Diverse Biotechnological Applications Research Articles

Expressing recombinant selenoproteins using redefinition of a single UAG codon in an RF1-depleted E. coli host strain.

Selenoproteins containing the rare amino acid selenocysteine (Sec), typically being enzymes utilizing the selenium atom of Sec for promoted catalysis of redox reactions, are challenging to obtain at high amounts in pure form. The technical challenges limiting selenoprotein supply derive from intricacies in their translation, necessitating the recoding of a UGA stop codon to a sense codon for Sec. This, in turn, involves the interactions of a Sec-dedicated elongation factor, either directly or indirectly, with a structure in the selenoprotein-encoding mRNA called a SECIS element (Selenocysteine Insertion Sequence), a dedicated tRNA species for Sec with an anticodon for the UGA, and several accessory enzymes and proteins involved in the selenoprotein synthesis. Here, we describe an alternative method for recombinant selenoprotein production using UAG as the Sec codon in a specific strain of E. coli lacking other UAG codons and lacking the release factor RF1 that normally terminates translation at UAG. We also describe how such recombinant selenoproteins can be purified and further analyzed for final Sec contents. The methodology can be used for production of natural selenoproteins in recombinant form as well as for production of synthetic selenoproteins that may be designed to use the unique biophysical properties of Sec for diverse biotechnological applications.

Haloarchaea have a high genomic diversity for the biosynthesis of carotenoids of biotechnological interest

Haloarchaea are mostly components of the microbial biomass of saline aquatic environments, where they can be a dietary source of heterotrophic metazoans or contribute to flamingo's plumage coloration. The diversity of secondary metabolites (SMs) produced by haloarchaea, which might play multiple ecological roles and have diverse biotechnological applications has been largely understudied. Herein, 67 haloarchaeal complete genomes were analyzed and 182 SMs biosynthetic gene clusters (BGCs) identified that encode the production of terpenes (including carotenoids), RiPPs and siderophores. Terpene BGCs were further analysed and it was concluded that all haloarchaea might produce squalene and bacterioruberin, which one a strong antioxidant. Most of them have other carotenoid BGCs that include a putative β-carotene ketolase that was not characterized so far in haloarchaea, but may be involved with canthaxanthin's biosynthesis. The production of bacterioruberin by Haloferax mediterranei ATCC 33500 was found to be not related to its antimicrobial activity.

Antimicrobial Activity of the Heterocytous Cyanobacterium Westiellopsis prolifica and Assessment of Its Microcystin–LR Biosynthesis

THE HETEROCYTOUS cyanobacterium Westiellopsis prolifica has been recently documented in Egypt; however, our understanding of its diverse biotechnological and biomedical applications remains a limited and challenging topic. In this study, a W. prolifica strain isolated from hyper-arid desert habitats was investigated to test its antimicrobial potential against six foodborne pathogenic bacteria (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, and Klebsiella pneumoniae) and eight mycotoxigenic fungi (Aspergillus carbonarius, A. flavus, A. niger, A. ochraceus, A. parasiticus, A. westerdijikia, Fusarium proliferatum, and Penicillium verrucosum), as well as to evaluate its probable toxicity and biosynthesis of the most common microcystin variant (MC–LR) using high-performance liquid chromatography (HPLC). The antimicrobial activity of various W. prolifica extracts was assessed using the disk–diffusion method, and the minimum inhibitory concentrations (MICs) were also determined. Our findings revealed that the extracts of dryland-dwelling W. prolifica had significant antimicrobial activities against all tested bacterial and fungal strains, with average MIC values of 0.4–6.7 and 0.3–9.2mg.mL−1, respectively. This antimicrobial potential can be ascribed to the combined synergistic effects of its biologically active and structurally diverse metabolites. Moreover, this species was a non-producer of MC–LR and was non-toxic based on the brine shrimp bioassay. Thus, the cyanoprokaryote W. prolifica can be considered a sustainable multifunctional candidate in the development of the pharmaceutical industry in Egypt.

