Large-Scale Production of Specialized Metabolites In Vitro Cultures.

For centuries plants have been intensively utilized as reliable sources of food, flavoring, and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to the climate change and agriculture. Plant biotechnology offers a sustainable approach for the bioproduction of specialized plant metabolites. The unique structural features of plant-derived specialized metabolites, such as their safety profile and multi-target spectrum, have led to the establishment of many plant-derived drugs. However, there are still many challenges to overcome regarding the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant specialized metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this book chapter, we attempted to focus on the advantages of plant in vitro systems and in particular plant cell suspensions for their cultivation as a source of plant-derived specialized metabolites. A state-of-the-art technological platform for plant cell suspension cultivation from callus induction to lab-scale cultivation, extraction, and purification is presented. Possibilities for bioreactor cultivation of plant cell suspensions in benchtop and large-scale volumes are highlighted, including several examples and patents for industrial production of specialized metabolites.

Structural diversification of vitamin D using microbial biotransformations.

Vitamin D deficiencies are linked to multiple human diseases. Optimizing its synthesis, physicochemical properties, and delivery systems while minimizing side effects is of clinical relevance and is of great medical and industrial interest. Biotechnological techniques may render new modified forms of vitamin D that may exhibit improved absorption, stability, or targeted physiological effects. Novel modified vitamin D derivatives hold promise for developing future therapeutic approaches and addressing specific health concerns related to vitamin D deficiency or impaired metabolism, such as avoiding hypercalcemic effects. Identifying and engineering key enzymes and biosynthetic pathways involved, as well as developing efficient cultures, are therefore of outmost importance and subject of intense research. Moreover, we elaborate on the critical role that microbial bioconversions might play in the a la carte design, synthesis, and production of novel, more efficient, and safer forms of vitamin D and its analogs. In summary, the novelty of this work resides in the detailed description of the physiological, medical, biochemical, and epidemiological aspects of vitamin D supplementation and the steps towards the enhanced and simplified industrial production of this family of bioactives relying on microbial enzymes. KEY POINTS: • Liver or kidney pathologies may hamper vitamin D biosynthesis • Actinomycetes are able to carry out 1α- or 25-hydroxylation on vitamin D precursors.

The Microalgae Chlamydomonas for Bioremediation and Bioproduct Production.

The extensive metabolic diversity of microalgae, coupled with their rapid growth rates and cost-effective production, position these organisms as highly promising resources for a wide range of biotechnological applications. These characteristics allow microalgae to address crucial needs in the agricultural, medical, and industrial sectors. Microalgae are proving to be valuable in various fields, including the remediation of diverse wastewater types, the production of biofuels and biofertilizers, and the extraction of various products from their biomass. For decades, the microalga Chlamydomonas has been widely used as a fundamental research model organism in various areas such as photosynthesis, respiration, sulfur and phosphorus metabolism, nitrogen metabolism, and flagella synthesis, among others. However, in recent years, the potential of Chlamydomonas as a biotechnological tool for bioremediation, biofertilization, biomass, and bioproducts production has been increasingly recognized. Bioremediation of wastewater using Chlamydomonas presents significant potential for sustainable reduction in contaminants and facilitates resource recovery and valorization of microalgal biomass, offering important economic benefits. Chlamydomonas has also established itself as a platform for the production of a wide variety of biotechnologically interesting products, such as different types of biofuels, and high-value-added products. The aim of this review is to achieve a comprehensive understanding of the potential of Chlamydomonas in these aspects, and to explore their interrelationship, which would offer significant environmental and biotechnological advantages.

The History of Agrobacterium Rhizogenes: From Pathogen to a Multitasking Platform for Biotechnology.

Agrobacterium's journey has been a roller coaster, from being a pathogen to becoming a powerful biotechnological tool. While A. tumefaciens has provided the scientific community with a versatile tool for plant transformation, Agrobacterium rhizogenes has given researchers a Swiss army knife for developing many applications. These applications range from a methodology to regenerate plants, often recalcitrant, to establish bioremediation protocols to a valuable system to produce secondary metabolites. This chapter reviews its discovery, biology, controversies over its nomenclature, and some of the multiple applications developed using A. rhizogenes as a platform.

Sustainable production of a biotechnologically relevant ß-galactosidase in Escherichia coli cells using crude glycerol and cheese whey permeate.

