Biotechnological Production Research Articles

Towards biotechnological production of bio-based low molecular weight esters: a patent review.

Low molecular weight (LMW) esters, like ethyl acetate, methyl formate or butyl acetate, are widespread bulk chemicals in many industries. Each of them is currently produced in huge amounts (millions of tons per year scale) starting from fossil-based feedstock and they are used mainly because of their low toxicity and complete biodegradability. Energy transition is just half of the story on the path of fighting climate change: 45% of the global greenhouse gas emissions are caused by the production and use of all the products, materials and food necessary for modern human life. If the world is to reach its climate goals, there is the need to leave underground a significant proportion of the fossil feedstock and minimize environmental impacts of chemical manufacturing. This is the reason why a lot of efforts have been made to find novel routes for LMW esters production starting from renewable raw materials (e.g. biomasses or off-gases) and exploiting low-impact manufacturing, such as microbial fermentation or enzymatic reactions. This review reports the most significant patents, in the field of white biotechnology, that will hopefully lead to the commercialization of bio-based LMW esters as well as novel strategies, current problems to be solved, newer technologies, and some patent applications aiming at possible future developments.

Proceedings of the 2023 Viral Clearance Symposium, Session 1: Regulatory Updates.

At the time of the 2023 Viral Clearance Symposium in Vienna, the ongoing revision of ICH Guideline Q5A(R1) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin clearly was the dominant regulatory topic. At the symposium, the changes expected for Q5A(R2) to mirror advances of scientific knowledge, for example, the inclusion of new products, including viral-vector-derived ones, that can be subject to virus clearance, deliberations around continuous manufacturing processes, the use of prior knowledge to supplement or in part replace virus validation studies, and new molecular methods for detection of adventitious viruses, were discussed by a European and a US regulator as well as representatives from industry associations that had been involved with the drafting process.

УПРАВЛІННЯ ІННОВАЦІЙНИМ РОЗВИТКОМ АГРАРНОГО СЕКТОРУ АПК В СУЧАСНИХ УМОВАХ

The article states that the development of the agrarian sector of the agro-industrial complex on an innovative basis in the future should take place at the expense of the use of new types of machinery, equipment, machines, tools, varieties of plants and animal breeds, new production technologies, ways of searching, accumulating, recording and transmitting information. raising the level of qualification of employees, production of new types or with new properties of agricultural products, introduction of new forms of management and organization of production, development of new markets and methods of selling products, construction of new logistics systems, etc. At the state level: there is a huge economic need for biotechnological products among producers, processors, consumers and other participants of the agro-industrial complex, as well as for scientific knowledge related to its production and commercialization. At the regional level: among the main principles that form the methods of the innovation process of the regional agricultural industry, the following should be noted: the principle of scientificity, the principle of a systemic approach, the principle of responsibility, the principle of selection and placement of personnel. At the level of territorial communities: it should be noted that the innovation process of these communities is influenced by their specific features of location, management conditions, and differences in the provision of personnel capable of successfully conducting innovative activities. Innovative activity of an agribusiness is an activity related to the search and implementation of innovations that will allow the enterprise to have advantages over other market entities, which contributed to increasing the competitiveness and efficiency of their activities. It was determined that the management of innovative activities in the agricultural sector will take place with the wide implementation of digital technologies that allow obtaining the maximum effect from each unit of resources based on the most rational application in the right place, at the right time and in the right amount at the state and regional levels and at the levels of territorial communities and economic entities by strengthening the ties of rural commodity producers with advisory services, higher education institutions, scientific institutions and organizations engaged in innovative developments.

Principles of designing specialized agricultural bioengineering complexes

Purpose. Determination of the basic principles for the design of specialized agricultural bioengineering complexes to increase the efficiency of the processes of their creation and use. Methods. System analysis, ontological engineering, software environment Visual Understanding Environment. Results. The basic principles regarding the designing specialized agricultural of bioengineering complexes have been defined, in particular, for the production of entomological plant protection products, namely, intellectual, modular, multi-level, immutability of biotechnological equipment parameters, system analysis, ontological, synergistic, continuous development, are is defined. An ontology for the designing of bioengineering complexes in the production of entomophages has been developed. Conclusions. The basic principles of the design of specialized agricultural bioengineering complexes in the production of entomophages are proposed to increase the validity of decision-making in the processes of engineering and technical support of biotechnological production. The use of the ontological principle of designing bioengineering complexes in the production of entomophages (innovative intelligent control system for the production of the entomophage Habrobracon hebetor and the technological complex for the production of Chrysoperla carnea) makes it possible to structure declarative and procedural knowledge, to increase the efficiency of the processes of creating and using these complexes. The prospect of further research is the creation of ontological knowledge bases in intelligent decision-making support systems for greening the production of agricultural products. Keywords: bioengineering complexes, production of entomophages, ontologies, principles, designing, agriculture.