Incorporation of Functional Rubisco Activases into Engineered Carboxysomes to Enhance Carbon Fixation.

The carboxysome is a versatile paradigm of prokaryotic organelles and is a proteinaceous self-assembling microcompartment that plays essential roles in carbon fixation in all cyanobacteria and some chemoautotrophs. The carboxysome encapsulates the central CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), using a polyhedral protein shell that is selectively permeable to specific metabolites in favor of Rubisco carboxylation. There is tremendous interest in repurposing carboxysomes to boost carbon fixation in heterologous organisms. Here, we develop the design and engineering of α-carboxysomes by coexpressing the Rubisco activase components CbbQ and CbbO with α-carboxysomes in Escherichia coli. Our results show that CbbQ and CbbO could assemble into the reconstituted α-carboxysome as intrinsic components. Incorporation of both CbbQ and CbbO within the carboxysome promotes activation of Rubisco and enhances the CO2-fixation activities of recombinant carboxysomes. We also show that the structural composition of these carboxysomes could be modified in different expression systems, representing the plasticity of the carboxysome architecture. In translational terms, our study informs strategies for engineering and modulating carboxysomes in diverse biotechnological applications.

Biotechnological applications of marine bacteria in bioremediation of environments polluted with hydrocarbons and plastics.

Marine ecosystems are some of the most adverse environments on Earth and contain a considerable portion of the global bacterial population, and some of these bacterial species play pivotal roles in several biogeochemical cycles. Marine bacteria have developed different molecular mechanisms to address fluctuating environmental conditions, such as changes in nutrient availability, salinity, temperature, pH, and pressure, making them attractive for use in diverse biotechnology applications. Although more than 99% of marine bacteria cannot be cultivated with traditional microbiological techniques, several species have been successfully isolated and grown in the laboratory, facilitating investigations of their biotechnological potential. Some of these applications may contribute to addressing some current global problems, such as environmental contamination by hydrocarbons and synthetic plastics. In this review, we first summarize and analyze recently published information about marine bacterial diversity. Then, we discuss new literature regarding the isolation and characterization of marine bacterial strains able to degrade hydrocarbons and petroleum-based plastics, and species able to produce biosurfactants. We also describe some current limitations for the implementation of these biotechnological tools, but also we suggest some strategies that may contribute to overcoming them. KEY POINTS: • Marine bacteria have a great metabolic capacity to degrade hydrocarbons in harsh conditions. • Marine environments are an important source of new bacterial plastic-degrading enzymes. • Secondary metabolites from marine bacteria have diverse potential applications in biotechnology.

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications.

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

Diverse ATPase Proteins in Mobilomes Constitute a Large Potential Sink for Prokaryotic Host ATP.

Prokaryote mobilome genomes rely on host machineries for survival and replication. Given that mobile genetic elements (MGEs) derive their energy from host cells, we investigated the diversity of ATP-utilizing proteins in MGE genomes to determine whether they might be associated with proteins that could suppress related host proteins that consume energy. A comprehensive search of 353 huge phage genomes revealed that up to 9% of the proteins have ATPase domains. For example, ATPase proteins constitute ∼3% of the genomes of Lak phages with ∼550 kbp genomes that occur in the microbiomes of humans and other animals. Statistical analysis shows the number of ATPase proteins increases linearly with genome length, consistent with a large sink for host ATP during replication of megaphages. Using metagenomic data from diverse environments, we found 505 mobilome proteins with ATPase domains fused to diverse functional domains. Among these composite ATPase proteins, 61.6% have known functional domains that could contribute to host energy diversion during the mobilome infection cycle. As many have domains that are known to interact with nucleic acids and proteins, we infer that numerous ATPase proteins are used during replication and for protection from host immune systems. We found a set of uncharacterized ATPase proteins with nuclease and protease activities, displaying unique domain architectures that are energy intensive based on the presence of multiple ATPase domains. In many cases, these composite ATPase proteins genomically co-localize with small proteins in genomic contexts that are reminiscent of toxin-antitoxin systems and phage helicase-antibacterial helicase systems. Small proteins that function as inhibitors may be a common strategy for control of cellular processes, thus could inspire future biochemical experiments for the development of new nucleic acid and protein manipulation tools, with diverse biotechnological applications.

