Enzymatic properties of UDP-glycosyltransferase 89B1 from radish and modulation of enzyme catalytic activity via loop region mutation.

Glycosyltransferases (GTs), crucial enzymes in plants, alter natural substances through glycosylation, a process with extensive applications in pharmaceuticals, food, and cosmetics. This study narrows its focus to GT family 1, specifically UDP-glycosyltransferases (UGTs), which are known for glycosylating small phenolic compounds, especially hydroxybenzoates. We delve into the workings of Raphanus sativus glucosyltransferase (Rs89B1), a homolog of Arabidopsis thaliana UGT89B1, and its mutant to explore their glycosyltransferase activities toward hydroxybenzoates. Our findings reveal that Rs89B1 glycosylates primarily the para-position of mono-, di-, trihydroxy benzoic acids, and its substrate affinity is swayed by the presence and position of the hydroxyl group on the benzene ring of hydroxybenzoate. Moreover, mutations in the loop region of Rs89B1 impact both substrate affinity and catalytic activity. The study demonstrates that insertional/deletional mutations in non-conserved regions, which are distant from the UGT's recognition site, can have an effect on the UGT's substrate recognition site, which in turn affects acceptor substrate selectivity and glycosyltransferase activity. This research uncovers new insights suggesting that mutations in the loop region could potentially fine-tune enzyme properties and enhance its catalytic activity. These findings not only have significant implications for enzyme engineering in biotechnological applications but also contribute to a more profound understanding of this field.

Production of 3-Hydroxytyrosol from Glucose by Chromosomally Engineered Escherichia coli by Fed-Batch Cultivation in a Jar Fermenter.

3-Hydroxytyrosol (HT) is a super antioxidant possessing many physiological advantages for human health. However, the extraction of natural HT from olive (Olea europaea) is expensive, and its chemical synthesis presents an environmental burden. Therefore, microbial production of HT from renewable sources has been investigated over the past decade. In the present study, we modified the chromosome of a phenylalanine-producing strain of Escherichia coli to generate an HT-producing strain. The initial strain showed good HT production in tests performed by test tube cultivation, but this performance did not transfer to jar-fermenter cultivation. To grow well and achieve higher titers, the chromosome was further engineered and the cultivation conditions were further modified. The final strain achieved a higher HT titer (8.8 g/L) and yield (8.7%) from glucose in the defined synthetic medium. These yields are the best reported to date for the biosynthesis of HT from glucose.

Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria.

Many phenylalanine- and tyrosine-producing strains have used plasmid-based overexpression of pathway genes. The resulting strains achieved high titers and yields of phenylalanine and tyrosine. Chromosomally engineered, plasmid-free producers have shown lower titers and yields than plasmid-based strains, but the former are advantageous in terms of cultivation cost and public health/environmental risk. Therefore, we engineered here the Escherichia coli chromosome to create superior phenylalanine- and tyrosine-overproducing strains that did not depend on plasmid-based expression. Integration into the E. coli chromosome of two central metabolic pathway genes (ppsA and tktA) and eight shikimate pathway genes (aroA, aroB, aroC, aroD, aroE, aroGfbr , aroL, and pheAfbr ), controlled by the T7lac promoter, resulted in excellent titers and yields of phenylalanine; the superscript "fbr" indicates that the enzyme encoded by the gene was feedback resistant. The generated strain could be changed to be a superior tyrosine-producing strain by replacing pheAfbr with tyrAfbr A rational approach revealed that integration of seven genes (ppsA, tktA, aroA, aroB, aroC, aroGfbr , and pheAfbr ) was necessary as the minimum gene set for high-yield phenylalanine production in E. coli MG1655 (tyrR, adhE, ldhA, pykF, pflDC, and ascF deletant). The phenylalanine- and tyrosine-producing strains were further applied to generate phenyllactic acid-, 4-hydroxyphenyllactic acid-, tyramine-, and tyrosol-producing strains; yield of these aromatic compounds increased proportionally to the increase in phenylalanine and tyrosine yields.IMPORTANCE Plasmid-free strains for aromatic compound production are desired in the aspect of industrial application. However, the yields of phenylalanine and tyrosine have been considerably lower in plasmid-free strains than in plasmid-based strains. The significance of this research is that we succeeded in generating superior plasmid-free phenylalanine- and tyrosine-producing strains by engineering the E. coli chromosome, which was comparable to that in plasmid-based strains. The generated strains have a potential to generate superior strains for the production of aromatic compounds. Actually, we demonstrated that four kinds of aromatic compounds could be produced from glucose with high yields (e.g., 0.28 g tyrosol/g glucose).