Disposable Bioreactors Used in Process Development and Production Processes with Plant Cell and Tissue Cultures.

The bioreactor is the centerpiece of the upstream processing in any biotechnological production process. Its design, the cultivation parameters, the production cell line, and the culture medium all have a major influence on the efficiency of the process and the result of the cultivation. Disposable bioreactors have been used for the past 20 years, playing a major role in process development and commercial production of high-value substances at medium scales.Our review deals with scalable, disposable bioreactors that have proven to be useful for the cultivation of plant cell and tissue cultures. Based on the definitions of terms and a categorization approach, the most commonly used, commercially available, disposable bioreactor types are presented below. The focus is on wave-mixed, stirred, and orbitally shaken bioreactors. In addition to their instrumentation and bioengineering characteristics, cultivation results are discussed, and emerging trends for the development of disposable bioreactors for plant cell and tissue cultures are also addressed.

The influence of varying concentrations of CO2 on the buildup of carbohydrates in microalgae biomass

Microalgae have gained popularity over the century due to their numerous intrinsic properties superior to higher plants, making them a potential target and feedstock for the development of biotechnological products in various fields. The storage of carbohydrates within microalgae cells positions them as a promising reservoir for biofuel production and a range of other valuable bioproducts, encompassing biological compounds, nutritional supplements, and more. Key determinants influencing microalgae carbohydrate levels comprise nutrient availability, light intensity, temperature, and CO2 concentrations. In this context, the CO2 concentration plays a key role, as it is one of the main factors influencing the photosynthetic processes. This study aimed to explore the impact of different CO2 concentrations on the carbohydrate profile of biomass sourced from Chlorella vulgaris sp. BB-2 and Scenedesmus quadicauda B-1. The findings revealed that a CO2 concentration of 2% v/v notably enhanced carbohydrate accumulation, reaching 75.5% for Chlorella vulgaris sp. BB-2 and 72.0% for Scenedesmus quadicauda B-1.

Innovative strategies for biotechnological production of hyaluronic acid from different sources

Innovative strategies for biotechnological production of hyaluronic acid from different sources

Diversity of Carotenoid Pigmented Marine Photosynthetic Bacteria in the Dumai Sea Waters

Marine photosynthetic bacteria represent autotrophic microorganisms capable of synthesizing natural pigments. This study aims to explore the diversity of carotenoid-pigmented marine photosynthetic bacteria in the Dumai Sea waters, Riau Province, Indonesia. The research method was a survey using marine surface water as samples from six locations. The research was conducted in four primary phases: 1) measurement of environmental conditions; 2) enrichment and isolation of marine photosynthetic bacteria; 3) enumeration of total bacteria; and 4) analysis of pigments colour production. Isolation results and population density measurements of photosynthetic bacteria revealed fascinating patterns. The highest cell count was observed at fish auctions (sampling site 2), while the lowest cell count was recorded near a river estuary (sampling site 6). Detailed characterization studies identified ten isolates capable of producing natural carotenoid pigments exhibiting colours ranging from pink to deep red. These findings contribute to the understanding of marine microbial diversity and the potential of bacteria for biotechnology products, aquaculture, and medical applications.

Investigation of Antibacterial and Antifungal Efficacy of Zinc and Silver Nanoparticles Synthesized from Nasturtium officinale

Nanoparticles are nano-sized materials that can be widely used in fields such as medicine, pharmacology, and industry. The use of natural and easily available materials in nanoparticle synthesis is preferred because it is economical. Plants are extremely suitable for the synthesis of nanoparticles due to their natural and easy availability and the large number of components they contain with different properties. For this purpose, silver nanoparticles and zinc nanoparticles (AgNPs and ZnNPs), two different nanoparticles were synthesized from an edible plant, watercress (Nasturtium officinale). SEM (Scanning electron microscopy), SEM-EDX (Scanning electron microscopy-Energy dispersive X-ray), UV-VIS spectroscopy, XRD (X-ray crystallography), and FTIR (Fourier Transform Infrared Spectrophotometer) analyses of these nanoparticles were performed. In addition, the antimicrobial effects of these synthesized nanoparticles were determined by the disk diffusion method. As a result, nanoparticles obtained from Nasturtium officinale were effective on gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa), gram-positive bacteria (Staphylococcus aureus, Streptococcus pyogenes), and fungi (Candida albicans). In particular, AgNPs with broad-spectrum antimicrobial activity were obtained from the watercress. While ZnNPs showed inhibition effects of 49% on K. pneumoniae, 51% on S. aureus, and 62% on C. albicans, AgNPs showed inhibition effects of 93% on P. aeruginosa, 87% on S. aureus, 81% on E. coli, 80% on C. albicans, 72% on K. pneumoniae, and 56% on S. pyogenes. Thus, it has been shown that Nasturtium officinale can be used effectively in the production of new biotechnological products, especially with antimicrobial properties.

