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Erythritol is a four-carbon sugar alcohol naturally produced by microorganisms as an osmoprotectant. As a new sugar substitute, erythritol has recently been popular on the ingredient market because of its unique nutritional characteristics. Even though the history of erythritol biosynthesis dates from the turn of the twentieth century, scientific advancement has lagged behind other polyols due to the relative complexity of making it. In recent years, biosynthetic methods for erythritol have been rapidly developed due to an increase in market demand, a better understanding of metabolic pathways, and the rapid development of genetic engineering tools. This paper reviews the history of industrial strain development and focuses on the underlying mechanism of high erythritol production by strains gained through screening or mutagenesis. Meanwhile, we highlight the metabolic pathway knowledge of erythritol biosynthesis in microorganisms and summarize the metabolic engineering and research progress on critical genes involved in different stages of the synthetic pathway. Lastly, we talk about the still-contentious issues and promising future research directions that will help break the erythritol production bottleneck and make erythritol production greener and more sustainable. [ABSTRACT FROM AUTHOR]

BACKGROUND: Rhamnolipids are biosurfactants that have their main application in bioremediation processes. They are able to increase the biodegradation and detoxification rates from industrial effluents. The north of Peru and Iquitos have been not only major oil producers but also have been responsible for oil spills. This study therefore focused on the optimization of mineral nutrients such as nitrogen, phosphorous, calcium and iron to improve rhamnolipids production by Pseudomonas aeruginosa 6 K‐11, which was isolated from oil soil in Peru. RESULTS: Pseudomonas aeruginosa 6 K‐11 was reactivated in Trypticase Soy Broth by scaling from 5 to 200 mL of culture medium for 8 h at each stage. The culture was centrifuged and placed in 100 mL Siegmund–Wagner medium for 36 h in a 500 mL Erlenmeyer flask. First, it was determined that sodium nitrate, monopotassium phosphate, calcium chloride and iron sulfate were the best sources of mineral nutrients. Then, by using corn oil as carbon source, the ratios obtained for carbon‐to‐nitrogen and carbon‐to‐phosphorous were 21.172 and 16.279, respectively. In addition, it was found that the optimal concentration for calcium was 0.046 g L−1 and for iron was 0.003 g L−1. The most influential variables were carbon‐to‐nitrogen and carbon‐to‐phosphorus ratio. Finally, 35.124 g L−1 rhamnolipids was produced after 168 h of fermentation with the optimal parameters. CONCLUSION: The optimization of mineral nutrients was an important achieved goal for rhamnolipid production. In addition, this optimization can be used for modifying the culture medium to grow the biosurfactant producer strain. © 2024 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Even though industrial biotechnology is successfully used for the production of some chemicals, for many other chemicals it is not yet competitive with conventional petrochemical production. Usually, fermentation as well as downstream processing requires improvement. Downstream processing has to deal with low product concentrations, microorganisms, impurities and thermodynamic constraints (e.g., azeotropes), which often makes it very challenging and expensive, especially on a large scale. However, downstream processing of biochemicals has not attracted as much attention as upstream fermentation processes. In that context, this perspective paper offers a lightly referenced scholarly opinion about the downstream processing performance of different bioalcohols after fermentation. Due to the stronger toxicity effects on microbes, the achievable concentrations of monohydric aliphatic alcohols in the fermentation broth decrease with the increasing chain length. Specifically, the concentrations used here are 6.14, 5.00, 1.61 and 0.24 wt% of ethanol, isopropanol, isobutanol and hexanol, respectively. More dilute fermentation broths lead to more complex recovery processes. According to our previous work, the total purification costs increase from 0.080 USD kg−1 for ethanol, 0.109 USD kg−1 for isopropanol and 0.161 USD kg−1 for isobutanol to 0.529 USD kg−1 for hexanol. A similar trend is noticeable for the energy usage (0.960, 1.348, 2.018 and 3.069 kWthh kg−1, respectively) and the related CO2 emissions (0.164, 0.221, 0.449 and 0.555 kgCO2 kg−1, respectively). This work shows that advanced separation and purification based on process intensification principles are crucial for overall efficient production processes. The achievable product concentration in the fermentation broth – and not so much the alcohol chain length – has the biggest influence on the performance of downstream processing. Therefore, simultaneous development of both upstream and downstream processing is necessary to ensure the competitiveness and viability of industrial fermentation processes. © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Synthetic biology encompasses many kinds of ideas and techniques with the common theme of creating something novel. The industrially relevant microorganism, Ralstonia eutropha (also known as Cupriavidus necator), has long been a subject of metabolic engineering efforts to either enhance a product it naturally makes (polyhydroxyalkanoate) or produce novel bioproducts (e.g., biofuels and other small molecule compounds). Given the metabolic versatility of R. eutropha and the existence of multiple molecular genetic tools and techniques for the organism, development of a synthetic biology toolkit is underway. This toolkit will allow for novel, user-friendly design that can impart new capabilities to R. eutropha strains to be used for novel application. This article reviews the different synthetic biology techniques currently available for modifying and enhancing bioproduction in R. eutropha. Key points: • R. eutropha (C. necator) is a versatile organism that has been examined for many applications. • Synthetic biology is being used to design more powerful strains for bioproduction. • A diverse synthetic biology toolkit is being developed to enhance R. eutropha's capabilities. [ABSTRACT FROM AUTHOR]

