Biotransformation of 2-keto-4-hydroxybutyrate via aldol condensation using an efficient and thermostable carboligase from Deinococcus radiodurans.

The bioconversion of 4-hydroxy-2-keto acid derivatives via aldol condensation of formaldehyde and pyruvate has received substantial attention as potential source of chemicals for production of amino acids, hydroxy carboxylic acids, and chiral aldehydes. We developed an environmentally friendly biocatalyst consisting of a novel thermostable class II pyruvate aldolase from Deinococcus radiodurans with maltose-binding protein (MBP-DrADL), which has specific activity of 46.3 µmol min-1 mg-1. Surprisingly, MBP-DrADL maintained over 60% of enzyme activity for 4 days at 50 to 65 °C, we used MBP-DrADL as the best candidate enzyme to produce 2-keto-4-hydroxybutyrate (2-KHB) from formaldehyde and pyruvate via aldol condensation. The optimum reaction conditions for 2-KHB production were 50 °C, pH 8.0, 5 mM Mg2+, 100 mM formaldehyde, and 200 mM pyruvate. Under these optimized conditions, MBP-DrADL produced 76.5 mM (8.94 g L-1) 2-KHB over 60 min with a volumetric productivity of 8.94 g L-1 h-1 and a specific productivity of 357.6 mg mg-enzyme-1 h-1. Furthermore, 2-KHB production was improved by continuous addition of substrates, which produced approximately 124.8 mM (14.6 g L-1) of 2-KHB over 60 min with a volumetric productivity and specific productivity of 14.6 g L-1 h-1 and 583.4 mg mg-enzyme-1 h-1, respectively. MBP-DrADL showed the highest specific productivity for 2-KHB production yet reported. Our study provides a highly efficient biocatalyst for the synthesis of 2-KHB and lays the foundation for large-scale production and application of high-value compounds from formaldehyde.

Sustainable production of natural products using synthetic biology: Ginsenosides.

Synthetic biology approaches offer potential for large-scale and sustainable production of natural products with bioactive potency, including ginsenosides, providing a means to produce novel compounds with enhanced therapeutic properties. Ginseng, known for its non-toxic and potent qualities in traditional medicine, has been used for various medical needs. Ginseng has shown promise for its antioxidant and neuroprotective properties, and it has been used as a potential agent to boost immunity against various infections when used together with other drugs and vaccines. Given the increasing demand for ginsenosides and the challenges associated with traditional extraction methods, synthetic biology holds promise in the development of therapeutics. In this review, we discuss recent developments in microorganism producer engineering and ginsenoside production in microorganisms using synthetic biology approaches.

Secretory Production of the Hericium erinaceus Laccase from Saccharomyces cerevisiae.

Mushroom laccases play a crucial role in lignin depolymerization, one of the most critical challenges in lignin utilization. Importantly, laccases can utilize a wide range of substrates, such as toxicants and antibiotics. This study isolated a novel laccase, named HeLac4c, from endophytic white-rot fungi Hericium erinaceus mushrooms. The cDNAs for this enzyme were 1569 bp in length and encoded a protein of 523 amino acids, including a 20 amino-acid signal peptide. Active extracellular production of glycosylated laccases from Saccharomyces cerevisiae was successfully achieved by selecting an optimal translational fusion partner. We observed that 5 and 10 mM Ca2+, Zn2+, and K+ increased laccase activity, whereas 5 mM Fe2+ and Al3+ inhibited laccase activity. The laccase activity was inhibited by the addition of low concentrations of sodium azide and L-cysteine. The optimal pH for the 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was 4.4. Guaiacylglycerol-ß-guaiacyl ether, a lignin model compound, was polymerized by the HeLac4c enzyme. These results indicated that HeLac4c is a novel oxidase biocatalyst for the bioconversion of lignin into value-added products for environmental biotechnological applications.

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.

Modulation of Kex2p Cleavage Site for In Vitro Processing of Recombinant Proteins Produced by Saccharomyces cerevisiae.

