular fragments. They purify and renew environments by eliminating possible feedstocks of pollution. This review aims to examine the various types of marine cellulase producers and assess the ability of these microorganisms to produce these enzymes and their subsequent biotechnological applications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The genus Streptomyces is intensively studied due to its excellent ability to produce secondary metabolites with diverse bioactivities. In particular, adequate precursors of secondary metabolites as well as sophisticated post modification systems make some high-yield industrial strains of Streptomyces the promising chassis for the heterologous production of natural products. However, lack of efficient genetic tools for the manipulation of industrial strains, especially the episomal vector independent tools suitable for large DNA fragment deletion, makes it difficult to remold the metabolic pathways and streamline the genomes in these strains. In this respect, we developed an efficient deletion system independent of the episomal vector for large DNA fragment deletion. Based on this system, four large segments of DNA, ranging in length from 10 kb to 200 kb, were knocked out successfully from three industrial Streptomyces strains without any marker left. Notably, compared to the classical deletion system used in Streptomyces , this deletion system takes about 25% less time in our cases. This work provides a very effective tool for further genetic engineering of the industrial Streptomyces .

Baculoviruses have been applied for biocontrol of agricultural pests, such as velvetbean caterpillar (Anticarsia gemmatalis) and fall armyworm (Spodoptera frugiperda). Cell culture is an interesting approach for large-scale production of these viruses. Co-infection of a host cell with two distinct viruses can contribute to reduce costs due to saving cell culture media, bioreactor space and the resulting co-occluded polyhedra may help to reduce final biopesticide costs. The baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) were chosen to test a model for in vitro co-infection in SF21 cells. Different proportions of SfMNPV/AgMNPV were evaluated along three in vitro passages by optical microscopy analysis of cells and real-time PCR (qPCR) of DNA obtained from budded viruses (BVs) and occlusion bodies (OBs). The kinetics of viral protein synthesis was carried out for analysis of the co-infection in first passage and bioassays with the resulting OBs were performed against A. gemmatalis and S. frugiperda larvae. The results demonstrated successful co-infection in these cells. The quantity of SfMNPV and AgMNPV in supernatants and sediments tends to be maintained stable during the three passages, although the amount of AgMNPV was higher than SfMPNV in most of the experiments. Analysis of the kinetics of radiolabed proteins showed that the cell protein synthesis was shut off and two distinct bands of about 30 kDa, regarded to be the polyhedrin of each virus, were strongly detected at 48 and 72 hp.i. Although the pathogenicity of the produced viruses was not completely satisfactory, the bioassays confirmed occurrence of co-infected larvae with disproportional amount of each virus., (© 2021. Sociedade Brasileira de Microbiologia.)

The edible filamentous fungi are hot candidate for future supply of functional food and feed with e.g. protein, essential amino acids, and compounds with immunostimulant activity. L-carnitine that plays a crucial role in energy metabolism represents a functional compound normally produced by Zygomycetes filamentous fungus Rhizopus oligosporus in solid-state fermentation. The present study provides the first insights on production of L-carnitine-enriched edible fungal biomass through submerged cultivation of several Ascomycetes and Zygomycetes including Aspergillus oryzae , Neurospora intermedia , Rhizopus oryzae , and Rhizopus oligosporus . A. oryzae with 3 mg L-carnitine yield per gram of fungal biomass, indicates great potential on production of this bioactive compound which is remarkably higher than the other tested fungi in this work and also previous studies. In addition to fungal strain, other factors such as cultivation time and presence of yeast extract were found to play a role. Further studies on submerged growth optimization of A . oryzae in both high-quality recipes and in medium based on low-value substrates are proposed in order to clarify its potential for production of L-carnitine-enriched fungal biomass.