The enzyme CbA5H, a [FeFe]‐hydrogenase from Clostridium beijerinckii, has previously been shown to survive exposure to oxygen, making it a promising candidate for biotechnological applications. Thus far [NiFe]‐hydrogenases are typically considered for such applications, due to the superior O2‐tolerance and therefore simplified enzyme handling. However, methods for production of [FeFe]‐hydrogenases are generally more successful than for other classes of hydrogenases, therefore in this work we focus on demonstrating scalable CbA5H production, and report results with active enzyme prepared in bioreactors (up to 10 L) with >20‐fold improvement in purified enzyme yield. We then go on to confirm excellent H2/H+‐cycling activity of the air‐purified protein, highlighting that CbA5H can be prepared and isolated without the need for complex and expensive infrastructure. Next, we demonstrate good stability of the air‐purified CbA5H both in solution assays, and as a heterogenous catalyst system when immobilized on a carbon support. Finally, we successfully implement this enzyme within previously demonstrated biotechnologies for flavin and NADH recycling, highlighting its relevance in chemical synthesis, and we demonstrate production of an important API precursor, 3‐quinuclidinol at >0.4 g scale in standard benchtop hydrogenation infrastructure, with >100,000 CbA5H turnovers over 18 operational hours. [ABSTRACT FROM AUTHOR]

Industrial biotechnology and biocatalysis can provide very effective synthetic tools to increase the sustainability of the production of fine chemicals, especially flavour and fragrance (F&F) ingredients, the market demand of which has been constantly increasing in the last years. One of the most important transformations in F&F chemistry is the reduction of C C bonds, typically carried out with metal-catalysed hydrogenations or hydride-based reagents. Its biocatalytic counterpart is a competitive alternative, showcasing a range of advantages such as excellent chemo-, regio- and stereoselectivity, ease of implementation, mild reaction conditions and modest environmental impact. In the present review, the application of biocatalysed alkene reductions (from microbial fermentations with wild-type strains to engineered isolated ene-reductase enzymes) to synthetic processes useful for the F&F industry will be described, highlighting not only the exquisite stereoselectivity achieved, but also the overall improvement when chirality is not involved. Multi-enzymatic cascades involving C C bioreductions are also examined, which allow much greater chemical complexity to be built in one-pot biocatalytic systems. [Display omitted] • C C reduction is a key transformation in the synthesis of flavours and fragrances. • Biocatalytic alternatives involve ene-reductases or microorganisms habouring them. • Many examples of industrially relevant molecules are provided and discussed. • Multi-enzymatic systems involving C C bioreductions are also examined. [ABSTRACT FROM AUTHOR]

Abstract The present study deals with the kinetics of improved poly(3-hydroxybutyrate) (PHB) production by an L-cysteine HCl-depressed mutant of Bacillus licheniformis. Production of biodegradable polymers is to eliminate use of materials derived from petrochemicals and also because of their environmental impact. For the current study, mutant strain (NA-21) & wild-type (IIB-isl19) were used for PHB production. Submerged culture with two-stage fermentation technique was used for PHB production. Results indicated that PHB production was improved with 300 mM of –HNO2. The superior mutant strain (NA-21) resulted in 2-fold more PHB as compared to the wild-type (IIB-isl9). It was selected, and resistance against L-cysteine HCl was developed. At 4 ppm concentration of L-cysteine HCl, PHB production by mutant strain (NA-cys4) was higher than its wild counterpart by 5.7-fold. Kinetic study of parameters including specific growth rate (µ h− 1), growth (Yx/s,Ys/x), product yield coefficients (Yp/s,Yp/x), volumetric rate constants (Qp, Qs, Qx) and specific rate constants (qp, qs, qx), were also accomplished. Moreover, Yp/x, Qp and qp = µ × Yp/x were found to be very significant as 1.254 ± 0.06 (g/g biomass), 0.134 ± 0.01 (g/l/h) and 0.168 ± 0.01 (g/g/h), respectively. The effect of fatty acids on PHB production highlighted the improvement in PHB production by 1.94-fold. The highest PHB production during the study was 16.35 ± 3.12 g/l which highlighted its significance (p ≤ 0.05) and impact on the overall process. The variation in PBH yield between wild-type and mutant B. licheniformis is possibly because of induced DNA interstrand thus making unstable thymidine-thymidine dymers. From the results, it was concluded that improved PHB production on industrial scale is fairly possible and it holds the potential to contribute significantly to plastic circularity in the future.

The study of living microorganisms using resource allocation models has been key in elucidating natural behaviors of bacteria, by allowing allocation of microbial resources to be represented through optimal control strategies. The approach can also be applied to research in microbial cell factories, to investigate the optimal production of value-added compounds regulated by an external control. The latter is the subject of this paper, in which we study batch bioprocessing from a resource allocation perspective. Based on previous works, we propose a simple bacterial growth model accounting for the dynamics of the bioreactor and intracellular composition, and we analyze its asymptotic behavior and stability. Using optimization and optimal control theory, we study the production of biomass and metabolites of interest for infinite- and finite-time horizons. The resulting optimal control problems are studied using Pontryagin's maximum principle and numerical methods, and the solutions found are characterized by the presence of the Fuller phenomenon (producing an infinite set of switching points occurring in a finite-time window) at the junctions with a second-order singular arc. The approach, inspired by biotechnological engineering, aims to shed light upon the role of cellular composition and resource allocation during batch processing and, at the same time, poses very interesting and challenging mathematical problems. [ABSTRACT FROM AUTHOR]