Kex2 protease (Kex2p) is a membrane-bound serine protease responsible for the proteolytic maturation of various secretory proteins by cleaving after dibasic residues in the late Golgi network. In this study, we present an application of Kex2p as an alternative endoprotease for the in vitro processing of recombinant fusion proteins produced by the yeast Saccharomyces cerevisiae. The proteins were expressed with a fusion partner connected by a Kex2p cleavage sequence for enhanced expression and easy purification. To avoid in vivo processing of fusion proteins by Kex2p during secretion and to guarantee efficient removal of the fusion partners by in vitro Kex2p processing, P1', P2', P4, and P3 sites of Kex2p cleavage sites were elaborately manipulated. The general use of Kex2p in recombinant protein production was confirmed using several recombinant proteins.

Insights into Enzyme Reactions with Redox Cofactors in Biological Conversion of CO2.

Carbon dioxide (CO2) is the most abundant component of greenhouse gases (GHGs) and directly creates environmental issues such as global warming and climate change. Carbon capture and storage have been proposed mainly to solve the problem of increasing CO2 concentration in the atmosphere; however, more emphasis has recently been placed on its use. Among the many methods of using CO2, one of the key environmentally friendly technologies involves biologically converting CO2 into other organic substances such as biofuels, chemicals, and biomass via various metabolic pathways. Although an efficient biocatalyst for industrial applications has not yet been developed, biological CO2 conversion is the needed direction. To this end, this review briefly summarizes seven known natural CO2 fixation pathways according to carbon number and describes recent studies in which natural CO2 assimilation systems have been applied to heterogeneous in vivo and in vitro systems. In addition, studies on the production of methanol through the reduction of CO2 are introduced. The importance of redox cofactors, which are often overlooked in the CO2 assimilation reaction by enzymes, is presented; methods for their recycling are proposed. Although more research is needed, biological CO2 conversion will play an important role in reducing GHG emissions and producing useful substances in terms of resource cycling.

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.

Biocatalytic characterization of Hericium erinaceus laccase isoenzymes for the oxidation of lignin derivative substrates.

Mushroom laccases are biocatalysts that oxidize various substrates. To identify a novel enzyme involved in lignin valorization, we isolated and characterized laccase isoenzymes from the mushroom Hericium erinaceus. The laccase cDNAs (Lac1a and Lac1b) cloned from the mushroom mycelia consisted of 1536 bp and each encoded a protein with 511 amino acids, containing a 21-amino-acid signal peptide. Comparative phylogenetic analysis revealed high homology between the deduced amino acid sequences of Lac1a and Lac1b and those from basidiomycetous fungi. In the Pichia pastoris expression system, high extracellular production of Lac1a, a glycoprotein, was achieved, whereas Lac1b was not expressed as a secreted protein because of hyper-glycosylation. Biochemical characterization of the purified recombinant Lac1a (rLac1a) protein revealed its oxidizing efficacy toward 14 aromatic substrates. The highly substrate-specific rLac1a showed catalytic efficiencies of 877 s-1 mM-1, 829 s-1 mM-1, 520 s-1 mM-1, and 467 s-1 mM-1 toward 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), hydroquinone, guaiacol, and 2,6-dimethylphenol, respectively. Moreover, rLac1a showed approximately 10 % higher activity in non-ionic detergents and >50 % higher residual activity in various organic solvents. These results indicate that rLac1a is a novel oxidase biocatalyst for the bioconversion of lignin into value-added products.

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.

Dual-functional carboxymethyl levan-based protein carrier for cosmeceutical application of human epidermal growth factor.

