Transcriptomics aids in uncovering the metabolic shifts and molecular machinery of Schizochytrium limacinum during biotransformation of hydrophobic substrates to docosahexaenoic acid.

BACKGROUND: Biotransformation of waste oil into value-added nutraceuticals provides a sustainable strategy. Thraustochytrids are heterotrophic marine protists and promising producers of omega (ω) fatty acids. Although the metabolic routes for the assimilation of hydrophilic carbon substrates such as glucose are known for these microbes, the mechanisms employed for the conversion of hydrophobic substrates are not well established. Here, thraustochytrid Schizochytrium limacinum SR21 was investigated for its ability to convert oils (commercial oils with varying fatty acid composition and waste cooking oil) into ω-3 fatty acid; docosahexaenoic acid (DHA). RESULTS: Within 72 h SR21 consumed ~ 90% of the oils resulting in enhanced biomass (7.5 g L- 1) which was 2-fold higher as compared to glucose. Statistical analysis highlights C16 fatty acids as important precursors of DHA biosynthesis. Transcriptomic data indicated the upregulation of multiple lipases, predicted to possess signal peptides for secretory, membrane-anchored and cytoplasmic localization. Additionally, transcripts encoding for mitochondrial and peroxisomal ß-oxidation along with acyl-carnitine transporters were abundant for oil substrates that allowed complete degradation of fatty acids to acetyl CoA. Further, low levels of oxidative biomarkers (H2O2, malondialdehyde) and antioxidants were determined for hydrophobic substrates, suggesting that SR21 efficiently mitigates the metabolic load and diverts the acetyl CoA towards energy generation and DHA accumulation. CONCLUSIONS: The findings of this study contribute to uncovering the route of assimilation of oil substrates by SR21. The thraustochytrid employs an intricate crosstalk among the extracellular and intracellular molecular machinery favoring energy generation. The conversion of hydrophobic substrates to DHA can be further improved using synthetic biology tools, thereby providing a unique platform for the sustainable recycling of waste oil substrates.

Synthesis and Characterization of Lignin-Silver Nanoparticles.

Metal nanoparticle synthesis via environmentally friendly methods is gaining interest for their potential advantages over conventional physico-chemical approaches. Herein, we propose a robust green synthesis route for lignin-modified silver nanoparticles, utilizing the recovery of lignin as a renewable raw material and exploring its application in valuable areas. Through a systematic approach combining UV-Vis spectroscopy with AAS and DLS, we identified repeatable and scalable reaction conditions in an aqueous solution at pH 11 for homogeneous silver nanoparticles with high uniformity. The TEM median sizes ranged from 12 to 15 nm with circularity between 0.985 and 0.993. The silver nanoparticles yield exceeded 0.010 mol L-1, comparable with traditional physico-chemical methods, with a minimal loss of silver precursor ranging between 0.5 and 3.9%. Characterization by XRD and XPS revealed the presence of Ag-O bonding involving lignin functional groups on the pure face-centered cubic structure of metallic silver. Moreover, the lignin-modified silver nanoparticles generated a localized thermal effect upon near-infrared laser irradiation (808 nm), potentially allowing for targeted applications in the biomedical field. Our study showcases the potential of lignin as a renewable reducing and capping agent for silver nanoparticle synthesis, addressing some shortcomings of green synthesis approaches and contributing to the development of suitable nanomaterials.

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach.

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.

Spruce Bark-Extracted Lignin and Tannin-Based Bioresin-Adhesives: Effect of Curing Temperatures on the Thermal Properties of the Resins.

In this study, formaldehyde-free bioresin adhesives were synthesised from lignin and tannin, which were obtained from softwood bark. The extraction was done via organosolv treatment and hot water extraction, respectively. A non-volatile, non-toxic aldehyde, glyoxal, was used as a substitute for formaldehyde in order to modify the chemical structure of both the lignin and tannin. The glyoxal modification reaction was confirmed by ATR-FTIR spectroscopy. Three different resin formulations were prepared using modified lignin along with the modified tannin. The thermal properties of the modified lignin, tannin, and the bioresins were assessed by DSC and TGA. When the bioresins were cured at a high temperature (200 °C) by compression moulding, they exhibited higher thermal stability as well as an enhanced degree of cross-linking compared to the low temperature-cured bioresins. The thermal properties of the resins were strongly affected by the compositions of the resins as well as the curing temperatures.

