Optimized co-fermentation strategy for efficient production of Dihydro-β-ionone from β-Ionol using engineered Escherichia coli and Saccharomyces cerevisiae.

Metabolic flux analysis is a vital tool used to determine the ultimate output of cellular metabolism and thus detect biotechnologically relevant bottlenecks in productivity. 13C-based metabolic flux analysis (13C-MFA) and flux balance analysis (FBA) have many potential applications in biotechnology. However, noteworthy hurdles in fluxomics study are still present. First, several technical difficulties in both 13C-MFA and FBA severely limit the scope of fluxomics findings and the applicability of obtained metabolic information. Second, the complexity of metabolic regulation poses a great challenge for precise prediction and analysis of metabolic networks, as there are gaps between fluxomics results and other omics studies. Third, despite identified metabolic bottlenecks or sources of host stress from product synthesis, it remains difficult to overcome inherent metabolic robustness or to efficiently import and express nonnative pathways. Fourth, product yields often decrease as the number of enzymatic steps increases. Such decrease in yield may not be caused by rate-limiting enzymes, but rather is accumulated through each enzymatic reaction. Fifth, a high-throughput fluxomics tool hasnot been developed for characterizing nonmodel microorganisms and maximizing their application in industrial biotechnology. Refining fluxomics tools and understanding these obstacles will improve our ability to engineer highlyefficient metabolic pathways in microbial hosts.

Psychrophilic bacteria are a large group of microorganisms that prevail in low-temperature ecosystems. Psychrophilic bacteria have undergone a number of adaptations that help them exist in such conditions. One of such adaptations is the use of enzymes with a high specific activity at low temperatures. Such enzymes are usually called “cold-active.” These enzymes have potential applications in biotechnology and industry. In our review, we considered individual genera of psychrophilic bacteria, current global trends in the study of cold-active enzymes, their applications, and place in industrial biotechnology. Thus, the main goal of this study was to explore the diversity of psychrophilic bacteria, as well as opportunities of their application in biotechnology. The natural ecological sites of psychrophiles are numerous and varied. Psychrophiles form a permanent microflora of eternal cold regions, polar regions and oceans. Bacteria belonging to this group are found in soil, water or associated with plants and animals. An important site for psychrophilic microorganisms is a low-temperature water reservoir. At present, many new genera of psychrophiles and psychrotrophs have been derived from the bottom sediments and sea waters of the Arctic and Antarctic and described. Psychrophilic microorganisms are found in caves and in ancient ice crystal structures. The latter testifies to the very possibility of the super-long anabiosis phenomenon, as well as vital capacity preservation without division for a long period of time. Psychrophiles do not have a single form, they belong to at least several phylogenetic groups. Psychrophilic forms are found among the representatives of a large number of genera. There are no common physiological and biochemical parameters typical of psychrophilic bacteria. They comprise rods, cocci, vibrios, gram-negative and gram-positive bacteria, bacteria that produce and do not produce spores, strict aerobes, facultative and strict anaerobes. We lay greater emphasis on the diversity of psychrophilic bacteria capable of producing industrially important enzymes. The review considers bacteria belonging to the genera Vibrio and Aliivibrio, Pseudomonas, Achromobacter, Arthrobacter, Pseudoalteromonas, Bacillus, Clostridium, Micrococcus, Psychrobacter, Psychromonas, Flavobacterium, and psychrophilic methanotrophic microorganisms. These bacteria enzymes are used in agriculture, biotechnology, pharmaceuticals and household chemicals, as well as other sectors of the national economy. Psychrophilic bacteria produce a chemical compound that can be used in medicine. For example, Pseudomonas antarctica contains a cluster of genes encoding microcin B, R-type pyocins, adenosylcobalamin, and pyrroloquinoline quinone. Thus, P. antarctica has antibiotic activity. Psychrobacter proteolyticus also has an antineoplastic action and secrets an extracellular cold-adapted metalloproteinase being able to inhibit the space-occupying process. Cold-active metalloproteinases are also widely used as detergents, in currying, food sector and molecular biology. The immunogenic Pal conformable protein was derived from the psychrophilic strain of The representatives of the genus Arthrobacter capable of metabolizing diuron and petroleum products have an important property. A. agilis produces a red pigment, a bacterioruberin-type carotenoid being interesting as an antioxidant. A. psychrochitiniphilus is promising for cleaning water areas, oil-polluted coastlines, as it decomposes oil and petroleum products. Flavobacterium limicola is a potential source of cold-active protease. This bacterium is characterized by an increase in protease secretion as temperature decreases. Thus, F. limicola can be used in environmental biotransformations and bioremediations. The psychrophilic bacteria of the genus Bacillus are the participants of active studies. Their cold-active enzymes have a high potential in various areas of biomedicine, immunology, decontamination, and various industrial applications. The antifreeze proteins of psychrophilic Clostridia are considered a promising biotechnological product for use in medicine, food, beauty products, fuel, and other industries. This study reviews literary sources and indicates that at present obligate and facultative psychrophiles (psychrotrophs) and their cold-active enzymes are of scientific interest throughout the world. A significant part of the research is focused on a general understanding of the distribution of psychrophilic bacteria and a local study of enzymatic activity. A further study of psychrophilic microorganisms producing enzymes at low temperatures will reveal new ways for the development of biotechnologies in various sectors of the national economy. The paper contains 94 References. The Authors declare no conflict of interest.