Since the 20th century, plastics that are widely being used in general life and industries are causing enormous plastic waste problems since improperly discarded plastics barely degrade and decompose. Thus, the demand for polyhydroxyalkanoates (PHAs), biodegradable polymers with material properties similar to conventional petroleum-based plastics, has been increased so far. The microbial production of PHAs is an environment-friendly solution for the current plastic crisis, however, the carbon sources for the microbial PHA production is a crucial factor to be considered in terms of carbon-neutrality. One‑carbon (C1) resources, such as methane, carbon monoxide, and carbon dioxide, are greenhouse gases and are abundantly found in nature and industry. C1 resources as the carbon sources for PHA production have a completely closed carbon loop with much advances; i) fast carbon circulation with direct bioconversion process and ii) simple fermentation procedure without sterilization as non-preferable nutrients. This review discusses the biosynthesis of PHAs based on C1 resource utilization by wild-type and metabolically engineered microbial host strains via biorefinery processes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A strategy by exogenous addition of quorum sensing molecule farnesol to improve the production, antioxidant activity and antitumor activity of extracellular polysaccharide (EPS) of Grifola frondosa by liquid fermentation was proposed in the study. The highest yield of EPS induced by farnesol was 1.25 g/L, which was 150% higher than that of the control. Four polysaccharides including EPS-C-0M, EPS-C-0.2M, EPS-F-0M and EPS-F-0.2M were extracted and purified under the conditions of control and farnesol respectively. The physicochemical properties, antioxidant activities and antitumor activities were studied. Their chemical composition differed in sugar, protein and uronic acid contents, and they were composed of six constituent monosaccharides with different ratios, with the average molecular weights of 1.12 × 10 3 , 1.89 × 10 3 , 1.41 × 10 3 and 2.02 × 10 3 kDa, respectively. They presented similar FT-IR spectra, but different surface morphology. Antioxidant experiments showed that they had strong scavenging activities on ABTS + , hydroxyl radical, O 2- and DPPH radical. Antitumor experiments showed that they had strong inhibitory effects on human cervical cancer (HeLa) cells and human liver cancer cells (HepG 2 ) cells. Among the four polysaccharides, EPS-F-0.2M showed the highest antioxidant and antitumor activities, indicating that farnesol could regulate the biological activity of EPS by affecting structure and properties. These results demonstrated that appropriate adjustment of culture conditions had potential application in the development of polysaccharides with high antioxidant and antitumor activity. It provided a new strategy to enhance the production and bioactivity of edible and medicinal fungal polysaccharides by using quorum sensing molecules., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A cellulose-producing bacterium Komagataeibacter rhaeticus K15 was isolated from kombucha tea, and its metabolic pathways and cellulose synthesis operon were analyzed by genome sequencing. Different from the reported K. rhaeticus, the K15 produced little gluconic acid (2.26 g/L) when glucose was the sole carbon source and has the capacity for high cellulose production (4.76 g/L) with other carbon sources. Furthermore, six nitrogen-fixing genes were found to be responsible for the survival of K15 on a nitrogen-free medium. Based on its fermentation characteristics, K15 was cultured in a kitchen waste medium as a strategy for green and sustainable bacterial cellulose production. The SEM, XRD, and FTIR results indicated that synthesized cellulose has a mean diameter of 40-50 nm nanofiber, good crystallinity, and the same chemical structure. The K15 strain provides a highly viable alternative strategy to reduce the costs of bacterial cellulose production using agro-industrial residues as nutrient sources., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Polyhydroxybutyrate (PHB) production by the cyanobacterium cf. Anabaena sp. was here studied by varying the medium composition and the carbon source used to induce mixotrophic growth conditions. The highest PHB productivity (0.06 g PHB g biomass -1 d -1 ) was observed when cultivating cf. Anabaena sp. in phosphorus-free medium and in the presence of sodium acetate (5.0 g L -1 concentration), after an incubation period of 7 days. A content of 40% of PHB on biomass, a dry weight of 0.1 g L -1 , and a photosynthetic efficiency equal to the control were obtained. The cyanobacterium was then grown on a larger scale (10 L) to evaluate the characteristics of the produced PHB in relation to the main composition of the biomass (the content of proteins, polysaccharides, and lipids): after an incubation period of 7 days, a content of 6% of lipids (52% of which as unsaturated fatty acids with 18 carbon atoms), 12% of polysaccharides, 28% of proteins, and 46% of PHB was reached. The extracted PHB had a molecular weight of 3 MDa and a PDI of 1.7. These promising results demonstrated that cf. Anabaena sp. can be included among the Cyanobacteria species able to produce polyhydroxyalkanoates (PHAs) either in photoautotrophic or mixotrophic conditions, especially when it is grown under phosphorus-free conditions., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The green biocatalyst nitrile hydratase (NHase) is able to bio-transform 3-cyanopyridine into nicotinamide. As the NHase reaction is exothermic, an enzyme with high activity and stability is needed for nicotinamide production. In this study, we used sequence analysis and site-directed mutagenesis to generate a mutant of thermophilic NHase from Pseudonocardia thermophila JCM3095 with substantially enhanced activity and developed a powerful process for nicotinamide bio-production. The specific activity of αF126Y/αF168Y mutant was successfully increased by 3.98-fold over that of the wild-type enzyme. The half-life of such mutant was longer than 2 h, which was comparable to its parent enzyme. The relative activity of the αF126Y/αF168Y mutant after treatment with 1 M 3-cyanopyridine and 2 M nicotinamide was 73.2% and 63.7%, respectively, showing minor loss of its original stability. Structural analysis demonstrated that hydrogen bonds at the active site and α-β subunit interface of the NHase contribute to the improved activity and the maintenance of stability. Escherichia coli transformant harboring the mutant NHase was used for nicotinamide bio-production, yielding a nicotinamide productivity of 251.1 g/(L·h), which is higher than the productivity obtained using other NHase-containing strains and transformants. The newly established variant is therefore a promising alternative for the industrial production of nicotinamides., (Copyright © 2021. Published by Elsevier B.V.)