Organosolv Fractionation of Birch Sawdust: Establishing a Lignin-First Biorefinery.

The use of residual biomass for bioconversions makes it possible to decrease the output of fossil-based chemicals and pursue a greener economy. While the use of lignocellulosic material as sustainable feedstock has been tried at pilot scale, industrial production is not yet economically feasible, requiring further technology and feedstock optimization. The aim of this study was to examine the feasibility of replacing woodchips with residual sawdust in biorefinery applications. Woodchips can be used in value-added processes such as paper pulp production, whereas sawdust is currently used mainly for combustion. The main advantages of sawdust are its large supply and a particle size sufficiently small for the pretreatment process. Whereas, the main challenge is the higher complexity of the lignocellulosic biomass, as it can contain small amounts of bark and cambium. Here, we studied the fractionation of birch sawdust by organosolv pretreatment at two different temperatures and for two different durations. We evaluated the efficiency of fractionation into the three main fractions: lignin, cellulose, and hemicellulose. The cellulose content in pretreated biomass was as high as 69.2%, which was nearly double the amount in untreated biomass. The obtained lignin was of high purity, with a maximum 4.5% of contaminating sugars. Subsequent evaluation of the susceptibility of pretreated solids to enzymatic saccharification revealed glucose yields ranging from 75% to 90% after 48 h but reaching 100.0% under the best conditions. In summary, birch sawdust can be successfully utilized as a feedstock for organosolv fractionation and replace woodchips to simplify and lower the costs of biorefinery processes.

A New Approach for Evaluating Electron Transfer Dynamics by Using In Situ Resonance Raman Microscopy and Chronoamperometry in Conjunction with a Dynamic Model.

Geobacter sulfurreducens is a good candidate as a chassis organism due to its ability to form thick, conductive biofilms, enabling long-distance extracellular electron transfer (EET). Due to the complexity of EET pathways in G. sulfurreducens, a dynamic approach is required to study genetically modified EET rates in the biofilm. By coupling online resonance Raman microscopy with chronoamperometry, we were able to observe the dynamic discharge response in the biofilm's cytochromes to an increase in anode voltage. Measuring the heme redox state alongside the current allows for the fitting of a dynamic model using the current response and a subsequent validation of the model via the value of a reduced cytochrome c Raman peak. The modeled reduced cytochromes closely fitted the Raman response data from the G. sulfurreducens wild-type strain, showing the oxidation of heme groups in cytochromes until a new steady state was achieved. Furthermore, the use of a dynamic model also allows for the calculation of internal rates, such as acetate and NADH consumption rates. The Raman response of a mutant lacking OmcS showed a higher initial oxidation rate than predicted, followed by an almost linear decrease of the reduced mediators. The increased initial rate could be attributed to an increase in biofilm conductivity, previously observed in biofilms lacking OmcS. One explanation for this is that OmcS acts as a conduit between cytochromes; therefore, deleting the gene restricts the rate of electron transfer to the extracellular matrix. This could, however, be modeled assuming a linear oxidation rate of intercellular mediators.IMPORTANCE Bioelectrochemical systems can fill a vast array of application niches, due to the control of redox reactions that it offers. Although native microorganisms are preferred for applications such as bioremediation, more control is required for applications such as biosensors or biocomputing. The development of a chassis organism, in which the EET is well defined and readily controllable, is therefore essential. The combined approach in this work offers a unique way of monitoring and describing the reaction kinetics of a G. sulfurreducens biofilm, as well as offering a dynamic model that can be used in conjunction with applications such as biosensors.

Effect of Oligosaccharide Degree of Polymerization on the Induction of Xylan-Degrading Enzymes by Fusarium oxysporum f. sp. Lycopersici.

