The Progress of the Biotechnological Production of Class IIa Bacteriocins in Various Cell Factories and Its Future Challenges.

Class IIa bacteriocins produced in lactic acid bacteria are short cationic peptides with antimicrobial activity. In the search for new biopreservation agents, class IIa bacteriocins are considered to be the best potential candidates, not only due to their large abundance but also because of their high biological activity and excellent thermal stability. However, regulated by the biosynthetic regulatory system, the natural class IIa bacteriocin yield is low, and the extraction process is complicated. The biotechnological production of class IIa bacteriocins in various cell factories has been attempted to improve this situation. In this review, we focus on the application of biotechnological routes for class IIa bacteriocin production. The drawbacks and improvements in the production of class IIa bacteriocins in various cell factories are discussed. Furthermore, we present the main challenge of class IIa bacteriocins, focusing on increasing their production by constructing suitable cell factories. Recombinant bacteriocins have made considerable progress from inclusion body formation, dissolved form and low antibacterial activity to yield recovery. The development of prospective cell factories for the biotechnological production of bacteriocins is still required, which may facilitate the application of bacteriocins in the food industry.

Fighting pathogenic yeasts with plant defensins and anti-fungal proteins from fungi.

Fungal infections represent a significant health risk worldwide. Opportunistic infections caused by yeasts, particularly by Candida spp. and their virulent emerging isolates, have become a major threat to humans, with an increase in fatal cases of infections attributed to the lack of effective anti-yeast therapies and the emergence of fungal resistance to the currently applied drugs. In this regard, the need for novel anti-fungal agents with modes of action different from those currently available is undeniable. Anti-microbial peptides (AMPs) are promising candidates for the development of novel anti-fungal biomolecules to be applied in clinic. A class of AMPs that is of particular interest is the small cysteine-rich proteins (CRPs). Among CRPs, plant defensins and anti-fungal proteins (AFPs) of fungal origin constitute two of the largest and most promising groups of CRPs showing anti-fungal properties, including activity against multi-resistant pathogenic yeasts. In this review, we update and compare the sequence, structure, and properties of plant defensins and AFPs with anti-yeast activity, along with their in vitro and in vivo potency. We focus on the current knowledge about their mechanism of action that may lead the way to new anti-fungals, as well as on the developments for their effective biotechnological production. KEY POINTS: • Plant defensins and fungal AFPs are alternative anti-yeast agents • Their multi-faceted mode of action makes occurrence of resistance rather improbable • Safe and cost-effective biofactories remain crucial for clinical application.

Aurachins, Bacterial Antibiotics Interfering with Electron Transport Processes.

Aurachins are farnesylated quinolone alkaloids of bacterial origin and excellent inhibitors of the respiratory chain in pro- and eukaryotes. Therefore, they have become important tool compounds for the investigation of electron transport processes and they also serve as lead structures for the development of antibacterial and antiprotozoal drugs. Especially aurachin D proved to be a valuable starting point for structure-activity relationship studies. Aurachin D is a selective inhibitor of the cytochrome bd oxidase, which has received increasing attention as a target for the treatment of infectious diseases caused by mycobacteria. Moreover, aurachin D possesses remarkable activities against Leishmania donovani, the causative agent of leishmaniasis. Aurachins are naturally produced by myxobacteria of the genus Stigmatella as well as by some Streptomyces and Rhodococcus strains. The recombinant production of these antibiotics turned out to be challenging due to their complex biosynthesis and their inherent toxicity. Recently, the biotechnological production of aurachin D was established in E. coli with a titer which is higher than previously reported from natural producer organisms.

Natural Carotenoids: Recent Advances on Separation from Microbial Biomass and Methods of Analysis.