Polyhydroxyalkanoate (PHA), a class of biopolyester synthesized by various bacteria, is considered as an alternative to petroleum-based plastics because of its excellent physochemical and material properties. Pseudomonas putida KT2440 can produce medium-chain-length PHA (mcl-PHA) from glucose, fatty acid and glycerol, and its whole-genome sequences and cellular metabolic networks have been intensively researched. In this study, we aim to improve the PHA yield of P. putida KT2440 using a novel promoter engineering-based strategy. Unlike previous studies, endogenous strong promoters screening from P. putida KT2440 instead of synthetic or exogenous promoters was applied to the optimization of PHA biosynthesis pathway. Based on RNA-seq and promoter prediction, 30 putative strong promoters from P. putida KT2440 were identified. Subsequently, the strengths of these promoters were characterized by reporter gene assays. Furthermore, each of 10 strong promoters screened by transcriptional level and GFP fluorescence was independently inserted into upstream of PHA synthase gene (phaC1) on chromosome. As a result, the transcriptional levels of the phaC1 and phaC2 genes in almost all of the promoter-substituted strains were improved, and the relative PHA yields of the three promoter-substituted strains KTU-P1C1, KTU-P46C1 and KTU-P51C1 were improved obviously, reaching 30.62 wt%, 33.24 wt% and 33.29 wt% [the ratio of PHA weight to cell dry weight (CDW)], respectively. By further deletion of the glucose dehydrogenase gene in KTU-P1C1, KTU-P46C1 and KTU-P51C1, the relative PHA yield of the resulting mutant strain KTU-P46C1-∆gcd increased by 5.29% from 33.24% to 38.53%. Finally, by inserting P46 into upstream of pyruvate dehydrogenase gene in the genome of KTU-P46C1-∆gcd, the relative PHA yield and CDW of the resulting strain KTU-P46C1A-∆gcd reached nearly 42 wt% and 4.06 g/l, respectively, which increased by 90% and 40%, respectively, compared with the starting strain KTU. In particular, the absolute PHA yield of KTU-P46C1A-∆gcd reached 1.7 g/l, with a 165% improvement compared with the strain KTU. Herein, we report the highest PHA yield obtained by P. putida KT2440 in shake-flask fermentation to date. We demonstrate for the first time the effectiveness of endogenous strong promoters for improving the PHA yield and biomass of P. putida KT2440. More importantly, our findings highlight great potential of this strategy for enhanced production of secondary metabolites and heterologous proteins in P. putida KT2440., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nanocellulose derived from microorganism is crucial bio-based products due to its unique physicochemical and mechanical properties for material science. Thus, optimizing bacterial cellulose (BNC) production is essential to widen applications and reduce production cost. Using various carbon sources derive from fruits as alternatives for synthesizing BNC could produce a low-cost BNC with comparable properties. Although Komagataeibacter xylinus grown in different natural juices, including clarified juice (CJ), sugarcane juice (SC) and coconut juice (CN) demonstrated a lower yield than that of control medium (HS), FTIR confirmed no change in chemical functional groups of BNCs. Similarly, different sugar sources have slightly effects on mechanical and thermal properties of BNC. However, the internal morphology illustrated the pore structure in oval shape for HS and CN while CJ and SC resulted in irregular pores which could lead to the highest crystallinity index value for BNC from HS compared to that from alternative media., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chromosomal integration of exogenous genes is preferred for industrially related fermentation, as plasmid-mediated fermentation leads to extra metabolic burden and genetic instability. Moreover, with the development and advancement of genome engineering and gene editing technologies, inserting genes into chromosomes has become more convenient; integration expression is extensively utilized in microorganisms for industrial bioproduction and expected to become the trend of recombinant protein expression. However, in actual research and application, it is important to enhance the expression of heterologous genes at the host genome level. Herein, we summarized the basic principles and characteristics of genomic integration; furthermore, we highlighted strategies to improve the expression of chromosomal integration of genes and pathways in host strains from three aspects, including chassis cell optimization, regulation of expression elements in gene expression cassettes, optimization of gene dose level and integration sites on chromosomes. Moreover, we reviewed and summarized the relevant studies on the application of integrated expression in the exploration of gene function and the various types of industrial microorganism production. Consequently, this review would serve as a reference for the better application of integrated expression.