The Outlook of Tomatoes with extended Shelf Life in Nigeria and the Ensuing Safety Measures and Regulated Introduction by Nigerian Regulatory Authorities

Tomato (Solanum lycopersicum) is widely consumed worldwide. The primary chance to influence tomatoes' edibility and market value lies in regulating the synthesis and function of the plant hormone ethylene responsible for ripening. The usage of harmless substances on tomato surfaces, a reduction in storage temperature, the implementation of modified atmosphere packaging, or a combination of these approaches have been associated with increasing tomatoes’ shelf life. As crop plants containing a genetically modified trait through molecular techniques continue their transition from the laboratory to the field, the National Biotechnology Development Agency (NABDA) has been reported to be developing genetically modified (GM) tomatoes by deactivating polygalacturonase (PG) gene that is responsible for polygalacturonase which is the enzyme that aids fruit softening and subsequently increases the shelf life of the tomatoes to be released in the Nigerian market. This article looks at tomatoes (S. lycopersicum) and the methods used in increasing their shelf life with more focus on the genetically modified tomatoes and the needed regulations by the Nigerian regulatory bodies before its commercialization in the country. Environmental and health hazards that require careful consideration in the course of embarking on the genetical modification and introduction of the hybrid tomatoes into the Nigeria markets encompass potential biodiversity loss, adverse impacts on natural food chains, and the potential rise of more virulent pathogen populations. Nevertheless, it is crucial to emphasize that the biotechnological processes and products must be accompanied by effective regulations to mitigate these concerns, National Biosafety Management Agency is believed to be engaged with these needs.

Biotechnological Advancements in Streptokinase Production

This article offers a deep analysis of activating streptokinase which looks into the biotechnological challenges and future perspectives. One of the main problems associated with streptokinase is its short half-life, complex production process, and immunogenicity which hinders patients from getting a successful reperfusion. Yet, the development of biotechnology methods that would not face these drawbacks, such as the strategy of recombinant streptokinase production and enzyme engineering theories, is a promise. This paper aims to provide an overview of biotechnological breakthroughs in the production of streptokinase with a focus on discovering long-lasting formulations exhibiting less immunogenicity and in addition the economic viability and scalability of biotechnological production processes. This article also gives the comping image of known purification methods like chromatography, ultrafiltration, and I-A chromatography for the separation and purification of streptokinase. In addition, the author presents the advantage of such suitable production technologies as cell-free production systems and E. Coli system optimization, such as recombinant streptokinase synthesis in Yeast systems. Biotechnology is turning out to be a new area, having a future boom due to the rising innovation and demand that are associated with the prospects revealed by the market trends.

Fine-Tuned Gene Expression Elements from Hybrid Promoter Libraries in Pichia pastoris.

As a desirable microbial cell factory, Pichia pastoris has garnered extensive utilization in metabolic engineering. Nevertheless, the lack of fine-tuned gene expression components has significantly constrained the potential scope of applications. Here, a gradient strength promoter library was constructed by random hybridization and high-throughput screening. The hybrid promoter, phy47, performed best with 2.93-fold higher GFP expression levels than GAP. The broad applicability of the novel hybrid promoter variants in biotechnological production was further validated in the biosynthesis of pinene and rHuPH20 with higher titers. The upstream regulatory sequences (UASE and URSD) were identified and applied to promoters GAP and ENO1, resulting in a 34 and 43% increase and an 18 and 37% decrease in the expression level, respectively. Yeast one-hybrid analysis showed that transcription factor HAP2 activates the hybrid promoter through a direct interaction with the crucial regulatory region UASH. Furthermore, a short segment of tunable activation sequence (20 bp) was also screened, and artificial promoters were constructed in tandem with the addition of regulatory sequence, resulting in a 61% expansion of the expression range. This study provides a molecular tool and regulatory elements for further synthetic biology research in P. pastoris.

The Next Food Revolution Is Here: Recombinant Microbial Production of Milk and Egg Proteins by Precision Fermentation.