Ni2+-functionalized porous ceramic/agarose composite beads (Ni-NTA Cerose) can be used as carrier materials to immobilize enzymes harboring a metal affinity tag. Here, a 6×His-tag fusion alcohol dehydrogenase Mu-S5 and glucose dehydrogenase from Bacillus megaterium (BmGDH) were co-immobilized on Ni-NTA Cerose to construct a packed bed reactor (PBR) for the continuous synthesis of the chiral intermediate (S)-(4-chlorophenyl)-(pyridin-2-yl) methanol ((S)-CPMA) NADPH recycling, and in situ product adsorption was achieved simultaneously by assembling a D101 macroporous resin column after the PBR. Using an optimum enzyme activity ratio of 2:1 (Mu-S5: BmGDH) and hydroxypropyl-β-cyclodextrin as co-solvent, a space-time yield of 1560 g/(L·d) could be achieved in the first three days at a flow rate of 5 mL/min and substrate concentration of 10 mM. With simplified selective adsorption and extraction procedures, (S)-CPMA was obtained in 84% isolated yield.

Chapter 1 - Microbial screening for extremozymes

The spoilage of cassava by Aspergillus niger strain XJ42 poses significant challenges to food security and industrial biotechnology, necessitating an in-depth understanding of its enzymatic mechanisms. This study aimed to amplify and characterize the α-amylase gene, CDF_Amyl, from A. niger strain XJ42 and evaluate its biotechnological potential. The CDF_Amyl gene was successfully amplified and partially sequenced, revealing a protein consisting of 222 amino acids with a molecular weight of 25.13 kDa and an isoelectric point of 4.17. Sequence alignment demonstrated high homology with α-amylase genes from A. niger (98%) and A. oryzae RIB40 (99%), suggesting evolutionary conservation and potential functional similarities. Kinetic analysis of the enzyme revealed a maximum velocity (Vmax) of 6.90 U/mg protein and a Michaelis constant (Km) of 6.70 mg/ml, indicating its catalytic efficiency. These findings highlight the enzyme’s robust activity and potential applications in biotechnology, particularly in starch hydrolysis for industrial, food, and pharmaceutical processes. The unique properties of CDF_Amyl suggest that it may serve as a valuable biocatalyst for enhancing cassava processing and other starch-based industries. Further studies on its structural and functional properties could facilitate its commercial exploitation in biotechnological innovations.

Newly isolated microorganisms with potential application in biotechnology

Steroid compounds are produced by eukaryotes where they have a variety of chemical structures and play important physiological roles. Many bacteria are capable of transforming and completely degrading steroids under various growth conditions. The microbial metabolism of steroids has gained considerable interest due to its potential applications in industrial and environmental biotechnology. The oxic degradation pathways of steroids and some of the involved enzymes are well characterized. The key players in these pathways are oxygenases which depend on dioxygen as a co-substrate. On the contrary, much less is known about the mechanisms operating under anoxic conditions. Obviously, anoxic bacterial metabolism of steroids should proceed via oxygenase-independent reactions. So far, a few bacteria that can completely degrade steroids in the absence of oxygen were characterized. Surprisingly, all of them belong to denitrifying bacteria and utilize only nitrate as the alternative electron acceptor. Recent studies of anoxic metabolism of steroids using denitrifying bacteria revealed unique and interesting biochemical reactions and enzymes. Here we discuss the current understanding of the biochemistry and molecular biology of bacterial steroid metabolism under anoxic conditions. The aerobic metabolism of steroids is briefly presented for the sake of comparison. Future investigations on anoxic metabolism of steroids will unravel novel aspects of the regulation and evolution of catabolic pathways as well as unprecedented biocatalysts with useful applications in biotechnology.

Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription-translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription-translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.

Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnological applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mechanical model that accounts for the ensemble nature of folding. We validate the method by predicting the absolute changes in stability for 138 experimental mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing meosphile-thermophile protein pairs. In parallel, we devise a methodology to rapidly engineer mutations in silico which we benchmark against experimental mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnology industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs.

Enzyme-catalyzed asymmetric reduction of ethyl 4-chloro-3-oxobutanoate in an organic solvent-water diphasic system was studied. NADPH-dependent aldehyde reductase isolated from Sporobolomyces salmonicolor AKU4429 and glucose dehydrogenase were used as catalysts for reduction of ethyl 4-chloro-3-oxobutanoate and recycling of NADPH, respectively, in this system. In an aqueous system, the substrate was unstable. Inhibition of the reaction and inactivation of the enzymes by the substrate and the product were also observed. An n-butyl acetate-water diphasic system very efficiently overcame these limitations. In a 1,600-ml-1,600-ml scale diphasic reaction, ethyl (R)-4-chloro-3-hydroxybutanoate (0.80 mol; 86% enantiomeric excess) was produced from the corresponding oxoester in a molar yield of 95.4% with an NADPH turnover of 5,500 mol/mol.