Responsible use of natural resources and waste reduction are key concepts in bioeconomy. This study demonstrates that agro-food derived-biomasses from the Italian food industry, such as crude glycerol and cheese whey permeate (CWP), can be combined in a high-density fed-batch culture to produce a recombinant ß-galactosidase from Marinomonas sp. ef1 (M-ßGal). In a small-scale process (1.5 L) using 250 mL of crude glycerol and 300 mL of lactose-rich CWP, approximately 2000 kU of recombinant M-ßGal were successfully produced along with 30 g of galactose accumulated in the culture medium. The purified M-ßGal exhibited high hydrolysis efficiency in lactose-rich matrices, with hydrolysis yields of 82 % in skimmed milk at 4 °C and 94 % in CWP at 50 °C, highlighting its biotechnological potential. This approach demonstrates the effective use of crude glycerol and CWP in sustainable and cost-effective high-density Escherichia coli cultures, potentially applicable to recombinant production of various proteins.

Dilute acid-assisted microbubbles-mediated ozonolysis of Eucheuma denticulatum phycocolloid for biobased L-lactic acid production.

Biobased L-lactic acid (L-LA) appeals to industries; however, existing technologies are plagued by limited productivity and high energy consumption. This study established an integrated process for producing macroalgae-based L-LA from Eucheuma denticulatum phycocolloid (EDP). Dilute acid-assisted microbubbles-mediated ozonolysis (DAMMO) was selected for the ozonolysis of EDP to optimize D-galactose recovery. Through single-factor optimization of DAMMO treatment, a maximum D-galactose recovery efficiency (59.10 %) was achieved using 0.15 M H2SO4 at 80 °C for 75 min. Fermentation with 3 % (w/v) mixed microbial cells (Bacillus coagulans ATCC 7050 and Lactobacillus acidophilus-14) and fermented residues achieved a 97.67 % L-LA yield. Additionally, this culture approach was further evaluated in repeated-batch fermentation and showed an average L-LA yield of 93.30 %, providing a feasible concept for macroalgae-based L-LA production.

Design, construction and optimization of formaldehyde growth biosensors with broad application in biotechnology.

Formaldehyde is a key metabolite in natural and synthetic one-carbon metabolism. To facilitate the engineering of formaldehyde-producing enzymes, the development of sensitive, user-friendly, and cost-effective detection methods is required. In this study, we engineered Escherichia coli to serve as a cellular biosensor capable of detecting a broad range of formaldehyde concentrations. Using both natural and promiscuous formaldehyde assimilation enzymes, we designed three distinct E. coli growth biosensor strains that depend on formaldehyde for cell growth. These strains were engineered to be auxotrophic for one or several essential metabolites that could be produced through formaldehyde assimilation. The respective assimilating enzyme was expressed from the genome to compensate the auxotrophy in the presence of formaldehyde. We first predicted the formaldehyde dependency of the biosensors by flux balance analysis and then analysed it experimentally. Subsequent to strain engineering, we enhanced the formaldehyde sensitivity of two biosensors either through adaptive laboratory evolution or modifications at metabolic branch points. The final set of biosensors demonstrated the ability to detect formaldehyde concentrations ranging approximately from 30 µM to 13 mM. We demonstrated the application of the biosensors by assaying the in vivo activity of different methanol dehydrogenases in the most sensitive strain. The fully genomic nature of the biosensors allows them to be deployed as "plug-and-play" devices for high-throughput screenings of extensive enzyme libraries. The formaldehyde growth biosensors developed in this study hold significant promise for advancing the field of enzyme engineering, thereby supporting the establishment of a sustainable one-carbon bioeconomy.

Pigments from pathogenic bacteria: a comprehensive update on recent advances.

Bacterial pigments stand out as exceptional natural bioactive compounds with versatile functionalities. The pigments represent molecules from distinct chemical categories including terpenes, terpenoids, carotenoids, pyridine, pyrrole, indole, and phenazines, which are synthesized by diverse groups of bacteria. Their spectrum of physiological activities encompasses bioactive potentials that often confer fitness advantages to facilitate the survival of bacteria amid challenging environmental conditions. A large proportion of such pigments are produced by bacterial pathogens mostly as secondary metabolites. Their multifaceted properties augment potential applications in biomedical, food, pharmaceutical, textile, paint industries, bioremediation, and in biosensor development. Apart from possessing a less detrimental impact on health with environmentally beneficial attributes, tractable and scalable production strategies render bacterial pigments a sustainable option for novel biotechnological exploration for untapped discoveries. The review offers a comprehensive account of physiological role of pigments from bacterial pathogens, production strategies, and potential applications in various biomedical and biotechnological fields. Alongside, the prospect of combining bacterial pigment research with cutting-edge approaches like nanotechnology has been discussed to highlight future endeavours.

