Enhanced theanine production with reduced ATP supply by alginate entrapped Escherichia coli co-expressing γ-glutamylmethylamide synthetase and polyphosphate kinase.

L-theanine is an amino acid with a unique flavor and many therapeutic effects. Its enzymatic synthesis has been actively studied and γ-Glutamylmethylamide synthetase (GMAS) is one of the promising enzymes in the biological synthesis of theanine. However, the theanine biosynthetic pathway with GMAS is highly ATP-dependent and the supply of external ATP was needed to achieve high concentration of theanine production. As a result, this study aimed to investigate polyphosphate kinase 2 (PPK2) as ATP regeneration system with hexametaphosphate. Furthermore, the alginate entrapment method was employed to immobilize whole cells containing both gmas and ppk2 together resulting in enhanced reusability of the theanine production system with reduced supply of ATP. After immobilization, theanine production was increased to 239 mM (41.6 g/L) with a conversion rate of 79.7% using 15 mM ATP and the reusability was enhanced, maintaining a 100% conversion rate up to the fifth cycles and 60% of conversion up to eighth cycles. It could increase long-term storage property for future uses up to 35 days with 75% activity of initial activity. Overall, immobilization of both production and cofactor regeneration system could increase the stability and reusability of theanine production system.

Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay.

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Catalytic hydrolysis of agar using magnetic nanoparticles: optimization and characterization.

BACKGROUND: Agar is used as a gelling agent that possesses a variety of biological properties; it consists of the polysaccharides agarose and porphyrin. In addition, the monomeric sugars generated after agar hydrolysis can be functionalized for use in biorefineries and biofuel production. The main objective of this study was to develop a sustainable agar hydrolysis process for bioethanol production using nanotechnology. Peroxidase-mimicking Fe3O4-MNPs were applied for agar degradation to generate agar hydrolysate-soluble fractions amenable to Saccharomyces cerevisiae and Escherichia coli during fermentation. RESULTS: Fe3O4-MNP-treated (Fe3O4-MNPs, 1 g/L) agar exhibited 0.903 g/L of reducing sugar, which was 21-fold higher than that of the control (without Fe3O4-MNP-treated). Approximately 0.0181% and 0.0042% of ethanol from 1% of agar was achieved using Saccharomyces cerevisiae and Escherichia coli, respectively, after process optimization. Furthermore, different analytical techniques (FTIR, SEM, TEM, EDS, XRD, and TGA) were applied to validate the efficiency of Fe3O4-MNPs in agar degradation. CONCLUSIONS: To the best of our knowledge, Fe3O4-MNP-treated agar degradation for bioethanol production through process optimization is a simpler, easier, and novel method for commercialization.

Enhanced production of glutaric acid by biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption.

Product inhibition caused by organic acids is a serious issue in establishing economical biochemical production systems. Herein, for enhanced production of glutaric acid by overcoming product inhibition triggered by glutaric acid, a whole-cell bioconversion system equipped with biocatalyst recycling process and in situ product recovery by adsorption was developed successfully. From the whole-cell bioconversion reaction, we found that both dissociated and undissociated forms of glutaric acid acted as an inhibitor in the whole-cell bioconversion reaction, wherein bioconversion was hindered beyond 200 mM glutaric acid regardless of reaction pH. Therefore, as the promising solution for the inhibition issue by glutaric acid, the biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption was introduced in the whole-cell bioconversion. As a result, 592 mM glutaric acid was produced from 1000 mM 5-aminovaleric acid with 59.2% conversion. We believe that our system will be a promising candidate for economically producing organic acids with high titer.

Poly(3-hydroxybutyrate) Degradation by Bacillus infantis sp. Isolated from Soil and Identification of phaZ and bdhA Expressing PHB Depolymerase.

Poly(3-hydroxybutyrate) (PHB) is a biodegradable and biocompatible bioplastic. Effective PHB degradation in nutrient-poor environments is required for industrial and practical applications of PHB. To screen for PHB-degrading strains, PHB double-layer plates were prepared and three new Bacillus infantis species with PHB-degrading ability were isolated from the soil. In addition, phaZ and bdhA of all isolated B. infantis were confirmed using a Bacillus sp. universal primer set and established polymerase chain reaction conditions. To evaluate the effective PHB degradation ability under nutrient-deficient conditions, PHB film degradation was performed in mineral medium, resulting in a PHB degradation rate of 98.71% for B. infantis PD3, which was confirmed in 5 d. Physical changes in the degraded PHB films were analyzed. The decrease in molecular weight due to biodegradation was confirmed using gel permeation chromatography and surface erosion of the PHB film was observed using scanning electron microscopy. To the best of our knowledge, this is the first study on B. infantis showing its excellent PHB degradation ability and is expected to contribute to PHB commercialization and industrial composting.