Symbiotic Associations in Ascidians: Relevance for Functional Innovation and Bioactive Potential.

Associations between different organisms have been extensively described in terrestrial and marine environments. These associations are involved in roles as diverse as nutrient exchanges, shelter or adaptation to adverse conditions. Ascidians are widely dispersed marine invertebrates associated to invasive behaviours. Studying their microbiomes has interested the scientific community, mainly due to its potential for bioactive compounds production—e.g., ET-73 (trabectedin, Yondelis), an anticancer drug. However, these symbiotic interactions embrace several environmental and biological functions with high ecological relevance, inspiring diverse biotechnological applications. We thoroughly reviewed microbiome studies (microscopic to metagenomic approaches) of around 171 hosts, worldwide dispersed, occurring at different domains of life (Archaea, Bacteria, Eukarya), to illuminate the functions and bioactive potential of associated organisms in ascidians. Associations with Bacteria are the most prevalent, namely with Cyanobacteria, Proteobacteria, Bacteroidetes, Actinobacteria and Planctomycetes phyla. The microbiomes of ascidians belonging to Aplousobranchia order have been the most studied. The integration of worldwide studies characterizing ascidians’ microbiome composition revealed several functions including UV protection, bioaccumulation of heavy metals and defense against fouling or predators through production of natural products, chemical signals or competition. The critical assessment and characterization of these communities is extremely valuable to comprehend their biological/ecological role and biotechnological potential.

Mass spore production of Mucor circinelloides on rice.

The online version contains supplementary material available at 10.1007/s13205-021-02853-1.

Urease-Based Biocatalytic Platforms―A Modern View of a Classic Enzyme with Applied Perspectives

The biotechnology industry is currently seeking increased or improved enzyme efficiency and the emergence of new methods to maximize their shelf-life. Enzyme immobilization on solid supports offers characteristic features, such as recyclability, easy product recovery, improved enzyme stability, and biocatalytic properties. Biopolymers-based support materials provide cost-effective, and stability attributes to immobilized enzymes due to non-toxicity, biodegradability, biocompatibility, and the presence of multiple functional moieties. In addition, nanostructured materials with large specific surface areas and unique structural, physical, chemical, and operating characteristics have significantly contributed to biocatalytic systems for diverse biotechnological applications. Urease is a ubiquitous metalloenzyme that displays a high ability to catalyze urea's decomposition into ammonia and carbamate. This review provides information on urease immobilization using various biopolymers-based support matrices to develop nano-biocatalytic systems for biotechnological, medical, and industrial fields.

Membrane perturbation of fullerene and graphene oxide distinguished by pore-forming peptide melittin

Carbon nanomaterials such as fullerenes (C60) and graphene oxide (GO) are considered as promising candidates for diverse applications in biotechnology and biomedicine. However, their potential toxic effects are still under debate. Herein, by using melittin (Mel), a representative pore-forming peptide, as a testing molecule we demonstrated that even the low-concentrated (usually assumed non-toxic) C60 and GO could still mechanically perturb a cell membrane by adsorption and insertion, and consequently influence the function realization of membrane active proteins/peptides. Such perturbations, however, are particle-property and membrane-environment dependent. GO would sensitize both model bilayers and bacterial membranes to Mel, demonstrated as significantly enhanced membrane permeabilization ability or improved antibacterial performance of Mel. In contrast, C60 activates the permeabilization effect of Mel on model membranes, while produces exactly the reverse effect on living bacteria and mammalian cells. Simulations further provide molecular details of the structural disturbance and probe the residue-specific formation of C60-Mel complex in membrane. This work emphasizes the dependence of biological toxicity of nanomaterials on their physico-chemical properties, provides a facile method to detect the subtle structural perturbation of cell membranes at nanoscale, and suggests a necessity for a careful evaluation of the potential influences of nanomaterials on biological processes.