Animal-based agriculture and the production of protein-rich foods from animals, particularly from ruminants, are not sustainable and have serious climate effects. A new type of alternative proteins is now on the menu, namely animal proteins produced recombinantly by microbial fermentation. This new technology, precision fermentation, is projected to completely disrupt traditional animal-based agriculture. Certain milk and egg proteins along with specific meat substitute analog components produced by precision fermentation are already entering the market. This first wave of precision fermentation products targets the use of these proteins as protein additives, and several commercial players are already active in the field. The cost-efficiency requirements involve production titers above 50 g/L which are several orders of magnitude higher than those for pharmaceutical protein manufacture, making strain engineering, process optimization, and scale-up critical success factors. This new development within alternative proteins defines a new research direction integrating biotechnology, process engineering, and sustainable food protein production.

Biologically Active Complex Based on Fermented Fiber to Normalize and Ensure the Vital Microflora Activity

Nowadays, the intestinal microflora plays the important role in ensuring normal metabolism, no doubt. Therefore, biotechnological products and its metabolites are of particular importance. A man developed biotechnological product in the form of a biologically active additive based on fermented fiber (a mixture of dietary fibers bacterized with probiotic cultures of Bifidobacterium animalis, Bifidobacterium adolescentis, Lactobacillus acidophilus). The probiotic feature is the ability to ferment dietary fibers while staying alive in a specialized product. The formulation includes dietary fibers that stimulate the growth and bacteria reproduction, specifically, forming a target intestinal biotope. Along with traditional quality and safety research methods, the researchers used the method of high-performance liquid chromatography to determine the short-chain fatty acids content, playing a key role in the metabolic body processes, in fermented fiber. The formulation of the developed additive contains ingredients that form its properties. The determined regulated quality indicators are the following (in 30 g): dietary fiber – at least 20,000 mg; polypructosans – at least 2,200 mg; thiamine (B1) – 1.4 mg; riboflavin (B2) – 1.6 mg; pantothenic acid (B5) – 6 mg; pyridoxine (B6) – 2 mg; chromium – 50 mcg. The authors run the safety dietary supplements assessment, enabling to determine the shelf life – 12 months at a temperature of 25 °C and a relative humidity of no more than 60 %. Evidence-based medical studies confirm the functional orientation and effectiveness of the developed product. A man took the developed supplement one serving (15 g) twice a day (in the morning 30 minutes before meals and in the evening before bedtime). The administration course was 30 days. The data obtained indicate a positive impact of the biocomplex on the microbiota restoration, the local intestinal syndrome effect and the general condition of the body.

Indoles and the advances in their biotechnological production for industrial applications

AbstractIndole is a signalling molecule produced both by bacteria and plants. In this review its signalling role between microbes and in particular in the human gut is discussed. Besides the natural roles, indole also has value for flavour and fragrance applications, for example, in food industry or perfumery. Additionally, indole can be derivatized to several halogenated and oxygenated compounds that can be used as natural colourants or have promising bioactivity with therapeutic potential to treat human diseases. Indole is traditionally obtained from coal tar. Biocatalytic approaches have been developed to convert indole into halogenated and oxygenated derivatives. This review will discuss recent advances in production of indole from glucose or tryptophan by fermentation and the production of derived halogenated and oxygenated derivatives by microbial cell factories.

Molecular Identification and Characterization of Five Ganoderma Species from the Lower Volta River Basin of Ghana Based on Nuclear Ribosomal DNA (nrDNA) Sequences.

Ganoderma is a genus of biomedical fungus that is used in the development of numerous health products throughout the world. The Lower Volta River Basin of Ghana is an undulating land surface covered by extensive vegetation and water bodies and is rich in polypore mushrooms resembling various members of the Ganoderma genus. Despite the extensive biopharmaceutical benefits of Ganoderma spp., the isolates from the Lower Volta River Basin have not been properly characterized, thus limiting their use in the development of biotechnological products. In this study, Ganoderma spp. collected from the Lower Volta River Basin were genetically analyzed using the nuclear ribosomal sequences, the internal transcribed spacer 2 (ITS 2), the complete internal transcribed spacer (ITS), and the nuclear large subunit (nLSU). Blastn search and sequence analysis revealed that the sample we coded as Ganoderma LVRB-2 belongs to G. mbrekobenum, whereas Ganoderma LVRB-1, Ganoderma LVRB-14, and Ganoderma LVRB-16 belong to the species G. enigmaticum. Our analysis further demonstrates that Ganoderma LVRB-17 belongs to the species G. resinaceum. Thus, the five samples collected in the present study were positioned in three different distinct groups, namely G. mbrekobenum, G. enigmaticum, and G. resinaceum. The current data may serve as reference points for future studies.