ß-Ketoadipic acid production from poly(ethylene terephthalate) waste via chemobiological upcycling.

The upcycling of poly(ethylene terephthalate) (PET) waste can simultaneously produce value-added chemicals and reduce the growing environmental impact of plastic waste. In this study, we designed a chemobiological system to convert terephthalic acid (TPA), an aromatic monomer of PET, to ß-ketoadipic acid (ßKA), a C6 keto-diacid that functions as a building block for nylon-6,6 analogs. Using microwave-assisted hydrolysis in a neutral aqueous system, PET was converted to TPA with Amberlyst-15, a conventional catalyst with high conversion efficiency and reusability. The bioconversion process of TPA into ßKA used a recombinant Escherichia coli ßKA expressing two conversion modules for TPA degradation (tphAabc and tphB) and ßKA synthesis (aroY, catABC, and pcaD). To improve bioconversion, the formation of acetic acid, a deleterious factor for TPA conversion in flask cultivation, was efficiently regulated by deleting the poxB gene along with operating the bioreactor to supply oxygen. By applying two-stage fermentation consisting of the growth phase in pH 7 followed by the production phase in pH 5.5, a total of 13.61 mM ßKA was successfully produced with 96% conversion efficiency. This efficient chemobiological PET upcycling system provides a promising approach for the circular economy to acquire various chemicals from PET waste.

Continuous production of gamma aminobutyric acid by engineered and immobilized Escherichia coli whole-cells in a small-scale reactor system.

γ-Amino butyric acid (GABA) is a non-proteinogenic amino acid and a human neurotransmitter. Recently, increasing demand for food additives and biodegradable bioplastic monomers, such as nylon 4, has been reported. Consequently, considerable efforts have been made to produce GABA through fermentation and bioconversion. To realize bioconversion, wild-type or recombinant strains harboring glutamate decarboxylase were paired with the cheap starting material monosodium glutamate, resulting in less by-product formation and faster production compared to fermentation. To increase the reusability and stability of whole-cell production systems, this study used an immobilization and continuous production system with a small-scale continuous reactor for gram-scale production. The cation type, alginate concentration, barium concentration, and whole-cell concentration in the beads were optimized and this optimization resulted in more than 95 % conversion of 600 mM monosodium glutamate to GABA in 3 h and reuse of the immobilized cells 15 times, whereas free cells lost all activity after the ninth reaction. When a continuous production system was applied after optimizing the buffer concentration, substrate concentration, and flow rate, 165 g of GABA was produced after 96 h of continuous operation in a 14-mL scale reactor. Our work demonstrates the efficient and economical production of GABA by immobilization and continuous production in a small-scale reactor.

A Genetically Encoded Biosensor for the Detection of Levulinic Acid.

Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.

Recent Advances in the Chemobiological Upcycling of Polyethylene Terephthalate (PET) into Value-Added Chemicals.

Polyethylene terephthalate (PET) is a plastic material commonly applied to beverage packaging used in everyday life. Owing to PET's versatility and ease of use, its consumption has continuously increased, resulting in considerable waste generation. Several physical and chemical recycling processes have been developed to address this problem. Recently, biological upcycling is being actively studied and has come to be regarded as a powerful technology for overcoming the economic issues associated with conventional recycling methods. For upcycling, PET should be degraded into small molecules, such as terephthalic acid and ethylene glycol, which are utilized as substrates for bioconversion, through various degradation processes, including gasification, pyrolysis, and chemical/biological depolymerization. Furthermore, biological upcycling methods have been applied to biosynthesize value-added chemicals, such as adipic acid, muconic acid, catechol, vanillin, and glycolic acid. In this review, we introduce and discuss various degradation methods that yield substrates for bioconversion and biological upcycling processes to produce value-added biochemicals. These technologies encourage a circular economy, which reduces the amount of waste released into the environment.

Production of γ-Aminobutyrate (GABA) in Recombinant Corynebacterium glutamicum by Expression of Glutamate Decarboxylase Active at Neutral pH.