Xylan is one of the most abundant carbohydrates on Earth. Complete degradation of xylan is achieved by the collaborative action of endo-ß-1,4-xylanases and ß-d-xylosidases and a number of accessories enzymes. In filamentous fungi, the xylanolytic system is controlled through induction and repression. However, the exact mechanism remains unclear. Substrates containing xylan promote the induction of xylanases, which release xylooligosaccharides. These, in turn, induce expression of xylanase-encoding genes. Here, we aimed to determine which xylan degradation products acted as inducers, and whether the size of the released oligomer correlated with its induction strength. To this end, we compared xylanase production by different inducers, such as sophorose, lactose, cellooligosaccharides, and xylooligosaccharides in Fusarium oxysporum f. sp. lycopersici. Results indicate that xylooligosaccharides are more effective than other substrates at inducing endoxylanase and ß-xylosidases. Moreover, we report a correlation between the degree of xylooligosaccharide polymerization and induction efficiency of each enzyme. Specifically, xylotetraose is the best inducer of endoxylanase, xylohexaose of extracellular ß-xylosidase, and xylobiose of cell-bound ß-xylosidase.

Biosynthesis of Nutraceutical Fatty Acids by the Oleaginous Marine Microalgae Phaeodactylum tricornutum Utilizing Hydrolysates from Organosolv-Pretreated Birch and Spruce Biomass.

Polyunsaturated fatty acids (PUFAs) are essential for human function, however they have to be provided through the diet. As their production from fish oil is environmentally unsustainable, there is demand for new sources of PUFAs. The aim of the present work was to establish the microalgal platform to produce nutraceutical-value PUFAs from forest biomass. To this end, the growth of Phaeodactylum tricornutum on birch and spruce hydrolysates was compared to autotrophic cultivation and glucose synthetic media. Total lipid generated by P. tricornutum grown mixotrophically on glucose, birch, and spruce hydrolysates was 1.21, 1.26, and 1.29 g/L, respectively. The highest eicosapentaenoic acid (EPA) production (256 mg/L) and productivity (19.69 mg/L/d) were observed on spruce hydrolysates. These values were considerably higher than those obtained from the cultivation without glucose (79.80 mg/L and 6.14 mg/L/d, respectively) and also from the photoautotrophic cultivation (26.86 mg/L and 2.44 mg/L/d, respectively). To the best of our knowledge, this is the first report describing the use of forest biomass as raw material for EPA and docosapentaenoic acid (DHA) production.

On-Line Raman Spectroscopic Study of Cytochromes' Redox State of Biofilms in Microbial Fuel Cells.

Bio-electrochemical systems such as microbial fuel cells and microbial electrosynthesis cells depend on efficient electron transfer between the microorganisms and the electrodes. Understanding the mechanisms and dynamics of the electron transfer is important in order to design more efficient reactors, as well as modifying microorganisms for enhanced electricity production. Geobacter are well known for their ability to form thick biofilms and transfer electrons to the surfaces of electrodes. Currently, there are not many "on-line" systems for monitoring the activity of the biofilm and the electron transfer process without harming the biofilm. Raman microscopy was shown to be capable of providing biochemical information, i.e., the redox state of C-type cytochromes, which is integral to external electron transfer, without harming the biofilm. In the current study, a custom 3D printed flow-through cuvette was used in order to analyze the oxidation state of the C-type cytochromes of suspended cultures of three Geobacter sulfurreducens strains (PCA, KN400 and ΔpilA). It was found that the oxidation state is a good indicator of the metabolic state of the cells. Furthermore, an anaerobic fluidic system enabling in situ Raman measurements was designed and applied successfully to monitor and characterize G. sulfurreducens biofilms during electricity generation, for both a wild strain, PCA, and a mutant, ΔS. The cytochrome redox state, monitored by the Raman peak areas, could be modulated by applying different poise voltages to the electrodes. This also correlated with the modulation of current transferred from the cytochromes to the electrode. The Raman peak area changed in a predictable and reversible manner, indicating that the system could be used for analyzing the oxidation state of the proteins responsible for the electron transfer process and the kinetics thereof in-situ.

Simultaneous Quantification of L-arginine and Monosaccharides during Fermentation: An Advanced Chromatography Approach.