Unveiling Agricultural Biotechnological Prospects: The Draft Genome Sequence of Stenotrophomonas geniculata LGMB417.

Stenotrophomonas species are recognized as rhizobacteria that play a pivotal role in promoting plant growth by making substantial contributions to enhanced soil fertility, nutrient recycling, and phytopathogen control. Employing them as bioinputs constitutes an environmentally sound strategy, particularly within the rhizospheric community. This study revealed the draft genome sequence of Stenotrophomonas geniculata LGMB417, which was originally isolated from root samples of maize (Zea mays L.). This research assessed the potential of a bacterial strain at the molecular level through genome mining, aiming to identify genes with biotechnological significance for promoting plant growth and protection. The assembly findings indicate that strain LGMB417 possesses a genome size of 4,654,011 bp, with a G + C content of 66.50%. The draft genome sequence revealed the presence of gene clusters responsible for the synthesis of secondary metabolites and carbohydrate active enzymes (CAZymes), glycoside hydrolases (23), glycosyltransferases (18), carbohydrate esterases (5), polysaccharide lyases (2), carbohydrate-binding modules (2), and auxiliary activities (1). Several genes related to growth promotion were found in the genome, including those associated with phosphate transport and solubilization, nitrogen metabolism, siderophore production and iron transport, hormonal modulation, stress responses (such as to drought, temperature fluctuations, osmotic challenges, and oxidative conditions), and volatile organic compounds (VOCs). Subsequent phases will encompass investigations utilizing gene expression methodologies, with future explorations concentrating on facets pertinent to agricultural production, including comprehensive field studies.

Microbial Nanotechnology for Precision Nanobiosynthesis: Innovations, Current Opportunities and Future Perspectives for Industrial Sustainability.

A new area of biotechnology is nanotechnology. Nanotechnology is an emerging field that aims to develope various substances with nano-dimensions that have utilization in the various sectors of pharmaceuticals, bio prospecting, human activities and biomedical applications. An essential stage in the development of nanotechnology is the creation of nanoparticles. To increase their biological uses, eco-friendly material synthesis processes are becoming increasingly important. Recent years have shown a lot of interest in nanostructured materials due to their beneficial and unique characteristics compared to their polycrystalline counterparts. The fascinating performance of nanomaterials in electronics, optics, and photonics has generated a lot of interest. An eco-friendly approach of creating nanoparticles has emerged in order to get around the drawbacks of conventional techniques. Today, a wide range of nanoparticles have been created by employing various microbes, and their potential in numerous cutting-edge technological fields have been investigated. These particles have well-defined chemical compositions, sizes, and morphologies. The green production of nanoparticles mostly uses plants and microbes. Hence, the use of microbial nanotechnology in agriculture and plant science is the main emphasis of this review. The present review highlights the methods of biological synthesis of nanoparticles available with a major focus on microbially synthesized nanoparticles, parameters and biochemistry involved. Further, it takes into account the genetic engineering and synthetic biology involved in microbial nanobiosynthesis to the construction of microbial nanofactories.

A Novel Sustainable and Self-Sufficient Biotechnological Strategy for Directly Transforming Sewage Sludge into High-Value Liquid Biochemicals.

Sewage sludge, as a carbon-rich byproduct of wastewater treatment, holds significant untapped potential as a renewable resource. Upcycling this troublesome waste stream represents great promise in addressing global escalating energy demands through its wide practice of biochemical recovery concurrently. Here, we propose a biotechnological concept to gain value-added liquid bioproducts from sewage sludge in a self-sufficient manner by directly transforming sludge into medium-chain fatty acids (MCFAs). Our findings suggest that yeast, a cheap and readily available commercial powder, would involve ethanol-type fermentation in chain elongation to achieve abundant MCFA production from sewage sludge using electron donors (i.e., ethanol) and acceptors (i.e., short-chain fatty acids) produced in situ. The enhanced abundance and transcriptional activity of genes related to key enzymes, such as butyryl-CoA dehydrogenase and alcohol dehydrogenase, affirm the robust capacity for the self-sustained production of MCFAs. This is indicative of an effective metabolic network established between yeast and anaerobic microorganisms within this innovative sludge fermentation framework. Furthermore, life cycle assessment and techno-economic analysis evidence the sustainability and economic competitiveness of this biotechnological strategy. Overall, this work provides insights into sewage sludge upgrading independent of additional carbon input, which can be applied in existing anaerobic sludge fermentation infrastructure as well as to develop new applications in a diverse range of industries.