γ-Aminobutyrate (GABA) is an important chemical by itself and can be further used for the production of monomer used for the synthesis of biodegradable polyamides. Until now, GABA production usingCorynebacterium glutamicum harboring glutamate decarboxylases (GADs) has been limited due to the discrepancy between optimal pH for GAD activity (pH 4.0) and cell growth (pH 7.0). In this study, we developed recombinant C. glutamicum strains expressing mutated GAD from Escherichia coli (EcGADmut) and GADs from Lactococcus lactis CICC20209 (LlGAD) and Lactobacillus senmaizukei (LsGAD), all of which showed enhanced pH stability and adaptability at a pH of approximately 7.0. In shake flask cultivations, the GABA productions of C. glutamicum H36EcGADmut, C. glutamicum H36LsGAD, and C. glutamicum H36LlGAD were examined at pH 5.0, 6.0, and 7.0, respectively. Finally, C. glutamicum H36EcGADmut (40.3 and 39.3 g L-1), H36LlGAD (42.5 and 41.1 g L-1), and H36LsGAD (41.6 and 40.2 g L-1) produced improved GABA titers and yields in batch fermentation at pH 6.0 and pH 7.0, respectively, from 100 g L-1 glucose. The recombinant strains developed in this study could be used for the establishment of sustainable direct fermentative GABA production from renewable resources under mild culture conditions, thus increasing the availability of various GADs.

Development of a glutaric acid production system equipped with stepwise feeding of monosodium glutamate by whole-cell bioconversion.

In the bioproduction of glutaric acid, an emerging bioplastic monomer, α-ketoglutaric acid (α-KG) is required as an amine acceptor for 4-aminobutyrate aminotransferase (GabT)-driven conversion of 5-aminovalerate (5-AVA) to glutarate semialdehyde. Herein, instead of using expensive α-KG, an indirect α-KG supply system was developed using a relatively cheap alternative, monosodium glutamate (MSG), for l-glutamate oxidase (Gox)-based whole-cell conversion. Using 200 mM 5-AVA and 30 mM MSG initially with Gox, 67.1 mM of glutaric acid was produced. By applying the stepwise feeding strategy of MSG, the glutaric acid production capability was increased to 159.1 mM glutaric acid with a conversion yield of 79.6%. In addition, a buffer-free one-pot reaction from l-lysine was also applied in a 5 L bioreactor to evaluate its industrial applicability, resulting in a conversion yield of 54.2%. The system developed herein might have great potential for the large-scale, economically feasible production of glutaric acid by whole-cell conversion.

Microbial production of 2-pyrone-4,6-dicarboxylic acid from lignin derivatives in an engineered Pseudomonas putida and its application for the synthesis of bio-based polyester.

Lignin valorization depends on microbial upcycling of various aromatic compounds in the form of a complex mixture, including p-coumaric acid and ferulic acid. In this study, an engineered Pseudomonas putida strain utilizing lignin-derived monomeric compounds via biological funneling was developed to produce 2-pyrone-4,6-dicarboxylic acid (PDC), which has been considered a promising building block for bioplastics. The biosynthetic pathway for PDC production was established by introducing the heterologous ligABC genes under the promoter Ptac in a strain lacking pcaGH genes to accumulate a precursor of PDC, i.e., protocatechuic acid. Based on the culture optimization, fed-batch fermentation of the final strain resulted in 22.7 g/L PDC with a molar yield of 1.0 mol/mol and productivity of 0.21 g/L/h. Subsequent purification of PDC at high purity was successfully implemented, which was consequently applied for the novel polyester.

Gamma aminobutyric acid (GABA) production in Escherichia coli with pyridoxal kinase (pdxY) based regeneration system.

Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid act as a major neurotransmitter inhibitor in the nervous system of mammals. It also used as a precursor of bioplastics synthesis such as N-methylpyrolidone and polyamide 4. Chemical-based synthesis methods have many environmental-related issues, so efforts have been made to develop biosynthetic methods to produce GABA. Glutamate decarboxylase (GAD) transforms L-glutamate to GABA using pyridoxal 5'-phosphate (PLP) as a cofactor. Bioconversion of GABA with whole cells overexpressing the glutamate decarboxylase has advantages of fewer byproducts and rapid reaction. However, there is a bottleneck in the whole-cell bioconversion system i.e., higher GABA production require a large amount of cofactor PLP which make the process costly. Therefore, pyridoxal kinase (PdxY) able to regenerate PLP was introduced in the whole-cell system to construct a new GABA producing system. Culture and reaction conditions were optimized, and 100% conversion of 0.6 M MSG was obtained. This study reports that a competitive level of GABA production could be achieved without supplying additional PLPs.