Increasing demand for L-arginine by the food and pharmaceutical industries has sparked the search for sustainable ways of producing it. Microbial fermentation offers a suitable alternative; however, monitoring of arginine production and carbon source uptake during fermentation, requires simple and reliable quantitative methods compatible with the fermentation medium. Two methods for the simultaneous quantification of arginine and glucose or xylose are described here: high-performance anion-exchange chromatography coupled to integrated pulsed amperometric detection (HPAEC-IPAD) and reversed-phase ultra-high-performance liquid chromatography combined with charged aerosol detection (RP-UHPLC-CAD). Both were thoroughly validated in a lysogeny broth, a minimal medium, and a complex medium containing corn steep liquor. HPAEC-IPAD displayed an excellent specificity, accuracy, and precision for arginine, glucose, and xylose in minimal medium and lysogeny broth, whereas specificity and accuracy for arginine were somewhat lower in medium containing corn steep liquor. RP-UHPLC-CAD exhibited high accuracy and precision, and enabled successful monitoring of arginine and glucose or xylose in all media. The present study describes the first successful application of the above chromatographic methods for the determination and monitoring of L-arginine amounts during its fermentative production by a genetically modified Escherichia coli strain cultivated in various growth media.

Correction to: Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions.

After publication of the original article, authors found that there has been a minor mistake in the units of kcat and kcat/Km in Table 2. The units should be 103 min-1 g-1 FAE for kcat and mM-1 min-1 g-1 FAE for kcat/Km. This correction does not affect any conclusions drawn within the article.

Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Formation of Lignin Nanoparticles by Combining Organosolv Pretreatment of Birch Biomass and Homogenization Processes.

Valorization of lignocellulosic biomass into a biorefinery scheme requires the use of all biomass components; in this, the lignin fraction is often underutilized. Conversion of lignin to nanoparticles is an attractive solution. Here, we investigated the effect of different lignin isolation processes and a post-treatment homogenization step on particle formation. Lignin was isolated from birch chips by using two organosolv processes, traditional organosolv (OS) and hybrid organosolv-steam explosion (HOS-SE) at various ethanol contents. For post-treatment, lignin was homogenized at 500 bar using different ethanol:water ratios. Isolation of lignin with OS resulted in unshaped lignin particles, whereas after HOS-SE, lignin micro-particles were formed directly. Addition of an acidic catalyst during HOS-SE had a negative impact on the particle formation, and the optimal ethanol content was 50⁻60% v/v. Homogenization had a positive effect as it transformed initially unshaped lignin into spherical nanoparticles and reduced the size of the micro-particles isolated by HOS-SE. Ethanol content during homogenization affected the size of the particles, with the optimal results obtained at 75% v/v. We demonstrate that organosolv lignin can be used as an excellent starting material for nanoparticle preparation, with a simple method without the need for extensive chemical modification. It was also demonstrated that tuning of the operational parameters results in nanoparticles of smaller size and with better size homogeneity.

Acid Assisted Organosolv Delignification of Beechwood and Pulp Conversion towards High Concentrated Cellulosic Ethanol via High Gravity Enzymatic Hydrolysis and Fermentation.

BACKGROUND: Future biorefineries will focus on converting low value waste streams to chemical products that are derived from petroleum or refined sugars. Feedstock pretreatment in a simple, cost effective, agnostic manner is a major challenge. METHODS: In this work, beechwood sawdust was delignified via an organosolv process, assisted by homogeneous inorganic acid catalysis. Mixtures of water and several organic solvents were evaluated for their performance. Specifically, ethanol (EtOH), acetone (AC), and methyl- isobutyl- ketone (MIBK) were tested with or without the use of homogeneous acid catalysis employing sulfuric, phosphoric, and oxalic acids under relatively mild temperature of 175 °C for one hour. RESULTS: Delignification degrees (DD) higher than 90% were achieved, where both AC and EtOH proved to be suitable solvents for this process. Both oxalic and especially phosphoric acid proved to be good alternative catalysts for replacing sulfuric acid. High gravity simultaneous saccharification and fermentation with an enzyme loading of 8.4 mg/gsolids at 20 wt.% initial solids content reached an ethanol yield of 8.0 w/v%. CONCLUSIONS: Efficient delignification combining common volatile solvents and mild acid catalysis allowed for the production of ethanol at high concentration in an efficient manner.