Escherichia coli chromosome-based T7-dependent constitutive overexpression system and its application to generating a phenylalanine producing strain.

Many metabolic engineering approaches have been attempted to generate strains capable of producing valuable compounds. One of main goals is industrial application of these strains. Integration of synthetic pathway genes into the Escherichia coli chromosome enables generation of a plasmid-free strain that is stable and useful for industrial applications. Strains that do not require induction are advantageous in terms of cost. In the present study, we constructed a constitutive overexpression system in E. coli to generate plasmid-free and inducer-free strains. The T7 RNA polymerase/T7 promoter overexpression system, which is an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible gene overexpression system (T7-dependent inducible overexpression system), was modified to be a constitutive overexpression system. The constructed overexpression system, a "chromosome-based T7-dependent constitutive overexpression system", was applied in a metabolic engineering study to generate a plasmid-free and inducer-free phenylalanine producing strain of E. coli.

Production of P-aminobenzoic acid by metabolically engineered escherichia coli.

The production of chemical compounds from renewable resources is an important issue in building a sustainable society. In this study, Escherichia coli was metabolically engineered by introducing T7lac promoter-controlled aroF(fbr), pabA, pabB, and pabC genes into the chromosome to overproduce para-aminobenzoic acid (PABA) from glucose. Elevating the copy number of chromosomal PT7lac-pabA-pabB distinctly increased the PABA titer, indicating that elevation of 4-amino-4-deoxychorismic acid synthesis is a significant factor in PABA production. The introduction of a counterpart derived from Corynebacterium efficiens, pabAB (ce), encoding a fused PabA and PabB protein, resulted in a considerable increase in the PABA titer. The introduction of more than two copies of PT7lac-pabAB (ce-mod), a codon-optimized pabAB (ce), into the chromosome of a strain that simultaneously overexpressed aroF(fbr) and pabC resulted in 5.1 mM PABA from 55.6 mM glucose (yield 9.2%). The generated strain produced 35 mM (4.8 g L(-1)) PABA from 167 mM glucose (yield 21.0%) in fed-batch culture.

Expression and characterization of a thermostable acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis involved in the degradation of insoluble cellulose acetate.

A thermostable acetylxylan esterase gene, TTE0866, which catalyzes the deacetylation of cellulose acetate, was cloned from the genome of Caldanaerobacter subterraneus subsp. tengcongensis. The pH and temperature optima were 8.0 and 60 °C. The esterase was inhibited by phenylmethylsulfonyl fluoride. A mixture of the esterase and cellulolytic enzymes efficiently degraded insoluble cellulose acetate with a higher degree of substitution.

Production of aromatic compounds by metabolically engineered Escherichia coli with an expanded shikimate pathway.

Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.

Antimicrobial action of a unique phosphorus-adsorbent additive for resin, and the mechanism of its antimicrobial effect.

We found that an additive for a resin, which was comprised of collagen and aluminum (Al), showed a strong and stable antibacterial effect against various bacterium under certain conditions. We tried to clarify its mechanism of action, and investigated optimum conditions for its effects. This additive (Al cross-linked collagen powder: Al-COL) absorbed phosphorus in LB medium, gradually released aluminum in the phosphorus-reduced LB medium, and exhibited a bactericidal effect. Allophane was very suitable as the control subject, because it did not release Al in the medium, decreased phosphorus levels in the medium, and the phosphorus decrease led to a reduction in bacterial growth, though not to a bactericidal effect. On the other hand, the addition of Al to the phosphorus-reduced solution led to a bactericidal effect. These results suggested that Al can exert a strong antibacterial effect in the absence of phosphorus. This phenomenon was confirmed using film-shaped test items mixed with Al-COL powder. Furthermore, the reduction of phosphorus also synergistically led to the enhancement of the antibacterial effect of silver (Ag). The phosphorous absorption promoted the antibacterial action of Al and Ag, and Al, which has seldom been used as an antimicrobial agent, is available as an antibacterial agent in the absence of phosphorus.

A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome.

We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and ß-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF (fbr) and tyrA (fbr) or aroF (fbr) and pheA (fbr)) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.

Functional analysis of the carbohydrate-binding module of an esterase from Neisseria sicca SB involved in the degradation of cellulose acetate.

An esterase gene from Neisseria sicca SB encoding CaeA, which catalyzes the deacetylation of cellulose acetate, was cloned. CaeA contained a putative catalytic domain of carbohydrate esterase family 1 and a carbohydrate-binding module (CBM) family 2. We constructed two derivatives, with and without the CBM of CaeA. Binding assay indicated that the CBM of CaeA had an affinity for cellulose.