Biologically active secondary metabolites and biotechnological applications of species of the family Chaetomiaceae (Sordariales): an updated review from 2016 to 2021

Fungi are eukaryotic organisms that have the ability to synthesize an enormous number of metabolites with a vast diversity of chemical structures and bioactivities. Chaetomiaceae species are a wealthy source of enzymes with diverse biotechnological and industrial applications such as PMO (polysaccharide monooxygenase), L-methioninase, β-1,3-glucanase, laccase, dextranase, lipolytic, pectinolytic, amylolytic, chitinolytic, and proteolytic enzymes. Different classes of secondary metabolites have been reported from this family are derived from various biosynthetic pathways such as alkaloids, polyketides, peptides, terpenes, and polyketide-amino acid hybrid secondary metabolites. These metabolites have attracted research interest due to their fascinating structural frameworks and bioactivities. Therefore, these compounds can be taken into account as candidates for the development of effective and novel lead compounds for medicine, as well as for plant protection. The current review represents the relevant information for Chaetomiaceae species, in particular, its new and first reported secondary metabolites and their pharmacological activities, as well as the biotechnological applications published from 2016 until the beginning of 2021.

Genome Sequence Analysis of the Oleaginous Yeast, Rhodotorula diobovata, and Comparison of the Carotenogenic and Oleaginous Pathway Genes and Gene Products with Other Oleaginous Yeasts.

Rhodotorula diobovata is an oleaginous and carotenogenic yeast, useful for diverse biotechnological applications. To understand the molecular basis of its potential applications, the genome was sequenced using the Illumina MiSeq and Ion Torrent platforms, assembled by AbySS, and annotated using the JGI annotation pipeline. The genome size, 21.1 MB, was similar to that of the biotechnological “workhorse”, R. toruloides. Comparative analyses of the R. diobovata genome sequence with those of other Rhodotorula species, Yarrowia lipolytica, Phaffia rhodozyma, Lipomyces starkeyi, and Sporidiobolus salmonicolor, were conducted, with emphasis on the carotenoid and neutral lipid biosynthesis pathways. Amino acid sequence alignments of key enzymes in the lipid biosynthesis pathway revealed why the activity of malic enzyme and ATP-citrate lyase may be ambiguous in Y. lipolytica and L. starkeyi. Phylogenetic analysis showed a close relationship between R. diobovata and R. graminis WP1. Dot-plot analysis of the coding sequences of the genes crtYB and ME1 corroborated sequence homologies between sequences from R. diobovata and R. graminis. There was, however, nonsequential alignment between crtYB CDS sequences from R. diobovata and those from X. dendrorhous. This research presents the first genome analysis of R. diobovata with a focus on its biotechnological potential as a lipid and carotenoid producer.

Novel methanotrophic and methanogenic bacterial communities from diverse ecosystems and their impact on environment

Methane oxidizing and methane releasing bacterial communities are very pioneer microbial population, abundantly present in several ecosystems. The microbial dynamics and metabolic assemblages are positively affected by these peculiar groups of bacteria, playing very important role in sustaining carbon geochemical cycle. A number of conventional methanotrophic and methanogenic communities are reported from marine and coastal environment, forest/desert ecosystem, paddy field soil, fresh water lakes, swamps and other soil ecosystems. In rice fields, both bacterial populations are complementary to each other. Methane gas is released by one community while utilized by another community through oxidation process. The present review describe novel species belonging to methanotrophic and methanogenic bacterial communities obtained through culture dependent and culture independent approach mingled with polyphasic taxonomic methods. The novel species of methanogens and methanotrophic bacterial communities have been isolated from different ecosystems and characterized for diverse biotechnological applications. To name a few, Methylobacterium phyllosphaerae sp. nov., Methanococcus aeolicus sp. nov. and Methylocella silvestris sp. nov. have been isolated from paddy field, marine environment and forest ecosystem respectively. The present review deals with biodiversity of novel species of methanogens and methanotrophs and their impact on environment.

Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions.

A versatile approach to entrap relatively small enzymes in hydrogels allows their diverse biotechnological applications. In the present work, bovine carbonic anhydrase (BCA) was efficiently entrapped in calcium alginate beads with the help of liposomes. A mixture of sodium alginate (3 wt %) and carbonic anhydrase–liposome conjugates (BCALs) was dripped into a Tris-HCl buffer solution (pH = 7.5) containing 0.4 M CaCl2 to induce the gelation and curing of the dispersed alginate-rich droplets. The entrapment efficiency of BCALs, which was defined as the amount of catalysts entrapped in alginate beads relative to that initially charged, was 98.7 ± 0.2% as determined through quantifying BCALs in the filtrate being separated from the beads. When free BCA was employed, on the other hand, a significantly lower entrapment efficiency of 27.2 ± 4.1% was obtained because free BCA could pass through alginate matrices. Because the volume of a cured alginate bead (10 μL) entrapped with BCALs was about 2.5 times smaller than that of an original droplet, BCALs were densely present in the beads to give the concentrations of lipids and BCA of 4.6–8.3 mM and 1.1–1.8 mg/mL, respectively. Alginate beads entrapped with BCALs were used to catalyze the hydrolysis of 1.0 mM p-nitrophenyl acetate (p-NA) at pH = 7.5 using the wells of a microplate or 10 mL glass beakers as batch reactors. Furthermore, the beads were confined in a column for continuous-flow hydrolysis of 1.0 mM p-NA for 1 h at a mean residence time of 8.5 or 4.3 min. The results obtained demonstrate that the conjugation of BCA to liposomes gave an opportunity to achieve efficient and stable entrapment of BCA in alginate hydrogels for applying to catalytic reactions in bioreactors.

A Comparative Study on DMSO-Induced Modulation of the Structural and Dynamical Properties of Model Bilayer Membranes

Modulating the structures and properties of biomembranes via permeation of small amphiphilic molecules is immensely important, having diverse applications in cell biology, biotechnology, and pharmaceuticals, because their physiochemical and biological interactions lead to new pathways for transdermal drug delivery and administration. In this work, we have elucidated the role of dimethyl sulfoxide (DMSO), broadly used as a penetration-enhancing agent and cryoprotective agent on model lipid membranes, using a combination of fluorescence microscopy and time-resolved fluorescence spectroscopy. Spatially resolved fluorescence lifetime imaging microscopy (FLIM) has been employed to unravel how the fluidity of the DMSO-induced bilayer regulates the structural alteration of the vesicles. Moreover, we have also shown that the dehydration effect of DMSO leads to weakening of the hydrogen bond between lipid headgroups and water molecules and results in faster solvation dynamics as demonstrated by femtosecond time-resolved fluorescence spectroscopy. It has been gleaned that the water dynamics becomes faster because bilayer rigidity decreases in the presence of DMSO, which is also supported by time-resolved rotational anisotropy measurements. The enhanced diffusivity and increased membrane fluidity in the presence of DMSO are further ratified at the single-molecule level through fluorescence correlation spectroscopy (FCS) measurements. Our results indicate that while the presence of DMSO significantly affects the 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-rac-glycero-3-phosphatidylcholine (DPPC) bilayers, it has a weak effect on 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG) vesicles, which might explain the preferential interaction of DMSO with the positively charged choline group present in DMPC and DPPC vesicles. The experimental findings have also been further verified with molecular dynamics simulation studies. Moreover, it has been observed that DMSO is likely to have a differential effect on heterogeneous bilayer membranes depending on the structure and composition of their headgroups. Our results illuminate the importance of probing the lipid structure and composition of cellular membranes in determining the effects of cryoprotective agents.