Biotechnologically produced carotenoids occupy an important place in the scientific research. Owing to their role as natural pigments and their high antioxidant properties, microbial carotenoids have been proposed as alternatives to their synthetic counterparts. To this end, many studies are focusing on their efficient and sustainable production from renewable substrates. Besides the development of an efficient upstream process, their separation and purification as well as their analysis from the microbial biomass confers another important aspect. Currently, the use of organic solvents constitutes the main extraction process; however, environmental concerns along with potential toxicity towards human health necessitate the employment of "greener" techniques. Hence, many research groups are focusing on applying emerging technologies such as ultrasounds, microwaves, ionic liquids or eutectic solvents for the separation of carotenoids from microbial cells. This review aims to summarize the progress on both the biotechnological production of carotenoids and the methods for their effective extraction. In the framework of circular economy and sustainability, the focus is given on green recovery methods targeting high-value applications such as novel functional foods and pharmaceuticals. Finally, methods for carotenoids identification and quantification are also discussed in order to create a roadmap for successful carotenoids analysis.

Advances and trends in biotechnological production of natural astaxanthin by Phaffia rhodozyma yeast.

Astaxanthin (AXT) is a natural xanthophyll with strong antioxidant, anticancer and antimicrobial activities, widely used in the food, feed, pharmaceutical and nutraceutical industries. So far, 95% of the AXT global market is produced by chemical synthesis, but growing customer preferences for natural products are currently changing the market for natural AXT, highlighting the production from microbially-based sources such as the yeast Phaffia rhodozyma. The AXT production by P. rhodozyma has been studied for a long time at a laboratory scale, but its use in industrial-scale processes is still very scarce. The optimization of growing conditions as well as an effective integration of upstream-downstream operations into P. rhodozyma-based AXT processes has not yet been fully achieved. With this critical review, we scrutinized the main approaches for producing AXT using P. rhodozyma strains, highlighting the impact of using conventional and non-conventional procedures for the extraction of AXT from yeast cells. In addition, we also pinpointed research directions, for example, the use of low-cost residues to improve the economic and environmental sustainability of the bioprocess, the use of environmentally/friendly and low-energetic integrative operations for the extraction and purification of AXT, as well as the need of further human clinical trials using yeast-based AXT.

Advances and applications of capillary electromigration methods in the analysis of therapeutic and diagnostic recombinant proteins - A Review.

This review provides a comprehensive overview of methodological advances and applications of CE in the analysis and characterization of recombinant therapeutic and diagnostic proteins over the past two decades. The first part of the review discusses various aspects of biotechnological protein production and the related effects on the final product. This covers upstream processes, e.g., selection and transfection of host cells, up-scaling of cell cultures and cultivation conditions, as well as downstream processing and a discussion of future trends in biotechnological manufacturing. This part is essential for relating biotechnological production to analytical challenges and requirements in order to provide a holistic insight. In this context, the influence of manufacturing steps on the quality of the final drug substance/product is discussed in terms of related post-translational modifications of the target molecule with a major focus on glycosylation pattern and conformational effects. Particular attention is given to host cell specific and non-human modifications affecting the efficacy and safety of recombinant products. Endowed with this propaedeutic knowledge, the major part of the review discusses the manifold contributions of different CE techniques to the development and optimization of the manufacturing process, to the evaluation and characterization of the final drug product and their role in quality control. Different CE techniques, such as CZE, capillary gel electrophoresis (CGE), (imaged) capillary isoelectric focusing ((i)CIEF), µChipCE, CE-Western blot, affinity CE (ACE), and CE-MS are discussed including a brief introduction in the respective separation and hyphenation principle as well as their applications in the analysis of different recombinant biologics together with recent strategies. The addressed analyte portfolio comprises a vast variety of recombinant proteins with molecular masses from 4.1 kDa up to 20.3 MDa (for recombinant virus-like particles), and a pI range from 2.0 to 11.2. Antibodies are not explicitly covered in the survey. The review is complemented by compiling validation aspects and proposed suitability tests in order to assure the feasibility of methods to industrial and pharmaceutical needs.

Biological production of xylitol by using nonconventional microbial strains.

Xylitol (C5H12O5), an amorphous sugar alcohol of crystalline texture has received great interest on the global market due to its numerous applications in different industries. In addition to its high anticariogenic and sweetening properties, characteristics such as high solubility, stability and low glycemic index confer xylitol its fame in the food and odontological industries. Moreover, it also serves as a building-block in the production of polymers. As a result of the harmful effects of the chemical production of xylitol, the biotechnological means of producing this polyol have evolved over the decades. In contrast to the high consumption of energy, long periods of purification, specialized equipment and high production cost encountered during its chemical synthesis, the biotechnological production of xylitol offers advantages both to the economy and the environment. Non-Saccharomyces yeast strains, also termed as nonconventional, possess the inherent capacity to utilize D-xylose as a sole carbon source, unlike Saccharomyces species.