Optimization of Transesterification Reactions with CLEA-Immobilized Feruloyl Esterases from Thermothelomyces thermophila and Talaromyces wortmannii.

Feruloyl esterases (FAEs, E.C. 3.1.1.73) are biotechnologically important enzymes with several applications in ferulic acid production from biomass, but also in synthesis of hydroxycinnamic acid derivatives. The use of such biocatalysts in commercial processes can become feasible by their immobilization, providing the advantages of isolation and recycling. In this work, eight feruloyl esterases, immobilized in cross-linked enzyme aggregates (CLEAs) were tested in regard to their transesterification performance, towards the production of prenyl ferulate (PFA) and arabinose ferulate (AFA). After solvent screening, comparison with the activity of respective soluble enzymes, and operational stability tests, FAE125 was selected as the most promising biocatalyst. A central composite design revealed the optimum conditions for each transesterification product, in terms of water content, time, and substrate ratio for both products, and temperature and enzyme load additionally for prenyl ferulate. The optimum product yields obtained were 83.7% for PFA and 58.1% for AFA. FAE125 CLEAs are stable in the optimum conditions of transesterification reactions, maintaining 70% residual activity after five consecutive reactions. Overall, FAE125 CLEAs seem to be able to perform as a robust biocatalyst, offering satisfactory yields and stability, and thus showing significant potential for industrial applications.

Lignin from Hardwood and Softwood Biomass as a Lubricating Additive to Ethylene Glycol.

Ethylene glycol (EG)-based lubricant was prepared with dissolved organosolv lignin from birch wood (BL) and softwood (SL) biomass. The effects of different lignin types on the rheological, thermal, and tribological properties of the lignin/EG lubricants were comprehensively investigated by various characterization techniques. Dissolving organosolv lignin in EG results in outstanding lubricating properties. Specifically, the wear volume of the disc by EG-44BL is only 8.9% of that lubricated by pure EG. The enhanced anti-wear property of the EG/lignin system could be attributed to the formation of a robust lubrication film and the strong adhesion of the lubricant on the contacting metal surface due to the presence of a dense hydrogen bonding (H-bonding) network. The lubricating performance of EG-BL outperforms EG-SL, which could be attributed to the denser H-bonding sites in BL and its broader molecular weight distribution. The disc wear loss of EG-44BL is only 45.7% of that lubricated by EG-44SL. Overall, H-bonding is the major contributor to the different tribological properties of BL and SL in EG-based lubricants.

Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions.

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 µM for PFA, 329.9 µM for FA). PFA was not cytotoxic at 0.8-100 µM (IC50 220.23 µM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 µM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application.

Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflammatory, skin whitening, anti-aging, anti-wrinkling, or photoprotective activity, among others. During the past years, there has been an increased interest on the enzymatic synthesis of bioactive esters and glycosides based on (trans)esterification, (trans)glycosylation, or oxidation reactions. Natural bioactive compounds with exceptional theurapeutic properties and low toxicity may offer a new insight into the design and development of potent and beneficial cosmetics. This review gives an overview of the enzymatic modifications which are performed currently for the synthesis of products with attractive properties for the cosmeceutical industry.

Effect of Different Pretreatment Methods on Birch Outer Bark: New Biorefinery Routes.

A comparative study among different pretreatment methods used for the fractionation of the birch outer bark components, including steam explosion, hydrothermal and organosolv treatments based on the use of ethanol/water media, is reported. The residual solid fractions have been characterized by ATR-FTIR, (13)C-solid-state NMR and morphological alterations after pretreatment were detected by scanning electron microscopy. The general chemical composition of the untreated and treated bark including determination of extractives, suberin, lignin and monosaccharides was also studied. Composition of the residual solid fraction and relative proportions of different components, as a function of the processing conditions, could be established. Organosolv treatment produces a suberin-rich solid fraction, while during hydrothermal and steam explosion treatment cleavage of polysaccharide bonds occurs. This work will provide a deeper fundamental knowledge of the bark chemical composition, thus increasing the utilization efficiency of birch outer bark and may create possibilities to up-scale the fractionation processes.

Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis.

BACKGROUND: Arginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants. RESULTS: Firstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation. CONCLUSIONS: In this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA, ∆speC, ∆speF, ∆argR, ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.