Hideo Fukuda

Photosynthetic conversion of carbon dioxide to ethylene by the recombinant cyanobacterium, Synechococcus sp. PCC 7942, which harbors a gene for the ethylene-forming enzyme of Pseudomonas syringae

Abstract Photosynthetic conversion of carbon dioxide to ethylene was studied using the recombinant cyanobacterium, Synechococcus sp. strain PCC 7942 R2-SPc which expresses the ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding the EFE ( efe gene) from P. syringae was introduced into the cyanobacterium utilizing the pUC303 shuttle vector into which the efe gene was placed under the control of various transcriptional signals, i.e. , the native promoter and terminator of the efe gene (pUC303-EFE03), the Escherichia coli lacZ promoter and the efe gene terminator (pUC303-EFE10 and pUC303-EFE30), or the promoter and terminator of the psbAI gene from Synechococcus sp. PCC7942 which codes for the D1 protein in photosystem II (pEXE3). Among these configurations, EFE activity measured in the cell-free extracts of transformants that harbored the pEXE3 was highest. However, ethylene production in vivo of the transformants carrying pEXE3 declined with the number of generations, because homologous recombination of DNA sequences on the pEXE3 plasmid and host chromosomal psbAI locus took place. Deletion of the 5′-upstream region of the psbAI promoter and the 3′-downstream region of the psbAI terminator in pEXE3 resulted in pEXE3Δ8 which showed the highest level of ethylene-forming activity, although the latter plasmid was still unstable with a half-life of only 12 generations. The amount of carbon incorporated into ethylene was calculated as a percentage of the total carbon fixed, the maximum value of which was 5.84% in the recombinant cyanobacterium harboring pUC303-EFE03.

Two reactions are simultaneously catalyzed by a single enzyme: The arginine-dependent simultaneous formation of two products, ethylene and succinate, from 2-oxoglutarate by an enzyme from Pseudomonas syringae

A single enzyme isolated from Pseudomonas syringae pv. phaseolicola PK2 simultaneously catalyzed two reactions, namely, the formation of ethylene and succinate from 2-oxoglutarate, at a molar ratio of 2:1. In the main reaction, 2-oxoglutarate was dioxygenated to produce one molecule of ethylene and three molecules of carbon dioxide. In the sub-reaction, both 2-oxoglutarate and L-arginine were mono-oxygenated to yield succinate plus carbon dioxide and L-hydroxyarginine, respectively, the latter being further transformed to guanidine and L-delta 1-pyrroline-5-carboxylate. We propose a dual-circuit mechanism for the entire reaction, in which the binding of L-arginine and 2-oxoglutarate in a Schiff-base structure generates a common intermediate for two reactions.

Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2

The gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 was found to be encoded by an indigenous plasmid, designated pPSP1. The gene for the ethylene-forming enzyme was cloned and expressed in Escherichia coli JM109. Nucleotide sequence analysis of the clone revealed an open reading frame that encodes 350 amino acids (mol. wt. 39,444). In a comparison with other proteins, the homology score for the entire amino-acid sequence of the ethylene-forming enzyme of Pseudomonas syringae versus ethylene-forming enzymes from plants and 2-oxoglutarate-dependent dioxygenases was low. However, functionally significant regions are conserved.

Classification of ethylene-producing bacteria in terms of biosynthetic pathways to ethylene

Abstract Two hundred and twenty-nine ethylene-producing strains of bacteria were identified among 757 bacterial strains which included 13 strains of chemolithotrophs. The ethylene-producing bacetria were classified into three groups, namely, l -methionine-dependent, 2-ketoglutarate-dependent and meat extract-dependent, with reference to their respective biosynthetic pathways to ethylene. Two hundred and twenty-five l -methionine-dependent strains were obtained, while the only 2-ketoglutarate-dependent strain was Pseudomonas syringae pv. phaseolicola PK2. Three strains of chemolithotrophs had ethylene-forming capacity, and Thiobacillus novellus IFO 12443 had a novel ethylene-forming system which was dependent upon the addition of meat extract into the culture medium. The ethylene-forming systems of two of the strains of Thiobacillus ferrooxidans have not yet been characterized. Several strains of non-ethylene-producing bacteria failed to produce ethylene, even when l -methionine was added to the culture medium. We examined the causes of their lack of ethylene-producing ability and judged that these strains are either NADH:Fe(III)EDTA oxidoreductase-less or methionine-uptake activity-less.