Two multi-enzymatic systems for the production of optically active compounds are discussed. One is a system for deracemization via enzymatic stereoinversion useful for chiral 1,2-diol synthesis, which involves stereoselective oxidation and stereoselective reduction using an alcohol dehydrogenase and reductase with opposite stereospecificities. The recycling of NAD+ was carried out by a water-forming NADH oxidase. The recycling of NADPH was carried out by an NADPH-dependent glucose dehydrogenase. The recombinant Escherichia coli capable of overproducing the four enzymes was constructed for purposes of generating catalysts useful for the deracemization of racemic diols. Optically active 3-chloro-1,2-propanediol and (S)-1,2-pentanediol could be prepared by using the recombinant E. coli cells in a one-pot reaction with high yield. Second is a transamination system for chiral secondary amine synthesis. Two novel bacterial ω-transaminases capable of catalyzing a stereoselective transamination between a ketone and amine were discovered by enrichment culturing. An (R)-specific ω-transaminase was isolated from a microorganism and characterized, and the relevant gene was cloned. Addition of lactate dehydrogenase into the transaminase reaction mixture was effective at increasing the yield by ensuring the reversibility of the reaction. A recombinant E. coli system capable of overproducing the ω-transaminase, lactate dehydrogenase, and glucose dehydrogenase used for regenerating NADH was constructed. By using the recombinant enzyme systems, various kinds of useful optically active amines could be prepared in good yields and high optical purity without using high-pressure and high-temperature conditions.

Here we present a metabolic profiling strategy employing direct infusion Orbitrap mass spectrometry (MS) and gas chromatography-mass spectrometry (GC/MS) for the monitoring of soybean's (Glycine max L.) global metabolism regulation in response to Rhizoctonia solani infection in a time-course. Key elements in the approach are the construction of a comprehensive metabolite library for soybean, which accelerates the steps of metabolite identification and biological interpretation of results, and bioinformatics tools for the visualization and analysis of its metabolome. The study of metabolic networks revealed that infection results in the mobilization of carbohydrates, disturbance of the amino acid pool, and activation of isoflavonoid, α-linolenate, and phenylpropanoid biosynthetic pathways of the plant. Components of these pathways include phytoalexins, coumarins, flavonoids, signaling molecules, and hormones, many of which exhibit antioxidant properties and bioactivity helping the plant to counterattack the pathogen's invasion. Unraveling the biochemical mechanism operating during soybean-Rhizoctonia interaction, in addition to its significance towards the understanding of the plant's metabolism regulation under biotic stress, provides valuable insights with potential for applications in biotechnology, crop breeding, and agrochemical and food industries.

Biocatalysts are a biomolecule of interest for various biotechnological applications. Non-reusability and poor stability of especially enzymes has always limited their applications in large-scale processing units. Nanotechnology paves a way by conjugating the biocatalysts on different matrices. It predominantly enables nanomaterials to overcome the limited efficacy of conventional biocatalysts. Nanomaterial conjugated nanobiocatalyst have enhanced catalytic properties, selectivity, and stability. Nanotechnology extended the flexibility to engineer biocatalysts for various innovative and predictive catalyses. So developed nanobiocatalyst harbors remarkable properties and has potential applications in diverse biotechnological sectors. This article summaries various developments made in the area of nanobiocatalyst towards their applications in biotechnological industries. Novel nanobiocatalyst engineering is an area of critical importance for harnessing the biotechnological potential.

The tale of a versatile enzyme: Alpha-amylase evolution, structure, and potential biotechnological applications for the bioremediation of n-alkanes

Interests remain in searching for cofactor regeneration system with higher efficiency at lower substrate cost. Glucose dehydrogenase (GDH) system has been dominant in NADH regeneration, but it only has a theoretical yield of one NADH per glucose molecule. This work sought to explore the utility of a two-step ethanol utilization pathway (EUP) in pathway-based NADH regeneration. The pathway runs from ethanol to acetaldehyde and to acetyl-CoA with each step generating one NADH, that together results in a higher theoretical yield of two NADH per ethanol molecule. In this project, anaerobic biotransformation of ketone (acetophenone or butanone) to alcohol by cpsADH from Candida parapsilosis was used as readout for evaluating relative efficacy and operating modes for EUP cofactor regeneration in Escherichia coli BL21 (DE3). Experiment tests validated that EUP was more efficient than GDH in NADH regeneration. Further, growing cell delivered higher biotransformation efficiency compared to resting cell due to the driving force generated by cell growth. Finally, preculture or cultivation in M9 + 10 g/L ethanol medium delivered higher biotransformation efficiency compared to LB medium. Overall, EUP could help regenerate NADH in support of a biocatalytic reaction, and is more efficient in cofactor regeneration than GDH.