Phage-free production of artificial ssDNA with Escherichia coli.

Artificial single-stranded DNA (ssDNA) with user-defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass-producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross-contaminating manufacturer plants with self-replicating phages. Here we overcome this problem with an efficient, scalable, and cross-contamination-free method for the phage-free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self-replication in the absence of the helper plasmid. This enables cross-contamination-free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by-products and increased the maximal product concentration up to 83 mg L-1 of ssDNA in a stirred-tank bioreactor, thus realizing up to a 40-fold increase in maximal product concentration over previous scalable phage-free ssDNA production methods.

Biotechnological production of ß-carotene using plant in vitro cultures.

MAIN CONCLUSION: ß-carotene is biologically active compound widely distributed in plants. The use of plant in vitro cultures and genetic engineering is a promising strategy for its sustainable production. ß-carotene is an orange carotenoid often found in leaves as well as in fruits, flowers, and roots. A member of the tetraterpene family, this 40-carbon isoprenoid has a conjugated double-bond structure, which is responsible for some of its most remarkable properties. In plants, ß-carotene functions as an antenna pigment and antioxidant, providing protection against photooxidative damage caused by strong UV-B light. In humans, ß-carotene acts as a precursor of vitamin A, prevents skin damage by solar radiation, and protects against several types of cancer such as oral, colon and prostate. Due to its wide spectrum of applications, the global market for ß-carotene is expanding, and the demand can no longer be met by extraction from plant raw materials. Considerable research has been dedicated to finding more efficient production alternatives based on biotechnological systems. This review provides a detailed overview of the strategies used to increase the production of ß-carotene in plant in vitro cultures, with particular focus on culture conditions, precursor feeding and elicitation, and the application of metabolic engineering.

Emerging Trends in Genetic Engineering of Microalgae for Commercial Applications.

Recently, microalgal biotechnology has received increasing interests in producing valuable, sustainable and environmentally friendly bioproducts. The development of economically viable production processes entails resolving certain limitations of microalgal biotechnology, and fast evolving genetic engineering technologies have emerged as new tools to overcome these limitations. This review provides a synopsis of recent progress, current trends and emerging approaches of genetic engineering of microalgae for commercial applications, including production of pharmaceutical protein, lipid, carotenoids and biohydrogen, etc. Photochemistry improvement in microalgae and CO2 sequestration by microalgae via genetic engineering were also discussed since these subjects are closely entangled with commercial production of the above mentioned products. Although genetic engineering of microalgae is proved to be very effective in boosting performance of production in laboratory conditions, only limited success was achieved to be applicable to industry so far. With genetic engineering technologies advancing rapidly and intensive investigations going on, more bioproducts are expected to be produced by genetically modified microalgae and even much more to be prospected.

Biotechnological production and application of epsilon-poly-L-lysine (ε-PL): biosynthesis and its metabolic regulation.

Epsilon-poly-L-lysine (ε-PL) is an unusual biopolymer composed of L-lysine produced by several microorganisms, especially by the genus Streptomyces. Due to its excellent antimicrobial activity, good water solubility, high safety, and biodegradable nature, ε-PL with a GRAS status has been widely used in food and pharmaceutical industries. In the past years, studies have focused on the biotechnological production of ɛ-PL, the biosynthetic mechanism of microbial ɛ-PL, and its application. To provide new perspectives from recent advances, the review introduced the methods for the isolation of ɛ-PL producing strains and the biosynthetic mechanism of microbial ɛ-PL. We summarized the strategies for the improvement of ɛ-PL producing strains, including physical and chemical mutagenesis, ribosome engineering and gene engineering, and compared the different metabolic regulation strategies for improving ɛ-PL production, including medium optimization, nutrient supply, pH control, and dissolved oxygen control. Then, the downstream purification methods of ɛ-PL and its recent applications in food and medicine industries were introduced. Finally, we also proposed the potential challenges and the perspectives for the production of ε-PL.

