Kazuhiko Tabata

ywfE in Bacillus subtilis Codes for a Novel Enzyme, l-Amino Acid Ligase

The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as d-alanyl-d-alanine, polyglutamate, and gamma-peptide, but, curiously, no enzyme synthesizing alpha-dipeptides of l-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming l-alanyl-l-glutamine from l-alanine and l-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of l-amino acids. Among 231 combinations of l-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of alpha-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the d-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, l-amino acid ligase.

Large-scale production of N-acetyllactosamine through bacterial coupling.

A large-scale production system of N-acetyllactosamine, a core structure of various oligosaccharides, was established by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains over-expressed the UDP-Gal biosynthetic genes and the beta-(1-->4)-galactosyltransferase gene of Neisseria gonorrhoeae, respectively. C. ammoniagenes contributed the production of UTP from orotic acid. N-Acetyllactosamine was accumulated at 279 mM (107 g L-1) after a 38 h reaction (2.5 L in volume) starting from orotic acid, D-galactose, and 2-acetamido-2-deoxy-D-glucose.

Fermentative Production of l-Alanyl-l-Glutamine by a Metabolically Engineered Escherichia coli Strain Expressing l-Amino Acid α-Ligase

ABSTRACT In spite of its clinical and nutritional importance, l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing l-amino acid α-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous l-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing d-amino acids were formed.

Production of N-acetyl-d-neuraminic acid by coupling bacteria expressing N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic acid synthetase

Abstract N -acetyl- d -glucosamine (GlcNAc) 2-epimerase catalyzes the interconversion between GlcNAc and N -acetyl- d -mannosamine (ManNAc) that is a precursor of N -acetyl- d -neuraminic acid (NeuAc). Homology search using the sequence of the porcine GlcNAc 2-epimerase as a query revealed that a gene product (Slr1975) of Synechocystis sp. PCC6803 showed significant homology. When the gene of slr1975 was cloned by PCR and expressed in Escherichia coli , the recombinant E. coli showed GlcNAc 2-epimerase activity. This is the first example of the cloning of the gene for GlcNAc 2-epimerase from prokaryotes. GlcNAc 2-epimerase was purified from E. coli overexpressing slr1975 , and the enzymatic properties were determined. Molecular weight by SDS-PAGE was 45 kDa, similar to that predicted by the sequence. Km values for GlcNAc and ManNAc were 6.94 mM and 4.76 mM, respectively, and ATP was essential for the activity. Microbial production of NeuAc was carried out using E. coli cells overexpressing GlcNAc 2-epimerase and NeuAc synthetase as enzyme sources. Phosphoenolpyruvate and ATP, required as a substrate or a cofactor of the enzymes, were supplied by the activities of E. coli and Corynebacterium ammoniagenes cells. Starting with 800 mM GlcNAc and 360 mM glucose, NeuAc accumulated at 39.7 mM (12.3 g l −1 ) after 22 h.

Large-scale production of the carbohydrate portion of the sialyl–Tn epitope, α-Neup5Ac-(2→6)-d-GalpNAc, through bacterial coupling

Alpha-Neup5Ac-(2-->6)-D-GalpNAc, the carbohydrate portion of sialyl-Tn epitope of the tumor-associated carbohydrate antigen, was prepared by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains overexpressed the CMP-Neup5Ac biosynthetic genes and the alpha-(2-->6)-sialyltransferase gene of Photobacterium damsela. C. ammoniagenes contributed to the production of UTP from orotic acid. Alpha-Neup5Ac-(2-->6)-D-GalpNAc was accumulated at 87 mM (45 g/L) after a 25-h reaction starting from orotic acid, N-acetylneuraminic acid, and 2-acetamide-2-deoxy-D-galactose.

Production of UDP-N-acetylglucosamine by coupling metabolically engineered bacteria

A production system of UDP-N-acetylglucosamine (UDP-GlcNAc) was established by using recombinant Escherichia coli and Corynebacterium ammoniagenes in combination. E. coli overexpressed the UDP-GlcNAc biosynthetic genes, glmM, glmU, glk, ppa, ack, and pta, whereas C. ammoniagenes contributed to the formation of UTP from orotic acid. Glucose 1,6-diphosphate (Glc-1,6-P2), which was required for the activity of phosphoglucosamine mutase involved in UDP-GlcNAc biosynthesis, was supplied by phosphoglucomutase and phosphofructokinase. Starting with orotic acid (65 mM) and glucosamine (400 mM), UDP-GlcNAc accumulated at 11.4 mM (7.4 g l−1) after 8 h.

Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli

ABSTRACT l-Homophenylalanine (l-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While the chemoenzymatic route of synthesis is fully developed, we investigated microbial production of l-Hph to explore the possibility of a more efficient and sustainable approach to l-Hph production. We hypothesized that l-Hph is synthesized from l-Phe via a mechanism homologous to 3-methyl-2-oxobutanoic acid conversion to 4-methyl-2-oxopentanoic acid during leucine biosynthesis. Based on bioinformatics analysis, we found three putative homophenylalanine biosynthesis genes, hphA (Npun_F2464), hphB (Npun_F2457), and hphCD (Npun_F2458), in the cyanobacterium Nostoc punctiforme PCC73102, located around the gene cluster responsible for anabaenopeptin biosynthesis. We constructed Escherichia coli strains harboring hphABCD-expressing plasmids and achieved the fermentative production of l-Hph from l-Phe. To our knowledge, this is the first identification of the genes responsible for homophenylalanine synthesis in any organism. Furthermore, to improve the low conversion efficiency of the initial strain, we optimized the expression of hphA, hphB, and hphCD, which increased the yield to ∼630 mg/liter. The l-Hph biosynthesis and l-Leu biosynthesis genes from E. coli were also compared. This analysis revealed that HphB has comparatively relaxed substrate specificity and can perform the function of LeuB, but HphA and HphCD show tight substrate specificity and cannot complement the LeuA and LeuC/LeuD functions, and vice versa. Finally, the range of substrate tolerance of the l-Hph-producing strain was examined, which showed that m-fluorophenylalanine, o-fluorophenylalanine, and l-tyrosine were accepted as substrates and that the corresponding homoamino acids were generated.

Metabolic Engineering for l-Glutamine Overproduction by Using DNA Gyrase Mutations in Escherichia coli

ABSTRACT An l-glutamine-overproducing mutant of an Escherichia coli K-12-derived strain was selected from randomly mutagenized cells in the course of l-alanyl-l-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism for l-glutamine overproduction in this mutant. Three mutations were identified that are related to the l-glutamine overproduction phenotype, namely, an intergenic mutation in the 5′-flanking region of yeiG and two nonsynonymous mutations in gyrA (Gly821Ser and Asp830Asn). Expression of yeiG, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations in gyrA, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (topA) and topoisomerase IV (parC and parE), an increase in l-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase in l-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed an l-glutamine-hyperproducing strain by attenuating cellular l-glutamine degradation activity. Although the reconstituted mutant (with yeiG together with gyrA) produced 200 mM l-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mM l-glutamine.