Endosome-escaping micelle complexes dually equipped with cell-penetrating and endosome-disrupting peptides for efficient DNA delivery into intact plants.

The delivery of DNA to plants is crucial for enhancing their ability to produce valuable compounds and adapt to climate change. Peptides can provide a versatile tool for delivering DNA to a specific target organelle in various plant species without the use of specialized equipment. However, peptide-mediated DNA delivery suffers from endosomal entrapment and subsequent vacuolar degradation of the DNA cargo, which leads to poor transfection efficiency. To overcome the lack of a reliable approach for bypassing vacuolar degradation in plants, we herein present an endosome-escaping micelle. The micelle surface is dually modified with cell-penetrating (CPP) and endosome-disrupting peptides (EDP) and the core is composed of plasmid DNA condensed with cationic peptides. Due to the functions of CPP and EDP, the dual peptide-modified micelles efficiently undergo endocytic internalization and escape from endosomes to the cytosol, thereby achieving significantly enhanced transfection of intact plants with negligible cytotoxicity. The present study offers a robust strategy for efficient intracellular DNA delivery to plants without vacuolar degradation, and can facilitate plant bioengineering for diverse biotechnological applications.

A nano-biomimetic transformation system enables in planta expression of a reporter gene in mature plants and seeds.

Plant genetic engineering will be essential to decipher the genomic basis of complex traits, optimize crop genomics, and enable plant-based production of recombinant proteins. However, established plant transformation approaches for bioengineering are fraught with limitations. Although nanoparticle-mediated methods show great promise for advancing plant biotechnology, many engineered nanomaterials can have cytotoxic and ecological effects. Here, we demonstrate the efficient uptake of a nano-biomimetic carrier of plasmid DNA and transient expression of a reporter gene in leaves of Arabidopsis, common ice plant and tobacco, as well as in the developing seed tissues of Arabidopsis, field mustard, barley, and wheat. The nano-biomimetic transformation system described here has all the advantages of other nanoparticle-mediated approaches for passive delivery of genetic cargo into a variety of plant species and is also nontoxic to cells and to the environment for diverse biotechnological applications in plant biology and crop science.

The rise and shine of yeast optogenetics.

Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared with traditional chemically inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost, and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, and protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.

GeneReg: a constraint-based approach for design of feasible metabolic engineering strategies at the gene level.

MotivationLarge-scale metabolic models are widely used to design metabolic engineering strategies for diverse biotechnological applications. However, the existing computational approaches focus on alteration of reaction fluxes and often neglect the manipulations of gene expression to implement these strategies.ResultsHere, we find that the association of genes with multiple reactions leads to infeasibility of engineering strategies at the flux level, since they require contradicting manipulations of gene expression. Moreover, we identify that all of the existing approaches to design gene knockout strategies do not ensure that the resulting design may also require other gene alterations, such as up- or downregulations, to match the desired flux distribution. To address these issues, we propose a constraint-based approach, termed GeneReg, that facilitates the design of feasible metabolic engineering strategies at the gene level and that is readily applicable to large-scale metabolic networks. We show that GeneReg can identify feasible strategies to overproduce ethanol in Escherichia coli and lactate in Saccharomyces cerevisiae, but overproduction of the TCA cycle intermediates is not feasible in five organisms used as cell factories under default growth conditions. Therefore, GeneReg points at the need to couple gene regulation and metabolism to design rational metabolic engineering strategies.Availability and implementation https://github.com/MonaRazaghi/GeneReg Supplementary information Supplementary data are available at Bioinformatics online.