The present study deals with the kinetics of improved poly(3-hydroxybutyrate) (PHB) production by an L-cysteine HCl-depressed mutant of Bacillus licheniformis. Production of biodegradable polymers is to eliminate use of materials derived from petrochemicals and also because of their environmental impact. For the current study, mutant strain (NA-21) & wild-type (IIB-isl19) were used for PHB production. Submerged culture with two-stage fermentation technique was used for PHB production. Results indicated that PHB production was improved with 300 mM of –HNO2. The superior mutant strain (NA-21) resulted in 2-fold more PHB as compared to the wild-type (IIB-isl9). It was selected, and resistance against L-cysteine HCl was developed. At 4 ppm concentration of L-cysteine HCl, PHB production by mutant strain (NA-cys4) was higher than its wild counterpart by 5.7-fold. Kinetic study of parameters including specific growth rate (µ h− 1), growth (Yx/s,Ys/x), product yield coefficients (Yp/s,Yp/x), volumetric rate constants (Qp, Qs, Qx) and specific rate constants (qp, qs, qx), were also accomplished. Moreover, Yp/x, Qp and qp = µ × Yp/x were found to be very significant as 1.254 ± 0.06 (g/g biomass), 0.134 ± 0.01 (g/l/h) and 0.168 ± 0.01 (g/g/h), respectively. The effect of fatty acids on PHB production highlighted the improvement in PHB production by 1.94-fold. The highest PHB production during the study was 16.35 ± 3.12 g/l which highlighted its significance (p ≤ 0.05) and impact on the overall process. The variation in PBH yield between wild-type and mutant B. licheniformis is possibly because of induced DNA interstrand thus making unstable thymidine-thymidine dymers. From the results, it was concluded that improved PHB production on industrial scale is fairly possible and it holds the potential to contribute significantly to plastic circularity in the future. [ABSTRACT FROM AUTHOR]

Summary: Magnesium shows promise as a material for temporary fixation, yet its rapid corrosion poses health risks due to metal ion release. To mitigate these concerns, a biofunctionalization approach involving dicalcium phosphate dihydrate (DCPD) compounds and Chlorella sp. biomass was employed via electrodeposition, silanization, and dip-coating. Surface characterization using XRD, FTIR, and SEM confirmed successful deposition and immobilization. Corrosion behavior was assessed through electrochemical, immersion, and atomic absorption tests, revealing improved resistance and reduced Mg2+ ion release. The coatings demonstrated significant enhancement in corrosion resistance, guarding against pitting and cracks. The findings suggest the potential of Mg/DCPD and Mg/DCPD/microalgae coatings in addressing corrosion-related risks in temporary fixation applications, promising improved biocompatibility and longevity for medical implants.

Summary: Aiming to shed light on the biology of wild ruminants, we investigated the gut microbiome seasonal dynamics of the Alpine ibex (Capra ibex) from the Central Italian Alps. Feces were collected in spring, summer, and autumn during non-invasive sampling campaigns. Samples were analyzed by 16S rRNA amplicon sequencing, shotgun metagenomics, as well as targeted and untargeted metabolomics. Our findings revealed season-specific compositional and functional profiles of the ibex gut microbiome that may allow the host to adapt to seasonal changes in available forage, by fine-tuning the holobiont catabolic layout to fully exploit the available food. Besides confirming the importance of the host-associated microbiome in providing the phenotypic plasticity needed to buffer dietary changes, we obtained species-level genome bins and identified minimal gut microbiome community modules of 11–14 interacting strains as a possible microbiome-based solution for the bioconversion of lignocellulose to high-value compounds, such as volatile fatty acids.

Thanks to recent and continuing technological innovations, modern microfluidic systems are increasingly offering researchers working across all fields of biotechnology exciting new possibilities (especially with respect to facilitating high throughput analysis, portability, and parallelization). The advantages offered by microfluidic devices—namely, the substantially lowered chemical and sample consumption they require, the increased energy and mass transfer they offer, and their comparatively small size—can potentially be leveraged in every sub-field of biotechnology. However, to date, most of the reported devices have been deployed in furtherance of healthcare, pharmaceutical, and/or industrial applications. In this review, we consider examples of microfluidic and miniaturized systems across biotechnology sub-fields. In this context, we point out the advantages of microfluidics for various applications and highlight the common features of devices and the potential for transferability to other application areas. This will provide incentives for increased collaboration between researchers from different disciplines in the field of biotechnology. [ABSTRACT FROM AUTHOR]

Recent advancements in protein/enzyme engineering have enabled the production of a diverse array of high-value compounds in microbial systems with the potential for industrial applications. The goal of this review is to articulate some of the most recent protein engineering advances in bacteria, yeast, and other microbial systems to produce valuable substances. These high-value substances include α-farnesene, vitamin B12, fumaric acid, linalool, glucaric acid, carminic acid, mycosporine-like amino acids, patchoulol, orcinol glucoside, d-lactic acid, keratinase, α-glucanotransferases, β-glucosidase, seleno-methylselenocysteine, fatty acids, high-efficiency β-glucosidase enzymes, cellulase, β-carotene, physcion, and glucoamylase. Additionally, recent advances in enzyme engineering for enhancing thermostability will be discussed. These findings have the potential to revolutionize various industries, including biotechnology, food, pharmaceuticals, and biofuels.

The objectives set in the European Green Deal constitute the starting point of this review, which then focuses on the current implementation gap between agro-industrial wastes as resources for large-scale bioprocesses (e.g., baker’s yeast, bioethanol, citric acid, and amino acids). This review highlights the current lack of sustainability of the post-harvest processing of grapes and apples. In light of the European Green Deal, industrial biotechnology often lacks sustainability as well. We reviewed the recent progress reported in the literature to enhance the valorization of grape and apple pomace and the current failure to implement this research in technical processes. Nevertheless, selected recent papers show new perspectives to bridge this gap by establishing close collaborations between academic teams and industrial partners. As a final outcome, for the first time, we drew a circular flow diagram that connects agriculture post-harvest transformation with the industrial biotechnology and other industries through the substantial valorization of apple and grape pomace into renewable energy (solid biofuels) and sugar extracts as feedstock for large-scale bioprocesses (production of baker’s yeast industry, citric acid, bioethanol and amino acids). Finally, we discussed the requirements needed to achieve the successful bridging of the implementation gap between academic research and industrial innovation.

