Hayato Yamanaka

Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

ABSTRACT 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.

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.

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.

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 β-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 (aroFfbr and tyrAfbr or aroFfbr and pheAfbr) 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.

Enzymatic preparation of optically active silicon-containing amino acids and their application

Optically active 3-trimethyl silylalanine (TMS-Ala) was prepared by hydrolysis of N-acetyl-dl-TMS-Ala catalyzed by acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC3.5.1.14). Acylase I from porcine kidney (PKA) was found to be more effective than that from Aspergillus melleus in the preparation of l-TMS-Ala. Under the optimized conditions, optically pure l-TMS-Ala (>99% enantiomeric excess, ee) was obtained with a 72% yield. Furthermore, a highly optically pure d-TMS-Ala (96% ee) could also be obtained with a 76% yield by chemical hydrolysis of the residual substrate. Enzymatic synthesis of peptides containing TMS-Ala was also attempted in ethyl acetate. Benzyloxycarbonyl (Z)-l-TMS-Ala served as the substrate for thermolysin, whereas l-TMS-Ala-OMe was inactive as the amino component. In the case of inhibitory activity of dipeptides toward thermolysin, l-Leu-(l-TMS-Ala) was found to be a more potent inhibitor than l-Leu-l-Leu, which is known to be one of the most effective inhibitors of thermolysin among the dipeptides consisting of natural aminoacids.

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 aroFfbr, 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 aroFfbr 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. Graphical Abstract Escherichia coli was metabolically engineered by introducing several genes into the chromosome to overproduce para-aminobenzoic acid from glucose.

Efficient preparation of optically active p-trimethylsilylphenylalanine by using cell-free extract of Blastobacter sp. A17p-4

Abstract Optically active p -trimethylsilylphenylalanine (TMS-Phe) was prepared by enantioselective hydrolysis of N -carbamoyl- dl - p -trimethylsilylphenylalanine (C- dl -TMS-Phe) with the cell-free extract of Blastobacter sp. A17p-4, which is known to produce N -carbamoyl- d -amino acid amidohydrolase (DCase). Although this bacterium also produced N -carbamoyl- l -amino acid amidohydrolase (LCase), heat treatment of the cell-free extract for 40 min at 50°C and pH 7.0 was found to be effective in completely inactivating the LCase without loss of DCase activity, which provided a far simpler and more convenient method of preparing optically pure d -TMS-Phe (99% enantiomeric excess, ee). Furthermore, optically pure l -TMS-Phe (99% ee) could be obtained by LCase-catalyzed hydrolysis of the residual substrate with the non-treated cell-free extract as the enzyme source. The optimum pH for the hydrolysis of C- l -TMS-Phe was 7.0, and addition of 2 mM Mn 2+ and 5% N,N -dimethylformamide were effective in accelerating the activity of LCase and raising the substrate concentration, respectively.

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.

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.