Nutraceutical prospects of genetically engineered cyanobacteria- technological updates and significance.

Genetically engineered cyanobacterial strains that have improved growth rate, biomass productivity, and metabolite productivity could be a better option for sustainable bio-metabolite production. The global demand for biobased metabolites with nutraceuticals and health benefits has increased due to their safety and plausible therapeutic and nutritional utility. Cyanobacteria are solar-powered green cellular factories that can be genetically tuned to produce metabolites with nutraceutical and pharmaceutical benefits. The present review discusses biotechnological endeavors for producing bioprospective compounds from genetically engineered cyanobacteria and discusses the challenges and troubleshooting faced during metabolite production. This review explores the cyanobacterial versatility, the use of engineered strains, and the techno-economic challenges associated with scaling up metabolite production from cyanobacteria. Challenges to produce cyanobacterial bioactive compounds with remarkable nutraceutical values have been discussed. Additionally, this review also summarises the challenges and future prospects of metabolite production from genetically engineered cyanobacteria as a sustainable approach.

Regulatory framework for genetically modified organisms in the Kingdom of Eswatini.

The Kingdom of Eswatini is a Party to the Convention on Biological Diversity and to the Cartagena Protocol on Biosafety. As Party, Eswatini has domesticated these agreements by passing the Biosafety Act, of 2012 to provide for the safe handling, transfer, and use of living modified organisms (LMOs) in the country. The Act regulates living modified organisms to be used for confined field trials, commercial release, import, export, and transit, and for food, feed, and processing. Guidance is provided for prospective applicants before any application is made to the Competent Authority. This framework also provides for the regulation of emerging technologies such as synthetic biology and genome editing. The regulatory framework for living modified organisms aims to provide an enabling environment for the precautionary use of modern biotechnology and its products in the country in order to safeguard biological diversity and human health.

A novel closed-loop biotechnology for recovery of cobalt from a lithium-ion battery active cathode material.

In recent years, the demand for lithium-ion batteries (LIBs) has been increasing rapidly. Conventional recycling strategies (based on pyro- and hydrometallurgy) are damaging for the environment and more sustainable methods need to be developed. Bioleaching is a promising environmentally friendly approach that uses microorganisms to solubilize metals. However, a bioleaching-based technology has not yet been applied to recover valuable metals from waste LIBs on an industrial scale. A series of experiments was performed to improve metal recovery rates from an active cathode material (LiCoO2; LCO). (i) Direct bioleaching of ≤0.5 % LCO with two prokaryotic acidophilic consortia achieved >80 % Co and 90 % Li extraction. Significantly lower metal recovery rates were obtained at 30 °C than at 45 °C. (ii) In contrast, during direct bioleaching of 3 % LCO with consortia adapted to elevated LCO levels, the 30 °C consortium performed significantly better than the 45 °C consortium, solubilizing 73 and 93 % of the Co and Li, respectively, during one-step bioleaching, and 83 and 99 % of the Co and Li, respectively, during a two-step process. (iii) The adapted 30°C consortium was used for indirect leaching in a low-waste closed-loop system (with 10 % LCO). The process involved generation of sulfuric acid in an acid-generating bioreactor (AGB), 2-3 week leaching of LCO with the biogenic acid (pH 0.9), selective precipitation of Co as hydroxide, and recirculation of the metal-free liquor back into the AGB. In total, 58.2 % Co and 100 % Li were solubilized in seven phases, and >99.9 % of the dissolved Co was recovered after each phase as a high-purity Co hydroxide. Additionally, Co nanoparticles were generated from the obtained Co-rich leachates, using Desulfovibrio alaskensis, and Co electrowinning was optimized as an alternative recovery technique, yielding high recovery rates (91.1 and 73.6% on carbon felt and roughened steel, respectively) from bioleachates that contained significantly lower Co concentrations than industrial hydrometallurgical liquors. The closed-loop system was highly dominated by the mixotrophic archaeon Ferroplasma and sulfur-oxidizing bacteria Acidithiobacillus caldus and Acidithiobacillus thiooxidans. The developed system achieved high metal recovery rates and provided high-purity solid products suitable for a battery supply chain, while minimizing waste production and the inhibitory effects of elevated concentrations of dissolved metals on the leaching prokaryotes. The system is suitable for scale-up applications and has the potential to be adapted to different battery chemistries.