Recent advances in the microbial production of C4 alcohols by metabolically engineered microorganisms.

BACKGROUND: The heavy global dependence on petroleum-based industries has led to serious environmental problems, including climate change and global warming. As a result, there have been calls for a paradigm shift towards the use of biorefineries, which employ natural and engineered microorganisms that can utilize various carbon sources from renewable resources as host strains for the carbon-neutral production of target products. PURPOSE AND SCOPE: C4 alcohols are versatile chemicals that can be used directly as biofuels and bulk chemicals and in the production of value-added materials such as plastics, cosmetics, and pharmaceuticals. C4 alcohols can be effectively produced by microorganisms using DCEO biotechnology (tools to design, construct, evaluate, and optimize) and metabolic engineering strategies. SUMMARY OF NEW SYNTHESIS AND CONCLUSIONS: In this review, we summarize the production strategies and various synthetic tools available for the production of C4 alcohols and discuss the potential development of microbial cell factories, including the optimization of fermentation processes, that offer cost competitiveness and potential industrial commercialization.

Chemo-Biological Upcycling of Poly(ethylene terephthalate) to Multifunctional Coating Materials.

Chemo-biological upcycling of poly(ethylene terephthalate) (PET) developed in this study includes the following key steps: chemo-enzymatic PET depolymerization, biotransformation of terephthalic acid (TPA) into catechol, and its application as a coating agent. Monomeric units were first produced through PET glycolysis into bis(2-hydroxyethyl) terephthalate (BHET), mono(2-hydroxyethyl) terephthalate (MHET), and PET oligomers, and enzymatic hydrolysis of these glycolyzed products using Bacillus subtilis esterase (Bs2Est). Bs2Est efficiently hydrolyzed glycolyzed products into TPA as a key enzyme for chemo-enzymatic depolymerization. Furthermore, catechol solution produced from TPA via a whole-cell biotransformation (Escherichia coli) could be directly used for functional coating on various substrates after simple cell removal from the culture medium without further purification and water-evaporation. This work demonstrates a proof-of-concept of a PET upcycling strategy via a combination of chemo-biological conversion of PET waste into multifunctional coating materials.

Chemoautotroph Cupriavidus necator as a potential game-changer for global warming and plastic waste problem: A review.

Cupriavidus necator, a versatile microorganism found in both soil and water, can have both heterotrophic and lithoautotrophic metabolisms depending on environmental conditions. C. necator has been extensively examined for producing Polyhydroxyalkanoates (PHAs), the promising polyester alternatives to petroleum-based synthetic polymers because it has a superior ability for accumulating a considerable amount of PHAs from renewable resources. The development of metabolically engineered C. necator strains has led to their application for synthesizing biopolymers, biofuels and biochemicals such as ethanol, isobutanol and higher alcohols. Bio-based processes of recombinant C. necator have made much progress in production of these high-value products from biomass wastes, plastic wastes and even waste gases. In this review, we discuss the potential of C. necator as promising platform host strains that provide a great opportunity for developing a waste-based circular bioeconomy.

Improving the organic solvent resistance of lipase a from Bacillus subtilis in water-ethanol solvent through rational surface engineering.

Given that lipase is an enzyme applicable in various industrial fields and water-miscible organic solvents are important reaction media for developing industrial-scale biocatalysis, a structure-based strategy was explored to stabilize lipase A from Bacillus subtilis in a water-ethanol cosolvent. Site-directed mutagenesis of ethanol-interacting sites resulted in 4 mutants, i.e., Ser16Gly, Ala38Gly, Ala38Thr, and Leu108Asn, which were stable in 50% ethanol and had up to 1.8-fold higher stability than the wild-type. In addition, Leu108Asn was more thermostable at 45 °C than the wild type. The results discussed in this study not only provide insights into strategies for enzyme engineering to improve organic solvent resistance but also suggest perspectives on pioneering routes for constructing enzyme-based biorefineries to produce value-added fuels and chemicals.