Technology-based innovations in the bio-economy are expected to provide new market opportunities for agro-industrial growth in developing countries. This study explores how domestic entrepreneurs have been benefiting from the growing global demand for sustainable agribusiness by establishing themselves in the agricultural bio-inputs sector. Aiming to explore how developing countries can build agro-industrial capabilities that go beyond farming, a case study was conducted in Argentina giving the country’s large domestic market for agricultural inputs. The assessment was based on official data and a survey conducted with a sample of 14 Argentinean companies. Results revealed a growing number of domestic companies in the bio-inputs market, which accounted for 74% of the total number of companies by 2022. Contextual aspects that favored domestic players include private investments by local entrepreneurs, the use of locally available technologies, support from public innovation centers and assertive market strategies. Principal component analysis revealed different business models, which include companies focusing on the development of new products, companies with professional industrial plants for large-scale production and companies with a strong marketing component reaching out to farmers. The global growth of sustainable bio-based agricultural production offers areas of opportunities for domestic investments in cleaner agro-industrial sectors that go beyond the primary production of commodities and can build industrial capabilities in developing countries.

Isobutanol is a highly attractive renewable alternative to conventional fossil fuels, with superior fuel properties as compared to ethanol and 1-butanol. Even though the isobutanol production by fermentation has significant potential, complex downstream processing is limiting the wide-spreading of this technology. Accordingly, this original research significantly contributes to the advancement in industrial biofuel production by developing two eco-efficient downstream processes for the industrial-scale recovery of isobutanol (production capacity 50 ktonneIBUT/y), from a highly dilute fermentation broth (>98 wt% water). Vacuum distillation and a novel hybrid combination of gas stripping and vacuum evaporation were coupled with atmospheric azeotropic distillation to recover over 99.9 % of isobutanol as a high-purity product (100 wt%). Advanced heat pumping and heat integration techniques were further implemented to allow the complete electrification of these recovery processes. Furthermore, implementation of these techniques significantly decreased total annual costs (0.131–0.161 $/kgIBUT), reduced energy requirements (0.488–0.807 kWeh/kgIBUT) and lowered CO2 emissions (0.303–0.449 kgCO2/kgIBUT), resulting in highly competitive purification processes. In addition to efficiently recovering isobutanol, the designed downstream processes provide the potential to enhance the fermentation process by recycling all present microorganisms and reducing water demand. Therefore, the results of this original research substantially contribute to the advancement in industrial biotechnology and the wide-spreading of biofuel production.

The objectives set in the European Green Deal constitute the starting point of this review, which then focuses on the current implementation gap between agro-industrial wastes as resources for large-scale bioprocesses (e.g., baker's yeast, bioethanol, citric acid, and amino acids). This review highlights the current lack of sustainability of the post-harvest processing of grapes and apples. In light of the European Green Deal, industrial biotechnology often lacks sustainability as well. We reviewed the recent progress reported in the literature to enhance the valorization of grape and apple pomace and the current failure to implement this research in technical processes. Nevertheless, selected recent papers show new perspectives to bridge this gap by establishing close collaborations between academic teams and industrial partners. As a final outcome, for the first time, we drew a circular flow diagram that connects agriculture post-harvest transformation with the industrial biotechnology and other industries through the substantial valorization of apple and grape pomace into renewable energy (solid biofuels) and sugar extracts as feedstock for large-scale bioprocesses (production of baker's yeast industry, citric acid, bioethanol and amino acids). Finally, we discussed the requirements needed to achieve the successful bridging of the implementation gap between academic research and industrial innovation. [ABSTRACT FROM AUTHOR]

Technology-based innovations in the bio-economy are expected to provide new market opportunities for agro-industrial growth in developing countries. This study explores how domestic entrepreneurs have been benefiting from the growing global demand for sustainable agribusiness by establishing themselves in the agricultural bio-inputs sector. Aiming to explore how developing countries can build agro-industrial capabilities that go beyond farming, a case study was conducted in Argentina giving the country's large domestic market for agricultural inputs. The assessment was based on official data and a survey conducted with a sample of 14 Argentinean companies. Results revealed a growing number of domestic companies in the bio-inputs market, which accounted for 74% of the total number of companies by 2022. Contextual aspects that favored domestic players include private investments by local entrepreneurs, the use of locally available technologies, support from public innovation centers and assertive market strategies. Principal component analysis revealed different business models, which include companies focusing on the development of new products, companies with professional industrial plants for large-scale production and companies with a strong marketing component reaching out to farmers. The global growth of sustainable bio-based agricultural production offers areas of opportunities for domestic investments in cleaner agro-industrial sectors that go beyond the primary production of commodities and can build industrial capabilities in developing countries. [ABSTRACT FROM AUTHOR]

Heme enzymes are the most prominent category of iron-containing metalloenzymes with the capability of catalyzing an astonishingly wide range of reactions like epoxidation, hydroxylation, demethylation, desaturation, reduction, sulfoxidation, and decarboxylation. Various enzymes in this category are P450s, heme peroxidases, catalases, myoglobin, cytochrome C, and others. Besides this, the natural promiscuity and amenability of these enzymes to protein engineering and evolution have also added several non-native reactions such as C–H, N–H, S–H insertions, cyclopropanation, and other industrially important reactions to their capabilities. Surprisingly, all of these reactions and their wide substrate scopes are attributed to changes in the active site scaffold of different heme enzymes as the center of all enzymes is constituted by a porphyrin ring containing iron. Multiple prominent research groups across the world, including 2018, Nobel Laureate Frances Arnold's group, have shown keen interest in engineering and evolving these enzymes for utilizing their industrial potential. Besides engineering the active site, researchers have also explored the possibility of these enzymes catalyzing non-native reactions by replacing the center porphyrin ring with other cofactors or by changing the iron in the porphyrin ring with other metal ions along with engineering the active site and thereby creating novel artificial metalloenzymes. Thus, in this mini-review from our group, for the first time, we are trying to catalog various activities catalyzed by heme enzymes and their engineered variants and their active usage in various industries along with shedding light on their potential for use in various applications in the future. [ABSTRACT FROM AUTHOR]