Polyhydroxyalkanoates synthesis by halophiles and thermophiles: towards sustainable production of microbial bioplastics.

Polyhydroxyalkanoates (PHA) are microbial polyesters produced by numerous prokaryotes. These materials are generally considered to be renewable and biodegradable alternatives to petrochemical polymers in numerous applications. PHA are accumulated by microbial cells in form of intracellular granules primarily as storage compounds; nevertheless, numerous recent reports also highlight the importance of PHA for the stress robustness of bacteria. Therefore, in this review, we focus on summarizing current knowledge on PHA accumulation in halophiles and thermophiles - prokaryotic microorganisms adapted to high salinity and high temperature, respectively. Utilization of extremophiles for PHA production brings numerous benefits stemming especially from the enhanced robustness of the process against contamination by common mesophilic microflora as a basement of the Next-Generation Industrial Biotechnology concept. Further, recent advances and future perspectives in metabolic engineering and synthetic biology of halophiles and thermophiles for PHA production improvement are also summarized and suggested. Facts and ideas gathered in this review hold a promise that biotechnological production of PHA by extremophiles can be sustainable and economically feasible enabling PHA to enter the market massively and compete with non-biodegradable petrochemical polymers in suitable applications.

Expanding the lysine industry: biotechnological production of l-lysine and its derivatives.

l-lysine is an essential amino acid that contains various functional groups including α-amino, ω-amino, and α-carboxyl groups, exhibiting high reaction potential. The derivatization of these functional groups produces a series of value-added chemicals, such as cadaverine, glutarate, and d-lysine, that are widely applied in the chemical synthesis, cosmetics, food, and pharmaceutical industries. Here, we review recent advances in the biotechnological production of l-lysine and its derivatives and expatiate key technological strategies. Furthermore, we also discuss the existing challenges and potential strategies for more efficient production of these chemicals.

Biotechnological production and high potential of furan-based renewable monomers and polymers.

Of the 25 million tons of plastic waste produced every year in Europe, 40% of these are not reused or recycled, thus contributing to environmental pollution, one of the major challenges of the 21st century. Most of these plastics are made of petrochemical-derived polymers which are very difficult to degrade and as a result, a lot of research efforts have been made on more environmentally friendly alternatives. Bio-based monomers, derived from renewable raw materials, constitute a possible solution for the replacement of oil-derived monomers, with furan derivatives that emerged as platform molecules having a great potential for the synthesis of biobased polyesters, polyamides and their copolymers. This review article summarizes the latest developments in biotechnological production of furan compounds that can be used in polymer chemistry as well as in their conversion into polymers. Moreover, the biodegradability of the resulting materials is discussed.

Advances in biotechnological production of santalenes and santalols.

Sandalwood essential oil has been widely used not only as natural medicines but also in perfumery and food industries, with sesquiterpenoids as its major components including (Z)- α-santalol and (Z)-ß-santalol and so on. The mature heartwoods of Santalum album, Santalum austrocaledonicum and Santalum spicatum are the major plant resources for extracting sandalwood essential oil, which have been overexploited. Synthetic biology approaches have been successfully applied to produce natural products on large scale. In this review, we summarize biosynthetic enzymes of santalenes and santalols, including various santalene synthases (STSs) and cytochrome P450 monooxygenases (CYPs), and then highlight the advances of biotechnological production of santalenes and santalols in heterologous hosts, especially metabolic engineering strategies for constructing santalene- and santalol-producing Saccharomyces cerevisiae.

Mannitol: physiological functionalities, determination methods, biotechnological production, and applications.

Mannitol is a naturally occurring six-carbon sugar alcohol that has wide applications in the food and pharmaceutical industry because of its many properties, namely being a natural sweetener with a low metabolism and no glycemic index. The increasing demand for mannitol has spurred many studies of its production. Compared with its chemical synthesis and extraction from plants, both of which are difficult to satisfy for industrial requirements, biotechnological production of mannitol has received considerably more attention and interest from scientists because of its known advantages over those two methods. Accordingly, in this review, we summarize recent advances made in the production of mannitol through various biotechnological methods. The physicochemical properties, sources, and physiological functionalities and applications of mannitol are systematically covered and presented. Then, different determination methods for mannitol are also described and compared. Furthermore, different biotechnological strategies for the production of mannitol via fermentation engineering, protein engineering, and metabolic engineering receive a detailed overview in terms of mannitol-producing strains, enzymes, and their key reaction parameters and conditions. KEY POINTS: • Physiological functionalities and applications of mannitol are presented in detail. • Different determination methods for mannitol are also described and compared. • Various biotechnological strategies for the production of mannitol are reviewed.