Methodology of Examination of Patients with Osteoarthritis and Aseptic Necrosis of Hip and Knee Joints when Using Regenerative Interventional Technologies

Background. Diagnosis of osteoarthritis of the hip and knee joints, as of today, would seem to be a well-studied issue. Radiography, magnetic resonance imaging, ultrasonography - each of these methods makes it possible to make a diagnosis. At the same time, the use of new methods of treatment of osteoarthritis requires new approaches to its diagnosis.
 Objective: to develop a methodology for examining patients with osteoarthritis and aseptic necrosis of the hip and knee joints during treatment with regenerative technologies.
 Material and Methods. The material for the study was case histories of 233 patients with osteoarthritis and 58 patients with osteoarthritis of the hip and knee joints. Clinical, laboratory, and instrumental research methods were used.
 Results. The methodology of clinical examination of patients when using regenerative technologies is based on filling out quality of life questionnaires. A feature of the methodology of laboratory examination of patients with osteoarthritis and aseptic necrosis of the hip and knee joints when using regenerative technologies is oncological screening in the case of the use of biotechnological products containing MSCs, with the determination of epigenetic markers of the risk of oncogenesis in the form of miRNA.
 Conclusions. The methodology of the instrumental examination of patients with osteoarthritis is as follows: the key is an X-ray examination with the establishment of the stage of osteoarthritis according to the Kellgren-Lawrence classification and the subsequent ultrasound and MRI examination in order to assess the extent of damage to the soft tissues of the hip and knee joints, subchondral bone and paraarticular structures. In patients with aseptic necrosis of the hip and knee joints, when using regenerative technologies, the key is an MRI study followed by X-ray and ultrasound examination.

Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using β-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

Integrating Chemocatalysis and Enzyme Engineering for Coproduction of Bioplastic Precursors Butanediol and Succinic Acid from Xylose

AbstractPolybutylene succinate (PBS) is one of the most important biobased plastics, based on the amount produced. Owing to its high melting point and resemblance to petroleum‐based plastics (i. e. PP), PBS becomes one of the emerging bioplastics with an array of applications. PBS is manufactured by polymerization of 1,4‐butanediol and succinic acid. Thus, it is of great importance to ensure the use of renewable resources to produce the PBS precursors. As the second most abundant carbohydrate monomer on Earth, D‐xylose will be a suitable candidate for this purpose. In this work, we combined protein engineering with chemical oxidation by gold catalyst to enable transformation of D‐xylose to 1,4‐butanediol and succinic acid simultaneously. In silico docking studies and semi rational design were employed to create variants of the key enzyme, branched chain α‐keto acid decarboxylase (KdcA) with higher affinity for the intermediates in the production of 1,4‐butanediol and succinic acid. Direct enzymatic biotransformation would result in a production of both monomers with 3:1 ratio, thus not readily suitable for a direct polymerization to PBS. By developing a one‐pot multi‐step chemo‐enzymatic approach with a gold catalyst to perform the first oxidation step, we could achieve a final product ratio of 1:1. Application of an engineered KdcA variant allowed us to achieve >98% yield after four hours transformation. In contrast, after 24 h transformation, >10% intermediate was still observed when the original variant was used. We anticipate this new approach could serve as an alternative route for biotechnological productions of PBS and its precursors.

CRISPR/Cas12a toolbox for genome editing in Methanosarcina acetivorans.

Methanogenic archaea play an important role in the global carbon cycle and may serve as host organisms for the biotechnological production of fuels and chemicals from CO2 and other one-carbon substrates. Methanosarcina acetivorans is extensively studied as a model methanogen due to its large genome, versatile substrate range, and available genetic tools. Genome editing in M. acetivorans via CRISPR/Cas9 has also been demonstrated. Here, we describe a user-friendly CRISPR/Cas12a toolbox that recognizes T-rich (5'-TTTV) PAM sequences. The toolbox can manage deletions of 3,500 bp (i.e., knocking out the entire frhADGB operon) and heterologous gene insertions with positive rates of over 80%. Cas12a-mediated multiplex genome editing was used to edit two separate sites on the chromosome in one round of editing. Double deletions of 100 bp were achieved, with 8/8 of transformants being edited correctly. Simultaneous deletion of 100 bp at one site and replacement of 100 bp with the 2,400 bp uidA expression cassette at a separate site yielded 5/6 correctly edited transformants. Our CRISPR/Cas12a toolbox enables reliable genome editing, and it can be used in parallel with the previously reported Cas9-based system for the genetic engineering of the Methanosarcina species.