Cambial meristematic cells (CMCs) culture has received a fair share of scientific and industrial attention among the trending topics of plant cell culture, especially their potential toward secondary metabolites production. However, the conventional plant cell culture is often not commercially feasible because of difficulties associated with culture dedifferentiated cells. Several reports have been published to culture CMCs and bypass the dedifferentiation process in plant cell culture. Numerous mitochondria, multiple vacuoles, genetic stability, self-renewal, higher biomass, and stable metabolites accumulation are the characteristics features of CMCs compared with dedifferentiated cells (DDCs) culture. The CMCs culture has a broader application to produce large-scale natural compounds for: pharmaceuticals, food, and cosmetic industries. Cutting-edge progress in plant cellular and molecular biology has allowed unprecedented insights into cambial stem cell culture and its fundamental processes. Therefore, regarding sustainability and natural compound production, cambial cell culture ranks among the most vital biotechnological interventions for industrial and economic perspectives. This review highlights the recent advances in plant stem cell culture and understands the cambial cells induction and culture mechanisms that affect the growth and natural compounds production. [ABSTRACT FROM AUTHOR]

Microbial metabolism offers a wide variety of opportunities to produce chemicals from renewable resources. Employing such processes of industrial biotechnology provides valuable means to fight climate change by replacing fossil feedstocks by renewable substrate to reduce or even revert carbon emission. Several yeast species are well suited chassis organisms for this purpose, illustrated by the fact that the still largest microbial production of a chemical, namely bioethanol is based on yeast. Although production of ethanol and some other chemicals is highly efficient, this is not the case for many desired bulk chemicals. One reason for low efficiency is carbon loss, which decreases the product yield and increases the share of total production costs that is taken by substrate costs. Here we discuss the causes for carbon loss in metabolic processes, approaches to avoid carbon loss, as well as opportunities to incorporate carbon from CO2, based on the electron balance of pathways. These aspects of carbon efficiency are illustrated for the production of succinic acid from a diversity of substrates using different pathways. [ABSTRACT FROM AUTHOR]

Currently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Thus, is essential developing robust bacteria for improved cell tolerance to process- specific parameters. In this context, metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness. Here, in order to build genetic constructs that increase bacterial resistance to diverse stress conditions, we recovered novel protein-encoding sequences related to stress-resistance from metagenomic databases using an in silico approach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized 10 novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes in Escherichia coli under several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed in E. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increased E. coli tolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance. [ABSTRACT FROM AUTHOR]

Mine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost‐effective solution to meet regulatory standards, it represents a lost opportunity. In this study, we engineered Escherichia coli to overexpress its native NikABCDE transporter and a heterologous metallothionein to capture nickel at concentrations in local effluent streams. We found the engineered strain had a 7‐fold improvement in the bioaccumulation performance for nickel compared to controls, but also observed a drastic decrease in cell viability due to metabolic burden or inducer (IPTG) toxicity. Growth kinetic analysis revealed the IPTG concentrations used based on past studies lead to growth inhibition, thus delineating future avenues for optimization of the engineered strain and its growth conditions to perform in more complex environments. [ABSTRACT FROM AUTHOR]

Amongst the most important outputs of the biopharmaceutical industry are recombinant proteins, many of which are produced by integrating transgenes into the genomes of mammalian cells. However, expression is highly variable and can be unstable during prolonged culture. This is often due to epigenetic mechanisms silencing the transgenes. To combat this problem, vectors have been engineered to include ubiquitous chromatin opening elements (UCOEs) that protect against silencing. Here, we recount the evidence that UCOEs can modify chromatin environments and benefit biomanufacturing.

Abstract Background Membrane proteins (MPs) are an important class of molecules with a wide array of cellular functions and are part of many metabolic pathways. Despite their great potential—as therapeutic drug targets or in microbial cell factory optimization—many challenges remain for efficient and functional expression in a host such as Escherichia coli. Results A dynamically regulated small RNA-based circuit was developed to counter membrane stress caused by overexpression of different MPs. The best performing small RNAs were able to enhance the maximum specific growth rate with 123%. On culture level, the total MP production was increased two-to three-fold compared to a system without dynamic control. This strategy not only improved cell growth and production of the studied MPs, it also suggested the potential use for countering metabolic burden in general. Conclusions A dynamically regulated feedback circuit was developed that can sense metabolic stress caused by, in casu, the overexpression of an MP and responds to it by balancing the metabolic state of the cell and more specifically by downregulating the expression of the MP of interest. This negative feedback mechanism was established by implementing and optimizing simple-to-use genetic control elements based on post-transcriptional regulation: small non-coding RNAs. In addition to membrane-related stress when the MP accumulated in the cytoplasm as aggregates, the sRNA-based feedback control system was still effective for improving cell growth but resulted in a decreased total protein production. This result suggests promiscuity of the MP sensor for more than solely membrane stress.