Recent Advances in Sustainable Plastic Upcycling and Biopolymers.

Advances in scientific technology in the early twentieth century have facilitated the development of synthetic plastics that are lightweight, rigid, and can be easily molded into a desirable shape without changing their material properties. Thus, plastics become ubiquitous and indispensable materials that are used in various manufacturing sectors, including clothing, automotive, medical, and electronic industries. However, strong physical durability and chemical stability of synthetic plastics, most of which are produced from fossil fuels, hinder their complete degradation when they are improperly discarded after use. In addition, accumulated plastic wastes without degradation have caused severe environmental problems, such as microplastics pollution and plastic islands. Thus, the usage and production of plastics is not free from environmental pollution or resource depletion. In order to lessen the impact of climate change and reduce plastic pollution, it is necessary to understand and address the current plastic life cycles. In this review, "sustainable biopolymers" are suggested as a promising solution to the current plastic crisis. The desired properties of sustainable biopolymers and bio-based and bio/chemical hybrid technologies for the development of sustainable biopolymers are mainly discussed.

Biosynthesis of polyhydroxyalkanoates from sucrose by metabolically engineered Escherichia coli strains.

Sucrose utilization has been established in Escherichia coli strains by expression of Mannheimia succiniciproducens ß-fructofuranosidase (SacC), which hydrolyzes sucrose into glucose and fructose. Recombinant E. coli strains that can utilize sucrose were examined for their abilities to produce poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from sucrose. When recombinant E. coli strains expressing Ralstonia eutropha PhaCAB and SacC were cultured in MR medium containing 20 g/L of sucrose, all recombinant E. coli strains could produce P(3HB) from sucrose. Also, recombinant E. coli strains expressing Pseudomonas sp. MBEL 6-19 PhaC1437, Clostridium propionicum Pct540, R. eutropha PhaAB enzymes along with SacC could produce P(3HB-co-LA) from sucrose. Among the examined E. coli strains, recombinant E. coli XL1-Blue produced the highest contents of P(3HB) (53.60 ± 2.55 wt%) and P(3HB-co-LA) (29.44 ± 0.39 wt%). In the batch fermentations, recombinant E. coli XL1-Blue strains completely consumed 20 g/L of sucrose as the sole carbon source and supported the production of 3.76 g/L of P(3HB) and 1.82 g/L of P(3HB-co-LA) with 38.21 wt% P(3HB) and 20.88 wt% P(3HB-co-LA) contents, respectively. Recombinant E. coli strains developed in this study can be used to establish a cost-efficient biorefinery for the production of polyhydroxyalkanoates (PHAs) from sucrose, which is an abundant and inexpensive carbon source.

Metabolic engineering for the synthesis of polyesters: A 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters.

As concerns increase regarding sustainable industries and environmental pollutions caused by the accumulation of non-degradable plastic wastes, bio-based polymers, particularly biodegradable plastics, have attracted considerable attention as potential candidates for solving these problems by substituting petroleum-based plastics. Among these candidates, polyhydroxyalkanoates (PHAs), natural polyesters that are synthesized and accumulated in a range of microorganisms, are considered as promising biopolymers since they have biocompatibility, biodegradability, and material properties similar to those of commodity plastics. Accordingly, substantial efforts have been made to gain a better understanding of mechanisms related to the biosynthesis and properties of PHAs and to develop natural and recombinant microorganisms that can efficiently produce PHAs comprising desired monomers with high titer and productivity for industrial applications. Recent advances in biotechnology, including those related to evolutionary engineering, synthetic biology, and systems biology, can provide efficient and effective tools and strategies that reduce time, labor, and costs to develop microbial platform strains that produce desired chemicals and materials. Adopting these technologies in a systematic manner has enabled microbial fermentative production of non-natural polyesters such as poly(lactate) [PLA], poly(lactate-co-glycolate) [PLGA], and even polyesters consisting of aromatic monomers from renewable biomass-derived carbohydrates, which can be widely used in current chemical industries. In this review, we present an overview of strain development for the production of various important natural PHAs, which will give the reader an insight into the recent advances and provide indicators for the future direction of engineering microorganisms as plastic cell factories. On the basis of our current understanding of PHA biosynthesis systems, we discuss recent advances in the approaches adopted for strain development in the production of non-natural polyesters, notably 2-hydroxycarboxylic acid-containing polymers, with particular reference to systems metabolic engineering strategies.