Background: Microbially induced calcite precipitation (MICP) is an ancient property of bacteria, which has recently gained considerable attention for biotechnological applications. It occurs as a by-product of bacterial metabolism and involves a combination of chemical changes in the extracellular environment, e.g. pH increase, and presence of nucleation sites on the cell surface or extracellular substances produced by the bacteria. However, the molecular mechanisms underpinning MICP and the interplay between the contributing factors remain poorly understood, thus placing barriers to the full biotechnological and synthetic biology exploitation of bacterial biomineralisation., Results: In this study, we adopted a bottom-up approach of systematically engineering Bacillus subtilis, which has no detectable intrinsic MICP activity, for biomineralisation. We showed that heterologous production of urease can induce MICP by local increases in extracellular pH, and this can be enhanced by co-expression of urease accessory genes for urea and nickel uptake, depending on environmental conditions. MICP can be strongly enhanced by biofilm-promoting conditions, which appeared to be mainly driven by production of exopolysaccharide, while the protein component of the biofilm matrix was dispensable. Attempts to modulate the cell surface charge of B. subtilis had surprisingly minor effects, and our results suggest this organism may intrinsically have a very negative cell surface, potentially predisposing it for MICP activity., Conclusions: Our findings give insights into the molecular mechanisms driving MICP in an application-relevant chassis organism and the genetic elements that can be used to engineer de novo or enhanced biomineralisation. This study also highlights mutual influences between the genetic drivers and the chemical composition of the surrounding environment in determining the speed, spatial distribution and resulting mineral crystals of MICP. Taken together, these data pave the way for future rational design of synthetic precipitator strains optimised for specific applications., (© 2021. The Author(s).)

Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency., (© 2021. The Author(s).)

Because contaminated livestock trailers are a significant risk for transmitting viruses between herds, various methods of washing, disinfecting, and thermo-assisted drying and decontamination (TADD) have been evaluated for their effectiveness in inactivating porcine reproductive and respiratory syndrome virus (PRRSV) on contaminated surfaces. Information on when to expect negative qRT-PCR results after adequate trailer sanitation is lacking. The objective of this study was to evaluate whether there are conditions associated with washing-disinfectant-TADD procedures that will consistently produce a negative qRT-PCR result for the purpose of monitoring compliance with trailer sanitation and decontamination protocols for PRRSV on metal surfaces. 144 diamond plate aluminum coupons were spiked with PRRSV or phosphate-buffered saline (PBS) and treated with a designated disinfectant protocol. Disinfectants evaluated included multiple accelerated® hydrogen peroxide (AHP) disinfectants and a quaternary ammonium and glutaraldehyde combination disinfectant. Disinfectant was applied for 5 or 60 minutes of contact time at either 20 °C or -10 °C in a matrix of feces or PBS. All coupons were heated until the surface temperature of the coupon reached 71 °C and then held for 10 minutes to simulate TADD under field conditions. Post-treatment swabs for all treatment groups, except negative control groups, were positive by PRRSV qRT-PCR. Under the conditions evaluated in this study, consistently negative qRT-PCR results after treatments were not found. Therefore, for the purpose of monitoring compliance with trailer sanitation and decontamination protocols for PRRSV, alternatives to qRT-PCR should be explored., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Optimising the function of a protein of length N amino acids by directed evolution involves navigating a 'search space' of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is 'making such biology predictable'. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing kcat in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering., (© 2021 The Author(s).)

Biohydrometallurgy recovers metals through microbially mediated processes and has been traditionally applied for the extraction of base metals from low-grade sulfidic ores. New investigations explore its potential for other types of critical resources, such as rare earth elements. In recent times, the interest in rare earth elements (REEs) is growing due to of their applications in novel technologies and green economy. The use of biohydrometallurgy for extracting resources from waste streams is also gaining attention to support innovative mining and promote a circular economy. The increase in wastes containing REEs turns them into a valuable alternative source. Most REE ores and industrial residues do not contain sulfides, and bioleaching processes use autotrophic or heterotrophic microorganisms to generate acids that dissolve the metals. This review gathers information towards the recycling of REE-bearing wastes (fluorescent lamp powder, spent cracking catalysts, e-wastes, etc.) using a more sustainable and environmentally friendly technology that reduces the impact on the environment.

Previous studies have shown MALDI-TOF MS to be a powerful tool in wine yeast identification and potential prediction of application. However, it is also established that substrate composition influences protein expression, but the degree to which this may affect MALDI-TOF spectra (and analytical results thereof) has not been fully explored. To further inform assay optimisation, the influence on MALDI-TOF spectra was determined using eight Saccharomyces strains of diverse origins cultivated on grape juices from Pinot Noir and Chardonnay varieties, synthetic grape juice, and laboratory-grade artificial culture media (YPD broth and agar). Our results demonstrated significant influences of culture media on strain MALDI-TOF spectra. Yeast culture on YPD agar is recommended for taxonomic studies, with YPD broth culture of S. cerevisiae offering improved intra-subspecific differentiation Furthermore, our data supported a correlation between MALDI spectra and the potential industrial application of individual yeast strains., (Copyright © 2021 Elsevier B.V. All rights reserved.)