Human epidermal growth factor (hEGF) has been a subject of extensive research as its wide range of physiological functions has many potential applications. However, due to the low stability of hEGF, its physiological effect is easily lost under conditions of use. To compensate for this, we developed a stable delivery system using levan-based nanoparticles. The entrapment yield of various tested proteins was significantly improved by employing carboxymethyl levan (CML) instead of levan; the entrapment yield of the CML-hEGF nanoparticles was 84.1 %. The size and zeta potential of the nanoparticles were identified as 199.9 ± 3.87 nm and -19.1 mV, respectively, using scanning electron microscopy (SEM) and particle size analysis. Dual biological functions of the nanoparticles (skin regeneration and moisturizing) were identified through collagen synthesis activity and aquaporin 3 expression level analysis. Stability of the prepared nanoparticles was also investigated via cell proliferation activity comparison under mimicked physiological conditions. The CML-hEGF nanoparticles maintained cell proliferation activity over 100 % for 6 weeks, while free hEGF was almost inactivated within 2 weeks. Taken together, our results indicate that the CML-based hEGF nanoparticles can be used in pharma- and cosmeceutical applications, guaranteeing a high entrapment capability, functionality, and stability.

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.

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation.

Microbial infections remain a global health concern, calling for the urgent need to implement effective prevention measures. Antimicrobial peptides (AMPs) have been extensively studied as potential antimicrobial coating agents. However, an efficient and economical method for AMP production is lacking. Here, we synthesized the direct coating adhesive AMP, NKC-DOPA5, composed of NKC, a potent AMP, and repeats of the adhesive amino acid 3,4-dihydroxyphenylalanine (DOPA) via an intein-mediated protein ligation strategy. NKC was expressed as a soluble fusion protein His-NKC-GyrA (HNG) in Escherichia coli, comprising an N-terminal 6× His-tag and a C-terminal Mxe GyrA intein. The HNG protein was efficiently produced in a 500-L fermenter, with a titer of 1.63 g/L. The NKC-thioester was released from the purified HNG fusion protein by thiol attack and subsequently ligated with chemically synthesized Cys-DOPA5. The ligated peptide His-NKC-Cys-DOPA5 was obtained at a yield of 88.7%. The purified His-NKC-Cys-DOPA5 possessed surface-binding and antimicrobial properties identical to those of the peptide obtained via solid-phase peptide synthesis. His-NKC-Cys-DOPA5 can be applied as a practical and functional antimicrobial coating to various materials, such as medical devices and home appliances.

Production of autolysis-proof Kex2 protease from Candida albicans in Saccharomyces cerevisiae for in vitro processing of fusion proteins.

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.

Fructan Biosynthesis by Yeast Cell Factories.

Fructan is a polysaccharide composed of fructose and can be classified into several types, such as inulin, levan, and fructo-oligosaccharides, based on their linkage patterns and degree of polymerization. Owing to its structural and functional diversity, fructan has been used in various fields including prebiotics, foods and beverages, cosmetics, and pharmaceutical applications. With increasing interest in fructans, efficient and straightforward production methods have been explored. Since the 1990s, yeast cells have been employed as producers of recombinant enzymes for enzymatic conversion of fructans including fructosyltransferases derived from various microbes and plants. More recently, yeast cell factories are highlighted as efficient workhorses for fructan production by direct fermentation. In this review, recent advances and strategies for fructan biosynthesis by yeast cell factories are discussed.

A new platform host for strong expression under GAL promoters without inducer in Saccharomyces cerevisiae.

The gal80 mutant of yeast Saccharomyces cerevisiae is used for the constitutive expression under strong GAL promoters without galactose induction. To enhance productivity of gal80 mutant, an alternative strain, allgal, was developed by removing all galactose-utilizing genes that consume significant cellular resources in the gal80 strain when cultured in non-galactose conditions. The efficacy of the allgal mutant (gal80, gal1, gal2, gal7, and gal10) was verified by assessing the secretory expression of three recombinant proteins, Candida antarctica lipase B (CalB), human serum albumin (HSA), and human epidermal growth factor (hEGF), using the GAL10 promoter. The growth of the allgal mutant was enhanced by 15-38% compared to the gal80 mutant, and the secretion of recombinant proteins also increased by 16-22% in fed-batch fermentation. Thus, the expression of recombinant proteins using GAL10 promoter in the allgal mutant is suitable for the economical production of recombinant proteins in S. cerevisiae.