The Valley of Death confronts industrial biotechnology with a significant challenge to the commercialization of products. Fortunately, with the integration of computation, automation and artificial intelligence (AI) technology, the industrial biotechnology accelerates to cross the Valley of Death. The Fourth Industrial Revolution (Industry 4.0) has spurred advanced development of intelligent biomanufacturing, which has evolved the industrial structures in line with the worldwide trend. To achieve this, intelligent biomanufacturing can be structured into three main parts that comprise digitalization, modeling and intellectualization, with modeling forming a crucial link between the other two components. This paper provides an overview of mechanistic models, data-driven models and their applications in bioprocess development. We provide a detailed elaboration of the hybrid model and its applications in bioprocess engineering, including strain design, process control and optimization, as well as bioreactor scale-up. Finally, the challenges and opportunities of biomanufacturing towards Industry 4.0 are also discussed. [ABSTRACT FROM AUTHOR]

A study on the ability of new microbial strains to assimilate biodiesel-derived glycerol at low purity (75% w/w) and produce extra-cellular platform chemical compounds of major interest was carried out. After screening several bacterial strains under different fermentation conditions (e.g., pH, O2 availability, glycerol purity), three of the screened strains stood out for their high potential to produce valued-added products such as 2,3-butanediol (BDO), 1,3-propanediol (PDO) and ethanol (EtOH). The results indicate that under aerobic conditions, Klebsiella oxytoca ACA-DC 1581 produced BDO in high yield (YBDO/Gly = 0.46 g/g, corresponding to 94% of the maximum theoretical yield; Ymt) and titer, while under anaerobic conditions, Citrobacter freundii NRRL-B 2645 and Enterobacter ludwigii FMCC-204 produced PDO (YPDO/Gly = 0.56 g/g, 93% of Ymt) and EtOH (YEtOH/Gly = 0.44 g/g, 88% of Ymt), respectively. In the case of C. freundii, the regulation of pH proved to be mandatory, due to lactic acid production and a subsequent drop of pH that resulted in fermentation ceasing. In the fed-batch culture of K. oxytoca, the BDO maximum titer reached almost 70 g/L, the YBDO/Gly and the mean productivity value (PrBDO) were 0.47 g/g and 0.4 g/L/h, respectively, while no optimization was imposed. The final BDO production obtained by this wild strain (K. oxytoca) is among the highest in the international literature, although the bioprocess requires optimization in terms of productivity and total cost. In addition, for the first time in the literature, a strain from the species Hafnia alvei (viz., Hafnia alvei ACA-DC 1196) was reported as a potential BDO producer. The strains as well as the methodology proposed in this study can contribute to the development of a biorefinery that complements the manufacture of biofuels with high-value biobased chemicals. [ABSTRACT FROM AUTHOR]

The Antarctic continent is a reservoir of new genetic resources to the bioprospection of microorganisms adapted to the polar conditions and capable to produce molecules with differentiated properties. Biosurfactants are a promising alternative to replace synthetic surfactants due to their eco-friendly characteristics and the possibility of being produced from raw materials, such as lignocellulosic biomass. The aim of the current study was to evaluate the biosurfactants produced by Antarctic yeast strains using detoxified sugarcane straw hemicellulosic hydrolysate (DSSHH). Therefore, the biosurfactant production, using xylose as the carbon source, was first evaluated in semi-defined medium and subsequently in DSSHH. The Naganishia adellienses L95 showed the highest emulsification index (52%) and total xylose consumption (40 g.L−1) in DSSHH. The biosurfactant produced by the yeast strain L95 was partially characterized, and its emulsion remained stable under low-temperature conditions (0 and 4 °C), at high salt concentration (10%), and alkaline condition. The screening of yeasts for the attainment of natural products that have potential biotechnological applications is of great importance. The results showed the potential of L95 to produce biosurfactants in DSSHH. [ABSTRACT FROM AUTHOR]

Synthetic biologists design and engineer organisms for a better and more sustainable future. While the manifold prospects are encouraging, concerns about the uncertain risks of genome editing affect public opinion as well as local regulations. As a consequence, biosafety and associated concepts, such as the Safe-by-design framework and genetic safeguard technologies, have gained notoriety and occupy a central position in the conversation about genetically modified organisms. Yet, as regulatory interest and academic research in genetic safeguard technologies advance, the implementation in industrial biotechnology, a sector that is already employing engineered microorganisms, lags behind. The main goal of this work is to explore the utilization of genetic safeguard technologies for designing biosafety in industrial biotechnology. Based on our results, we posit that biosafety is a case of a changing value, by means of further specification of how to realize biosafety. Our investigation is inspired by the Value Sensitive Design framework, to investigate scientific and technological choices in their appropriate social context. Our findings discuss stakeholder norms for biosafety, reasonings about genetic safeguards, and how these impact the practice of designing for biosafety. We show that tensions between stakeholders occur at the level of norms, and that prior stakeholder alignment is crucial for value specification to happen in practice. Finally, we elaborate in different reasonings about genetic safeguards for biosafety and conclude that, in absence of a common multi-stakeholder effort, the differences in informal biosafety norms and the disparity in biosafety thinking could end up leading to design requirements for compliance instead of for safety. [ABSTRACT FROM AUTHOR]

Currently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Thus, is essential developing robust bacteria for improved cell tolerance to process-specific parameters. In this context, metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness. Here, in order to build genetic constructs that increase bacterial resistance to diverse stress conditions, we recovered novel protein-encoding sequences related to stress-resistance from metagenomic databases using an in silico approach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized 10 novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes in Escherichia coli under several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed in E. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increased E. coli tolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance.

Abstract Mine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost‐effective solution to meet regulatory standards, it represents a lost opportunity. In this study, we engineered Escherichia coli to overexpress its native NikABCDE transporter and a heterologous metallothionein to capture nickel at concentrations in local effluent streams. We found the engineered strain had a 7‐fold improvement in the bioaccumulation performance for nickel compared to controls, but also observed a drastic decrease in cell viability due to metabolic burden or inducer (IPTG) toxicity. Growth kinetic analysis revealed the IPTG concentrations used based on past studies lead to growth inhibition, thus delineating future avenues for optimization of the engineered strain and its growth conditions to perform in more complex environments.