Efficient microbial degradation of bisphenol A in the presence of activated carbon.

The biodegradation of bisphenol A (BPA) was carried out with Sphingomonas sp. strain BP-7 and Sphingomonas yanoikuyae BP-11R in the presence of activated carbon (AC). When AC was present, both BPA-degrading bacteria efficiently degraded 300 mg/l BPA without releasing 4-hydroxyacetophenone, the major intermediate produced in BPA degradation, into the medium. The biological regeneration of AC was possible using the BPA-degrading bacteria, suggesting that an efficient system for BPA removal can be constructed by introducing BPA-degrading bacteria into an AC treatment system.

Degradation of bisphenol A by Bacillus pumilus isolated from kimchi, a traditionally fermented food.

Novel bisphenol A (BPA)-degrading bacterial strains, designated as BP-2CK, BP-21DK, and BP-22DK, were isolated from kimchi, a traditionally fermented food. These isolates were identified as Bacillus pumilus and efficiently degraded BPA in a medium supplemented with nutrients such as peptone, beef extract, and yeast extract. Strains BP-2CK, BP-21DK, and BP-22DK successfully degraded 25, 25, and 50 ppm of BPA, respectively, and all strains exhibited BPA-degrading activity in the presence of 10% NaCl. Accumulation of the metabolites including 4-hydroxyacetophenone, one of the intermediates produced by the other BPA-degrading bacteria, was not observed in BPA degradation by the isolated strains. These results indicate that the isolated food-derived bacteria are applicable for the construction of efficient and safer systems for the removal of BPA.

Biodegradation of bisphenol A and related compounds by Sphingomonas sp. strain BP-7 isolated from seawater.

A bacterium capable of assimilating 2,2-bis(4-hydroxyphenyl)propane (bisphenol A), strain BP-7, was isolated from offshore seawater samples on a medium containing bisphenol A as sole source of carbon and energy, and identified as Sphingomonas sp. strain BP-7. Other strains, Pseudomonas sp. strain BP-14, Pseudomonas sp. strain BP-15, and strain no. 24A, were also isolated from bisphenol A-enrichment culture of the seawater. These strains did not degrade bisphenol A, but accelerated the degradation of bisphenol A by Sphingomonas sp. strain BP-7. A mixed culture of Sphingomonas sp. strain BP-7 and Pseudomonas sp. strain BP-14 showed complete degradation of 100 ppm bisphenol A within 7 d in SSB-YE medium, while Sphingomonas sp. strain BP-7 alone took about 40 d for complete consumption of bisphenol A accompanied by accumulation of 4-hydroxyacetophenone. On a nutritional supplementary medium, Sphingomonas sp. strain BP-7 completely degraded bisphenol A and 4-hydroxyacetophenone within 20 h. The strain degraded a variety of bisphenols, such as 1,1-bis(4-hydroxyphenyl)ethane, 2,2-bis(4-hydroxy-3-methylphenyl)propane, 2,2-bis(4-hydroxyphenyl)butane, and 1,1-bis(4-hydroxyphenyl)cyclohexane, and hydroxy aromatic compounds such as 4-hydroxyacetophenone, 4-hydroxybenzoic acid, catechol, protocatechuic acid, and hydroquinone. The strain did not degrade bis(4-hydroxyphenyl)methane, bis(4-hydroxyphenyl)sulfone, or bis(4-hydroxyphenyl)sulfide.

Mode of action on deacetylation of acetylated methyl glycoside by cellulose acetate esterase from Neisseria sicca SB.

The regioselective deacetylation of purified cellulose acetate esterase from Neisseria sicca SB was investigated on methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside. The substrates were used as model compounds of cellulose acetate in order to estimate the mechanism for deacetylation of cellulose acetate by the enzyme. The enzyme rapidly deacetylated at position C-3 of methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside to accumulate 2,4,6-triacetate as the main initial reaction product in about 70% yield. Deacetylation was followed at position C-2, and generated 4,6-diacetate in 50% yield. The enzyme deacetylated the product at positions C-4 and C-6 at slower rates, and generated 4- and 6-monoacetates at a later reaction stage. Finally, it gave a completely deacetylated product. For 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, CA esterase deacetylated at positions C-3 and C-6 to give 2,4,6- and 2,3,4-triacetate. Deacetylation proceeded sequentially at positions C-3 and C-6 to accumulate 2,4-diacetate in 55% yield. The enzyme exhibited regioselectivity for the deacetylation of the acetylglycoside.