Characterization of Acyl-CoA Oxidases from the Lipolytic Yeast Candida aaseri SH14.

The lipolytic yeast Candida aaseri SH14 contains three Acyl-CoA oxidases (ACOXs) which are encoded by the CaAOX2, CaAOX4, and CaAOX5 genes and catalyze the first reaction in the ß-oxidation of fatty acids. Here, the respective functions of the three CaAOX isozymes were studied by growth analysis of mutant strains constructed by a combination of three CaAOX mutations in minimal medium containing fatty acid as the sole carbon source. Substrate specificity of the CaAOX isozymes was analyzed using recombinant C. aaseri SH14 strains overexpressing the respective genes. CaAOX2 isozyme showed substrate specificity toward short- and medium-chain fatty acids (C6-C12), while CaAOX5 isozyme preferred long-chain fatty acid longer than C12. CaAOX4 isozyme revealed a preference for a broad substrate spectrum from C6-C16. Although the substrate specificity of CaAOX2 and CaAOX5 covers medium- and long-chain fatty acids, these two isozymes were insufficient for complete ß-oxidation of long-chain fatty acids, and therefore CaAOX4 was indispensable.

Metabolite trafficking enables membrane-impermeable-terpene secretion by yeast.

Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells. We investigate whether engineered microbes can exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner. In this study, we demonstrate a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking is highly achievable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically couple a terpene-binding protein with an export signal peptide and subsequently demonstrate efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for ß-carotene). Other carrier proteins can also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the most efficient cognate pathway for metabolite secretion by microorganisms.

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.

New discovery on the nematode activity of aureothin and alloaureothin isolated from endophytic bacteria Streptomyces sp. AE170020.

Endophytic bacteria, a rich source of bioactive secondary metabolites, are ideal candidates for environmentally benign agents. In this study, an endophytic strain, Streptomyces sp. AE170020, was isolated and selected for the purification of nematicidal substances based on its high nematicidal activity. Two highly active components, aureothin and alloaureothin, were identified, and their chemical structures were determined using spectroscopic analysis. Both compounds suppressed the growth, reproduction, and behavior of Bursaphelenchus xylophilus. In in vivo experiments, the extracts of strain Streptomyces sp. AE170020 effectively suppressed the development of pine wilt disease in 4-year-old plants of Pinus densiflora. The potency of secondary metabolites isolated from endophytic strains suggests applications in controlling Bursaphelenchus xylophilus and opens an avenue for further research on exploring bioactive substances against the pine wood nematode.

Adaptive laboratory evolution of Escherichia coli W enhances gamma-aminobutyric acid production using glycerol as the carbon source.

The microbial conversion of glycerol into value-added commodity products has emerged as an attractive means to meet the demands of biosustainability. However, glycerol is a non-preferential carbon source for productive fermentation because of its low energy density. We employed evolutionary and metabolic engineering in tandem to construct an Escherichia coli strain with improved GABA production using glycerol as the feedstock carbon. Adaptive evolution of E. coli W under glycerol-limited conditions for 1300 generations harnessed an adapted strain with a metabolic system optimized for glycerol utilization. Mutation profiling, enzyme kinetic assays, and transcriptome analysis of the adapted strain allowed us to decipher the basis of glycerol adaptation at the molecular level. Importantly, increased substrate influx mediated by the mutant glpK and modulation of intracellular cAMP levels were the key drivers of improved fitness in the glycerol-limited condition. Leveraging the enhanced capability of glycerol utilization in the strain, we constructed a GABA-producing E. coli W-derivative with superior GABA production compared to the wild-type. Furthermore, rationally designed inactivation of the non-essential metabolic genes, including ackA, mgsA, and gabT, in the glycerol-adapted strain improved the final GABA titer and specific productivity by 3.9- and 4.3-fold, respectively, compared with the wild-type.