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.

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.

Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution.

BACKGROUND: Lignocellulosic raw materials have extensively been examined for the production of bio-based fuels, chemicals, and polymers using microbial platforms. Since xylose is one of the major components of the hydrolyzed lignocelluloses, it is being considered a promising substrate in lignocelluloses based fermentation process. Ralstonia eutropha, one of the most powerful and natural producers of polyhydroxyalkanoates (PHAs), has extensively been examined for the production of bio-based chemicals, fuels, and polymers. However, to the best of our knowledge, lignocellulosic feedstock has not been employed for R. eutropha probably due to its narrow spectrum of substrate utilization. Thus, R. eutropha engineered to utilize xylose should be useful in the development of microbial process for bio-based products from lignocellulosic feedstock. RESULTS: Recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes encoding xylose isomerase and xylulokinase respectively, was constructed and examined for the synthesis of poly(3-hydroxybutyrate) [P(3HB)] using xylose as a sole carbon source. It could produce 2.31 g/L of P(3HB) with a P(3HB) content of 30.95 wt% when it was cultured in a nitrogen limited chemically defined medium containing 20.18 g/L of xylose in a batch fermentation. Also, recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes produced 5.71 g/L of P(3HB) with a P(3HB) content of 78.11 wt% from a mixture of 10.05 g/L of glucose and 10.91 g/L of xylose in the same culture condition. The P(3HB) concentration and content could be increased to 8.79 g/L and 88.69 wt%, respectively, when it was cultured in the medium containing 16.74 g/L of glucose and 6.15 g/L of xylose. Further examination of recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes by fed-batch fermentation resulted in the production of 33.70 g/L of P(3HB) in 108 h with a P(3HB) content of 79.02 wt%. The concentration of xylose could be maintained as high as 6 g/L, which is similar to the initial concentration of xylose during the fed-batch fermentation suggesting that xylose consumption is not inhibited during fermentation. Finally, recombinant R. eutorpha NCIMB11599 expressing the E. coli xylAB gene was examined for the production of P(3HB) from the hydrolysate solution of sunflower stalk. The hydrolysate solution of sunflower stalk was prepared as a model lignocellulosic biomass, which contains 78.8 g/L of glucose, 26.9 g/L of xylose, and small amount of 4.8 g/L of galactose and mannose. When recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes was cultured in a nitrogen limited chemically defined medium containing 23.1 g/L of hydrolysate solution of sunflower stalk, which corresponds to 16.8 g/L of glucose and 5.9 g/L of xylose, it completely consumed glucose and xylose in the sunflower stalk based medium resulting in the production of 7.86 g/L of P(3HB) with a P(3HB) content of 72.53 wt%. CONCLUSIONS: Ralstonia eutropha was successfully engineered to utilize xylose as a sole carbon source as well as to co-utilize it in the presence of glucose for the synthesis of P(3HB). In addition, R. eutropha engineered to utilized xylose could synthesize P(3HB) from the sunflower stalk hydrolysate solution containing glucose and xylose as major sugars, which suggests that xylose utilizing R. eutropha developed in this study should be useful for development of lignocellulose based microbial processes.

Controlled production of a polyhydroxyalkanoate (PHA) tetramer containing different mole fraction of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3 HV), 4 HV and 5 HV units by engineered Cupriavidus necator.

Polyhydroxyalkanoates (PHAs) are promising alternatives to existing petrochemical-based plastics because of their bio-degradable properties. However, the limited structural diversity of PHAs has hindered their application. In this study, high mole-fractions of Poly (39 mol% 3HB-co-17 mol% 3 HV-co-44 mol% 4 HV) and Poly (25 mol% 3HB-co-75 mol% 5 HV) were produced from 4- hydroxyvaleric acid and 5-hydroxyvaleric acid, using Cupriavidus necator PHB-4 harboring the gene phaCBP-M-CPF4 with modified sequences. In addition, the complex toxicity of precursor mixtures was tested, and it was confirmed that the engineered C. necator was capable of synthesizing Poly (32 mol% 3HB-co-11 mol% 3 HV-co-25 mol% 4 HV-co-32 mol% 5 HV) at low mixture concentrations. Correlation analyses of the precursor ratio and the monomeric mole fractions indicated that each mole fractions could be precisely controlled using the precursor proportion. Physical property analysis confirmed that Poly (3HB-co-3 HV-co-4 HV) is a rubber-like amorphous polymer and Poly (3HB-co-5 HV) has a high tensile strength and elongation at break. Poly (3HB-co-3 HV-co-4 HV-co-5 HV) had a much lower glass transition temperature than the co-, terpolymers containing 3 HV, 4 HV and 5 HV. This study expands the range of possible physical properties of PHAs and contributes to the realization of custom PHA production by suggesting a method for producing PHAs with various physical properties through mole-fraction control of 3 HV, 4 HV and 5 HV.

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.

Development of novel recombinant peroxidase secretion system from Pseudomonas putida for lignin valorisation.

Pseudomonas putida is a promising strain for lignin valorisation. However, there is a dearth of stable and efficient systems for secreting enzymes to enhance the process. Therefore, a novel secretion system for recombinant lignin-depolymerising peroxidase was developed. By adopting a flagellar type III secretion system, P. putida KT-M2, a secretory host strain, was constructed and an optimal secretion signal fusion partner was identified. Application of the dye-decolourising peroxidase of P. putida to this system resulted in efficient oxidation activity of the cell-free supernatant against various chemicals, including lignin model compounds. This peroxidase-secreting strain was examined to confirm its lignin utilisation capability, resulting in the efficient assimilation of various lignin substrates with 2.6-fold higher growth than that of the wild-type strain after 72 h of cultivation. Finally, this novel system will lead efficient bacterial lignin breakdown and utilization through enzyme secretion, paving the way for sustainable lignin-consolidated bioprocessing.

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.

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.

Improvement of polyhydroxybutyrate (PHB) plate-based screening method for PHB degrading bacteria using cell-grown amorphous PHB and recovered by sodium dodecyl sulfate (SDS).

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable plastic. Considering the environmental issues of petroleum-based plastics, PHB is promising as it can be degraded in a relatively short time by bacteria to water and carbon dioxide. Substantial efforts have been made to identify PHB-degrading bacteria. To identify PHB-degrading bacteria, solid-based growth or clear zone assays using PHB as the sole carbon source are the easiest methods; however, PHB is difficult to dissolve and distribute evenly, and bacteria grow slowly on PHB plates. Here, we suggest an improved PHB plate assay using cell-grown PHB produced by Halomonas sp. and recovered by sodium dodecyl sulfate (SDS). Preparation using SDS resulted in evenly distributed PHB plates that could be used for sensitive depolymerase activity screening in less time compared with solvent-melted pellet or cell-grown PHB. With this method, we identified 15 new strains. One strain, Cutibacterium sp. SOL05 (98.4% 16S rRNA similarity to Cutibacterium acne), showed high PHB depolymerase activity in solid and liquid conditions. PHB degradation was confirmed by clear zone size, liquid culture, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The results indicate this method can be used to easily identify PHB-degrading bacteria from various sources to strengthen the benefits of bioplastics.

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.

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.