Since the mid-1960s, methylotrophic yeast Komagataella phaffii (previously described as Pichia pastoris) has received increasing scientific attention. The interest for the industrial production of proteins for different applications (e.g., feed, food additives, detergent, waste treatment processes, and textile) is a well-consolidated scientific topic, and the importance for this approach is rising in the current era of environmental transition in human societies. This review aims to summarize fundamental and specific information in this scientific field. Additionally, an updated description of the relevant products produced with K. phaffii at industrial levels by a variety of companies—describing how the industry has leveraged its key features, from products for the ingredients of meat-free burgers (e.g., IMPOSSIBLE™ FOODS, USA) to diabetes therapeutics (e.g., Biocon, India)—is provided. Furthermore, active patents and the typical workflow for industrial protein production with this strain are reported. [ABSTRACT FROM AUTHOR]

Many biotechnology applications tend to be for low production volumes and relatively high-value products such as insulin and vaccines. More difficult to perfect at scale are bioprocesses for high-volume products with lower value, especially if the target product is a reduced chemical such as a solvent or a plastic. Historically, industrial microbiology succeeded under special circumstances when fossil feedstocks were either unavailable or expensive. Inevitably, as these circumstances relaxed, bioprocesses struggled to compete with petrochemistry. Why try to compete? Fossil resources will be phased out in the coming decades in the struggle with climate change. To reach net-zero carbon by 2050 will require all sectors to transition, not only energy and transportation. This may herald a new opportunity for industrial bioprocesses with much better tools. Some of the issues around biotechnology in the first issue of Trends in Biotechnology remain issues today despite huge advances in technology. The Weizmann process and single-cell protein thrived, died, but may be on their way back. The bioeconomy envisages the gradual replacement of oil and gas, but the price of oil is very often a determining factor in the success of any competing technology. Net-zero carbon aspirations should be a huge opportunity for biotechnologies. The automation possible in the biofoundry allied to automated chemical synthesis seems to hold the key. Many of the hardest-to-abate industrial sectors cannot be decarbonized, but they might be 'defossilized'. Governments need to invest in bio-based manufacturing to bring them more quickly to the marketplace. [ABSTRACT FROM AUTHOR]

Enzymes are versatile and ecological biocatalysts that are biodegradable, which leads to lower environmental impacts. Peptidases are enzymes that catalyze the hydrolysis reaction of peptide bonds in proteins. Protein hydrolysis is fundamental in food processing, cleaning products, and pharmaceuticals, among others. Microbial peptidases have been the preferred source for developing new enzymes, besides their advantages compared to plant or animal sources. This is owing to their characteristics, e.g., fast growth, wide diversity of microbial enzymes, longer shelf life, and potential for genetic manipulation of microorganisms. Enzymes catalyze biochemical reactions and are highly specific. They generally work in mild conditions and depend on their native conditions, such as pH, temperature, and solvent characteristics. However, the protease used must tolerate operational adversities such as extreme pH and temperature in industrial processes. For this, it is necessary to discover new peptidases and improve existing ones. Methods of metagenomic selection and genome mining or through extremophile diversity (e.g., thermophilic, cold-active, alkali tolerant, acidophilic, and halophilic microorganisms) have been used in the discovery of new enzymes. Protein engineering (by directed evolution, rational design, semi-rational design, and de novo design) and enzyme immobilization are strategies used to improve the catalytic properties, efficiency, and stability of enzymes. This review aimed to critically discuss the current state of the art and future trends of microbial peptidases, highlighting their applications in food processing. [ABSTRACT FROM AUTHOR]

Abstract Background The need to mitigate and substitute the use of fossil fuels as the main energy matrix has led to the study and development of biofuels as an alternative. Second-generation (2G) ethanol arises as one biofuel with great potential, due to not only maintaining food security, but also as a product from economically interesting crops such as energy-cane. One of the main challenges of 2G ethanol is the inefficient uptake of pentose sugars by industrial yeast Saccharomyces cerevisiae, the main organism used for ethanol production. Understanding the main drivers for xylose assimilation and identify novel and efficient transporters is a key step to make the 2G process economically viable. Results By implementing a strategy of searching for present motifs that may be responsible for xylose transport and past adaptations of sugar transporters in xylose fermenting species, we obtained a classifying model which was successfully used to select four different candidate transporters for evaluation in the S. cerevisiae hxt-null strain, EBY.VW4000, harbouring the xylose consumption pathway. Yeast cells expressing the transporters SpX, SpH and SpG showed a superior uptake performance in xylose compared to traditional literature control Gxf1. Conclusions Modelling xylose transport with the small data available for yeast and bacteria proved a challenge that was overcome through different statistical strategies. Through this strategy, we present four novel xylose transporters which expands the repertoire of candidates targeting yeast genetic engineering for industrial fermentation. The repeated use of the model for characterizing new transporters will be useful both into finding the best candidates for industrial utilization and to increase the model’s predictive capabilities. Graphical Abstract

Background: Industrial Biotechnology (“White Biotechnology”) is the large-scale production of materials and chemicals using renewable raw materials along with biocatalysts like enzymes derived from microorganisms or by using microorganisms themselves (“whole cell biocatalysis”). While the production of ethanol has existed for several millennia and can be considered a product of Industrial Biotechnology, the application of complex and engineered biocatalysts to produce industrial scale products with acceptable economics is only a few decades old. Bioethanol as fuel, lactic acid as food and PolyHydroxyAlkanoates (PHA) as a processible material are some examples of products derived from Industrial Biotechnology.

Abstract Fatty acid hydratases are unique to microorganisms. Their native function is the oxidation of unsaturated C–C bonds to enable detoxification of environmental toxins. Within this enzyme family, the oleate hydratases (Ohys), which catalyze the hydroxylation of oleic acid to 10-(R)-hydroxy stearic acid (10-HSA) have recently gained particular industrial interest. 10-HSA is considered to be a replacement for 12-(R)-hydroxy stearic acid (12-HSA), which has a broad application in the chemical and pharmaceutical industry. As 12-HSA is obtained through an energy consuming synthesis process, the biotechnological route for sustainable 10-HSA production is of significant industrial interest. All Ohys identified to date have a non-redox active FAD bound in their active site. Ohys can be divided in several subfamilies, that differ in their oligomerization state and the decoration with amino acids in their active sites. The latter observation indicates a different reaction mechanism across those subfamilies. Despite intensive biotechnological, biochemical and structural investigations, surprising little is known about substrate binding and the reaction mechanism of this enzyme family. This review, summarizes our current understanding of Ohys with a focus on sustainable biotransformation.