A Novel Hydroxylation Step in the Taxane Biosynthetic Pathway: A New Approach to Paclitaxel Production by Synthetic Biology.

Engineered plant cell lines have the potential to achieve enhanced metabolite production rates, providing a high-yielding source of compounds of interest. Improving the production of taxanes, pharmacologically valuable secondary metabolites of Taxus spp., is hindered by an incomplete knowledge of the taxane biosynthetic pathway. Of the five unknown steps, three are thought to involve cytochrome P450-like hydroxylases. In the current work, after an in-depth in silico characterization of four candidate enzymes proposed in a previous cDNA-AFLP assay, TB506 was selected as a candidate for the hydroxylation of the taxane side chain. A docking assay indicated TB506 is active after the attachment of the side chain based on its affinity to the ligand 3'N-dehydroxydebenzoyltaxol. Finally, the involvement of TB506 in the last hydroxylation step of the paclitaxel biosynthetic pathway was confirmed by functional assays. The identification of this hydroxylase will contribute to the development of alternative sustainable paclitaxel production systems using synthetic biology techniques.

Microbial production of (2R,3S)-isocitric acid: state of the arts and prospects.

(2R,3S)-isocitric acid has long been used only as a specific biochemical reagent. However, there is ever increasing evidence that it can also be used as an original promising substance for prevention and treatment of some diseases. The review considers the results of longtime research in our laboratory and the data of other researchers related to microbial synthesis of (2R,3S)-isocitric acid, derivation and selection of active microbial producers, development of their cultivation conditions, as well as the results of study of the mechanism of acid overproduction, and regulation of enzymes involed in this process. The efficient processes of (2R,3S)-isocitric acid production with the aid of natural, mutant, and recombinant strains of Yarrowia lipolytica and methods of product isolation and purification to pharmacopeial standarts are also reviewed.

An overview on biological production of functional lactose derivatives.

Lactose is a natural disaccharide obtained from the milk of most mammals and a waste product of cheese and casein manufacturing. Over the past decades, lactose in whey has increasingly been promoted as an important resource, and an increasing number of significant advances have been made to investigate its healthy and functional properties. Lactose can be biotransformed into many kinds of derivatives, including galacto-oligosaccharides, epilactose, lactulose, lactosucrose, and D-tagatose. Biological efficiency and safety are critical for the enzymatic production of lactose derivatives from lactose. These lactose derivatives show a range of prominent physiological features and effects, such as prebiotic properties, indigestibility, and obesity prevention, which can be utilized in the pharmaceutical, health, and food industries. In this review, we present the properties and physiological effects of lactose derivatives, detailing their biological production by various enzymes and their applications in dairy products, especially directly in the milk industry.

Tropane Alkaloids: Chemistry, Pharmacology, Biosynthesis and Production.

Tropane alkaloids (TA) are valuable secondary plant metabolites which are mostly found in high concentrations in the Solanaceae and Erythroxylaceae families. The TAs, which are characterized by their unique bicyclic tropane ring system, can be divided into three major groups: hyoscyamine and scopolamine, cocaine and calystegines. Although all TAs have the same basic structure, they differ immensely in their biological, chemical and pharmacological properties. Scopolamine, also known as hyoscine, has the largest legitimate market as a pharmacological agent due to its treatment of nausea, vomiting, motion sickness, as well as smooth muscle spasms while cocaine is the 2nd most frequently consumed illicit drug globally. This review provides a comprehensive overview of TAs, highlighting their structural diversity, use in pharmaceutical therapy from both historical and modern perspectives, natural biosynthesis in planta and emerging production possibilities using tissue culture and microbial biosynthesis of these compounds.