Kohei Mase

Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes

Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of praR and praABEGFDCHI. Based on the enzyme activities of cell extracts of Escherichia coli carrying praI, praA, praH, praB, praC, and praD, these genes were found to code for 4HB 3-hydroxylase, PCA 2,3-dioxygenase, 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate (OCA) tautomerase, and OCA decarboxylase, respectively, which are involved in the conversion of 4HB into 2-hydroxypenta-2,4-dienoate (HPD). The praE, praF, and praG gene products exhibited 45 to 61% amino acid sequence identity to the corresponding enzymes responsible for the catabolism of HPD to pyruvate and acetyl coenzyme A. The deduced amino acid sequence of praR showed similarity with those of IclR-type transcriptional regulators. Reverse transcription-PCR analysis revealed that praABEGFDCHI constitute an operon, and these genes were expressed during the growth of JJ-1b on 4HB and PCA. praR-praABEGFDCHI conferred the ability to grow on 4HB to E. coli, suggesting that praEGF were functional for the conversion of HPD to pyruvate and acetyl coenzyme A. A promoter analysis suggested that praR encodes a repressor of the pra operon.

Characterization of the Protocatechuate 4,5-Cleavage Pathway Operon in Comamonas sp. Strain E6 and Discovery of a Novel Pathway Gene

ABSTRACT The protocatechuate (PCA) 4,5-cleavage (PCA45) pathway is the essential catabolic route for the degradation of various aromatic acids in the genus Comamonas. All of the PCA45 pathway genes, orf1-pmdKEFDABC, as well as another PCA 4,5-dioxygenase gene, pmdAIIBII, were isolated from a phthalate-degrading bacterium, Comamonas sp. strain E6. Disruption of pmdB and pmdD in E6, which code for the β subunit of PCA 4,5-dioxygenase and 2-pyrone-4,6-dicarboxylate (PDC) hydrolase, respectively, resulted in a growth defect on PCA, indicating that these genes are essential for the growth of E6 on PCA. On the other hand, inactivation of pmdBII did not affect the growth of E6 on PCA. Disruption of pmdK, which is related to a 4-hydroxybenzoate/PCA transporter of Pseudomonas putida, resulted in growth retardation on PCA. The insertional inactivation of orf1 in E6, whose deduced amino acid sequence has no similarity with proteins of known function, led to the complete loss of growth on PCA and the accumulation of PDC and 4-oxalomesaconate (OMA) from PCA. These results indicated the involvement of orf1 in the PCA45 pathway, and this gene, designated pmdU, was suggested to code for OMA tautomerase. Reverse transcription-PCR analysis suggested that the pmdUKEFDABC genes constitute an operon. The transcription start site of the pmd operon was mapped at 167 nucleotides upstream of the initiation codon of pmdU. The pmd promoter activity was enhanced 20-fold when the cells were grown in the presence of PCA. Inducers of the pmd operon were found to be PCA and PDC, but PDC was the more effective inducer.

The ectopic expression of phenylalanine ammonia lyase with ectopic accumulation of polysaccharide-linked hydroxycinnamoyl esters in internode parenchyma of rice mutant Fukei 71

Both polysaccharide-linked hydroxycinnamoyl esters (PHEs) and lignin are biosynthesized via the phenylpropanoid pathway. In the abnormal internode parenchyma of the rice (Oryza sativa L.) mutant Fukei 71, which has a defective recessive gene (d50), the biosynthesis of lignin and PHEs differs. . The polysaccharide-linked ferulate and p-coumarate have been shown to accumulate to high levels in the irregularly shaped and collapsed internode parenchyma cells of Fukei 71 without an accompanying overaccumulation of lignin as a result of the defective d50 gene. In the present study we demonstrated that in this abnormal parenchyma tissue of Fukei 71 the expression of phenylalanine ammonia lyase (PAL) and glutamine synthetase (GS) were ectopically induced with the ectopic accumulation of PHEs, suggesting that the d50 gene may play a role as a controlling element in the biosynthesis of PHEs during cell-wall formation in the grasses.

Microbial conversion of glucose to a novel chemical building block, 2-pyrone-4,6-dicarboxylic acid.

2-Pyrone-4,6-dicarboxylic acid (PDC) is a catabolic intermediate in Sphingobium sp. SYK-6 (previously characterized as Sphingomonas paucimobilis SYK-6), which is a degrader of lignin-derived aromatic compounds. Recently, PDC has been also characterized as a novel starting material for several potentially useful synthetic polymers. In a previous study, we constructed a biosynthetic system in which PDC was generated efficiently from a chemically synthesized compound, protocatechuate. In order to develop an alternative system for production of PDC, we tried to generate it from glucose, which is a low-cost sugar that can be obtained from abundant cellulosic wastes and biomass crops. We designed a metabolic bypass to PDC from the shikimate pathway in recombinant Escherichia coli cells. PDC accumulated in the medium of recombinant E. coli cells that had been transformed with genes isolated from Emericella niger, E. coli, Pseudomonas putida, and Sphingobium sp. SYK-6. The yield of PDC depended on the combination of genes that we introduced into the cells and on the specific of host strain. Under optimal conditions, the yield and titer of PDC were, respectively, 17.3% and 0.35 mg/l when the concentration of glucose was 2 g/l and the culture volume was 50 ml. Our results open up the possibility of novel utilization of biomass as the source of a useful chemical building block.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase is regulated for the accumulation of polysaccharide-linked hydroxycinnamoyl esters in rice (Oryza sativa L.) internode cell walls

Polysaccharide-linked hydroxycinnamoyl esters (PHEs) over-accumulate in the internodes of a rice (Oryza sativa L.) mutant, Fukei 71 (F71). This accumulation is accompanied by over-expression of phenylalanine ammonialyase (PAL). In this study, we show that only one member of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) family expresses in close correlation with PAL. Furthermore, substrate availability to DAHPS is promoted by down-regulating the expression of plastidic pyruvate kinase (PKp), a competitor of DAHPS. Since the over-production of PHEs is caused by D50 gene disruption, these results suggest that specific enzymes in the phenylpropanoid and shikimate pathways are coordinately up-regulated. In addition, the results indicate that carbon-flow into the shikimate pathway is modified for the synthesis of PHEs, and is probably controlled by D50.

Stable Chiral Carboxymuconolactone Production from a Lignin-Related Aromatic Compound, Protocatechuic Acid

Susumu Kondo1, Kosuke Sugimura1, Yuta Okamura1, Kohei Mase1, Kanna Sato-Izawa1, Yuichiro Otsuka2, Shinya Kajita1, Eiji Masai3, Masaya Nakamura2, Tomonori Sonoki4* and Yoshihiro Katayama1,5 1Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan 2Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan 3Graduate School of Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan 4Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, Japan 5Department of Forest Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan *Corresponding author: Tomonori Sonoki, Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, Japan. Tel: +81-172-39-3585; E-mail: sonoki@hirosaki-u.ac.jp