Haloarchaea comprise the extremely halophilic branch of the phylum Euryarchaeota and they are members of the prokaryotic domain Archaea. They thrive best in extremely saline habitats with salt concentrations of 2–5 mol L–1 NaCl, and thus under conditions of near salt saturation in water. These ancient organisms are among the oldest species on Earth, and are characterized by ether-linked lipids in the cytoplasmic membrane and murein-free cell walls. Haloarchaea are increasingly receiving attention as microbial cell factories for the bioproduction of diverse marketable products, such as bacterioruberin, bacteriorhodopsin, isoprenoids, and polyhydroxyalkanoates (PHAs). These biopolyesters serve as intracellular storage compounds from the secondary metabolism of haloarchaea and many other prokaryotes. Engineered or wild type PHA-producing haloarchaea that utilize inexpensive raw materials for bioproduction, and which only require clean-in-place procedures to run PHA production bioprocesses, are currently undergoing scale-up within the research and development field of Archaea Biotechnology. In addition, PHAs exhibit high potential as both bio-based and biodegradable plastic-like bulk products on the industrial biotechnology market (“White Biotechnology”) for biopolymers. However, PHA production by haloarchaea has not yet reached industrial maturity. The present review discusses the background, previous research, and biological role of PHA biosynthesis in haloarchaea, as well as current trends, and a critical discussion of its potential for broad industrialization.

ABSTRACTRecent years have seen the uptake of challenge and mission-oriented innovation policies in Europe, including the UK. These developments have been paralleled by variable support for responsible research and innovation (RRI) policies. This article explores the adoption of challenge-driven innovation strategies in the UK's industrial biotech (IB) sector alongside RRI expectations, examining the effects and implications. Drawing on analysis of IB policy documents and interview data, we argue that the articulation of innovation aims in terms of grand challenges provides innovation stakeholders with new possibilities to re-frame the value and purposes of controversial technologies such as synthetic biology, and to downplay or ignore the anticipation of potential impacts and risks. The conflation of innovation objectives and grand societal challenges has afforded new opportunities to construct representations of societal consensus and alignment, while avoiding the participatory practices on which shared understandings and nuanced exploration of societal expectations would normally be based.

Background: Membrane proteins (MPs) are an important class of molecules with a wide array of cellular functions and are part of many metabolic pathways. Despite their great potential—as therapeutic drug targets or in microbial cell factory optimization—many challenges remain for efficient and functional expression in a host such as Escherichia coli. Results: A dynamically regulated small RNA-based circuit was developed to counter membrane stress caused by overexpression of different MPs. The best performing small RNAs were able to enhance the maximum specific growth rate with 123%. On culture level, the total MP production was increased two-to three-fold compared to a system without dynamic control. This strategy not only improved cell growth and production of the studied MPs, it also suggested the potential use for countering metabolic burden in general. Conclusions: A dynamically regulated feedback circuit was developed that can sense metabolic stress caused by, in casu, the overexpression of an MP and responds to it by balancing the metabolic state of the cell and more specifically by downregulating the expression of the MP of interest. This negative feedback mechanism was established by implementing and optimizing simple-to-use genetic control elements based on post-transcriptional regulation: small non-coding RNAs. In addition to membrane-related stress when the MP accumulated in the cytoplasm as aggregates, the sRNA-based feedback control system was still effective for improving cell growth but resulted in a decreased total protein production. This result suggests promiscuity of the MP sensor for more than solely membrane stress. [ABSTRACT FROM AUTHOR]

The performance of large‐scale stirred tank and bubble column bioreactors is often hindered by insufficient macromixing of feeds, leading to heterogeneities in pH, substrate, and oxygen, which complicates process scale‐up. Appropriate feed placement or the use of multiple feed points could improve mixing. Here, theoretically optimal placement of feed points was derived using one‐dimensional diffusion equations. The utility of optimal multipoint feeds was evaluated with mixing, pH control, and bioreaction simulations using three‐dimensional compartment models of four industrially relevant bioreactors with working volumes ranging from 8 to 237 m3. Dividing the vessel axially in equal‐sized compartments and locating a feed point or multiple feed points symmetrically in each compartment reduced the mixing time substantially by more than a minute and mitigated gradients of pH, substrate, and oxygen. Performance of the large‐scale bioreactors was consequently restored to ideal, homogeneous reactor performance: oxygen consumption and biomass yield were recovered and the phenotypical heterogeneity of the biomass population was diminished. [ABSTRACT FROM AUTHOR]

The fast growth accompanied with high substrate consumption rates and a versatile metabolism paved the way to exploit Vibrio natriegens as unconventional host for biotechnological applications. Meanwhile, a wealth of knowledge on the physiology, the metabolism, and the regulation in this halophilic marine bacterium has been gathered. Sophisticated genetic engineering tools and metabolic models are available and have been applied to engineer production strains and first chassis variants of V. natriegens. In this review, we update the current knowledge on the physiology and the progress in the development of synthetic biology tools and provide an overview of recent advances in metabolic engineering of this promising host. We further discuss future challenges to enhance the application range of V. natriegens., Competing Interests: Additional Information All figures were created using BioRender. Author Contributions MH, CS, JH, and BB conceived and wrote the paper. All authors read and approved the manuscript. Funding This work was supported by the BL1408/2-1 grant (Deutsche Forschungsgemeinschaft) and by the TUM WACKER Institute for Industrial Biotechnology. Conflict of Interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)