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.

Biocatalytic valorization of lignin subunit: Screening a carboxylic acid reductase with high substrate preference to syringyl functional group.

Lignin is inexpensive and the most abundant source of biological aromatics. It can be decomposed to three types of subunits, 4-hydroxybenzoic, vanillic and syringic acids, each of which can be valorized to value added compounds. Syringaldehyde is a versatile phenolic aldehyde implicated with multiple bioactive properties as well as intermediates for biofuels. Herein, fourteen microbial carboxylic acid reductases (CARs) were screened for the biocatalysis of the energetically unfavorable reduction of syringic acid to syringaldehyde. Nine CARs were positive to syringic acid reduction, among which Mycobacterium abscessus CAR exhibited the highest analytical yield of the product. By the optimization of the reaction condition, the whole-cell biocatalyst (i.e., recombinant Escherichia coli expressing the gene) successfully converted syringic acid to syringaldehyde with a yield of 90%. Furthermore, structural features of the screened CAR responsible for the specificity toward the syringyl subunit were analyzed that helps to further engineer the biocatalyst for improved performances.

Physiological activity of E. coli engineered to produce butyric acid.

Faecalibacterium prausnitzii (F. prausnitzii) is one of the most abundant bacteria in the human intestine, with its anti-inflammatory effects establishing it as a major effector in human intestinal health. However, its extreme sensitivity to oxygen makes its cultivation and physiological study difficult. F. prausnitzii produces butyric acid, which is beneficial to human gut health. Butyric acid is a short-chain fatty acid (SCFA) produced by the fermentation of carbohydrates, such as dietary fibre in the large bowel. The genes encoding butyryl-CoA dehydrogenase (BCD) and butyryl-CoA:acetate CoA transferase (BUT) in F. prausnitzii were cloned and expressed in E. coli to determine the effect of butyric acid production on intestinal health using DSS-induced colitis model mice. The results from the E. coli Nissle 1917 strain, expressing BCD, BUT, or both, showed that BCD was essential, while BUT was dispensable for producing butyric acid. The effects of different carbon sources, such as glucose, N-acetylglucosamine (NAG), N-acetylgalactosamine (NAGA), and inulin, were compared with results showing that the optimal carbon sources for butyric acid production were NAG, a major component of mucin in the human intestine, and glucose. Furthermore, the anti-inflammatory effects of butyric acid production were tested by administering these strains to DSS-induced colitis model mice. The oral administration of the E. coli Nissle 1917 strain, carrying the expression vector for BCD and BUT (EcN-BCD-BUT), was found to prevent DSS-induced damage. Introduction of the BCD expression vector into E. coli Nissle 1917 led to increased butyric acid production, which improved the strain's health-beneficial effects.

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.

A novel hyperthermophilic methylglyoxal synthase: molecular dynamic analysis on the regional fluctuations.

Two putative methylglyoxal synthases, which catalyze the conversion of dihydroxyacetone phosphate to methylglyoxal, from Oceanithermus profundus DSM 14,977 and Clostridium difficile 630 have been characterized for activity and thermal stability. The enzyme from O. profundus was found to be hyperthermophilic, with the optimum activity at 80 °C and the residual activity up to 59% after incubation of 15 min at 95 °C, whereas the enzyme from C. difficile was mesophilic with the optimum activity at 40 °C and the residual activity less than 50% after the incubation at 55 °C or higher temperatures for 15 min. The structural analysis of the enzymes with molecular dynamics simulation indicated that the hyperthermophilic methylglyoxal synthase has a rigid protein structure with a lower overall root-mean-square-deviation value compared with the mesophilic or thermophilic counterparts. In addition, the simulation results identified distinct regions with high fluctuations throughout those of the mesophilic or thermophilic counterparts via root-mean-square-fluctuation analysis. Specific molecular interactions focusing on the hydrogen bonds and salt bridges in the distinct regions were analyzed in terms of interatomic distances and positions of the individual residues with respect to the secondary structures of the enzyme. Key interactions including specific salt bridges and hydrogen bonds between a rigid beta-sheet core and surrounding alpha helices were found to contribute to the stabilisation of the hyperthermophilic enzyme by reducing the regional fluctuations in the protein structure. The structural information and analysis approach in this study can be further exploited for the engineering and industrial application of the enzyme.

Engineering D-Lactate Dehydrogenase from Pediococcus acidilactici for Improved Activity on 2-Hydroxy Acids with Bulky C3 Functional Group.

Engineering D-lactic acid dehydrogenases for higher activity on various 2-oxo acids is important for the synthesis of 2-hydroxy acids that can be utilized in a wide range of industrial fields including the production of biopolymers, pharmaceuticals, and cosmetic compounds. Although there are many D-lactate dehydrogenases (D-LDH) available from a diverse range of sources, there is a lack of biocatalysts with high activities for 2-oxo acids with large functional group at C3. In this study, the D-LDH from Pediococcus acidilactici was rationally designed and further engineered by controlling the intermolecular interactions between substrates and the surrounding residues via analysis of the active site structure of D-LDH. As a result, Y51L mutant with the catalytic efficiency on phenylpyruvate of 2200 s-1 mM-1 and Y51F mutant on 2-oxobutryate and 3-methyl-2-oxobutyrate of 37.2 and 23.2 s-1 mM-1 were found, which were 138-, 8.5-, and 26-fold increases than the wild type on the substrates, respectively. Structural analysis revealed that the distance and the nature of the interactions between the side chain of residue 51 and the substrate C3 substituent group significantly affected the kinetic parameters. Bioconversion of phenyllactate as a practical example of production of the 2-hydroxy acids was investigated, and the Y51F mutant presented the highest productivity in in vitro conversion of D-PLA.

A novel d-2-hydroxy acid dehydrogenase with high substrate preference for phenylpyruvate originating from lactic acid bacteria: Structural analysis on the substrate specificity.

2-Hydroxy acid dehydrogenases (2-HADHs) have been implicated in the synthesis of 2-hydroxy acids from 2-oxo acids that are used in wide areas of industry. d-lactate dehydrogenases (d-LDHs), a subfamily of 2-HADH, have been utilized to this purpose, yet they exhibited relatively low catalytic activity to the 2-oxo acids with large functional groups at C3. In this report, four putative 2-HADHs from Oenococcus oeni, Weissella confusa, Weissella koreensis and Pediococcus claussenii were examined for activity on phenylpyruvate (PPA), a substrate to 3-phenyllactic acid (PLA) with a C3 phenyl group. The 2-HADH from P. claussenii was found to have the highest kcat/Km on PPA with 1,348.03 s-1 mM-1 among the four enzymes with higher substrate preference for PPA than pyruvate. Sequential, structural and mutational analysis of the enzyme revealed that it belonged to the d-LDH family, and phenylalanine at the position 51 was the key residue for the PPA binding to the active site via hydrophobic interaction, whereas in the 2-HADHs from O. oeni and W. confusa the hydrophilic tyrosine undermined the interaction. Because phenyllactate is a potential precursor for pharmaceutical compounds, antibiotics and biopolymers, the enzyme could increase the efficiency of bio-production of valuable chemicals. This study suggests a structural basis for the high substrate preference of the 2-HADH, and further engineering possibilities to synthesize versatile 2-hydroxy acids.