Ethylene production by strains of the plant-pathogenic bacterium Pseudomonas syringae depends upon the presence of indigenous plasmids carrying homologous genes for the ethylene-forming enzyme.

The molecular characteristics of the ethylene-forming enzymes of strains of Pseudomonas syringae were tested. The ethylene-producing activities of the nine strains as measured in vivo and in vitro were similar, except for that of P. syringae pv. mori M5. A polyclonal antibody and a DNA probe for the ethylene-forming enzyme from P. syringae pv. phaseolicola PK2 were prepared to investigate homologies among the proteins and genes for the ethylene-forming enzymes. With the exception of P. syringae pv. mori M5, eight strains tested expressed the same antigen as the ethylene-forming enzyme from P. syringae pv. phaseolicola PK2 and were homologous to DNA sequences on indigenous plasmids. Molecular masses of antigenic proteins from all ethylene-producing strains were 40 kDa. The N-terminal amino acid sequence of the purified ethylene-forming enzyme from P. syringae pv. glycinea KN130 was identical to that of the enzyme from P. syringae pv. phaseolicola PK2. These results show that the ethylene-forming enzymes encoded by the indigenous plasmid(s) in the pathogenic bacteria examined were similar.

L-Arginine is essential for the formation in vitro of ethylene by an extract of Pseudomonas syringae

Summary: A system was developed for the formation of ethylene in vitro by an extract of Pseudomonas syringae pv. phaseolicola PK2. The ethylene-forming activity of a cell-free extract of this bacterium measured in a system reported previously was almost completely lost when the cell-free extract was dialysed against potassium phosphate buffer for 24 h at 4 °C. When the fraction of cell-free extract with a molecular mass < 10 kDa (SupI) was added back to the enzyme fraction after gel filtration of the cell-free extract, the enzymic activity increased to about four times that of the gel-filtered crude enzyme. The action of SupI could be reproduced by the addition of l-arginine. The complete system for the formation of ethylene under aerobic conditions in vitro required 0·25 mm-2-oxoglutarate, 0·2 mm-FeSO4, 2 mm-DTT, 10 mm-l-histidine and 0·2 mm-l-arginine. The cofactor specificity was examined by replacing l-arginine or l-histidine with various analogues, but none of them were effective. The components of this system, with the exception of l-histidine, were similar to those of a system derived from the ethylene-producing, plant pathogenic fungus Penicillium digitatum which also produced ethylene in vitro in a reaction dependent on 2-oxoglutarate. The intermediates in the ethylene-forming reaction are postulated and the roles of l-arginine and l-histidine in the formation of ethylene by Ps. syringae are discussed.

The Role of NADH:Fe(III)EDTA Oxidoreductase in Ethylene Formation from 2-Keto-4-Methylthiobutyrate

Abstract A mechanism for the formation of ethylene from 2-keto-4-methylthiobutyric acid (KMBA), a deaminated derivative of l -methionine, was studied with NADH:Fe(III)EDTA oxidoreductase purified from Cryptococcus albidus IFO 0939. The characteristics of the reactions catalyzing the NADH:Fe(III)EDTA oxidoreduction and the formation of ethylene were compared and found to be almost identical. A chemical ethylene-forming system, composed of Fe(II)EDTA, KMBA and oxygen, was constructed and the characteristics of the formation of ethylene were compared with those of the enzymatic reaction. Both the enzymatic and the chemical ethylene-forming reactions were strongly inhibited by scavengers of free radicals, such as benzoic acid, hydroquinone and catalase, and both were activated by H2O2. From these results, we propose the following mechanism for the formation of ethylene from KMBA. The first step involves the NADH:Fe(III)EDTA oxidoreduction that is catalyzed by NADH:Fe(III)EDTA oxidoreductase, with Fe(II)EDTA always being supplied as the result. Oxidation of Fe(II)EDTA by molecular oxygen yields the superoxide radical anion (O2 . ) which can undergo the dismutation reaction to form hydrogen peroxide. Hydrogen peroxide in turn can react with Fe2+ via the Fenton reaction, generating the hydroxyl radical (OH•), which can serve as an oxidizing agent in the oxidation of KMBA to ethylene. The role of the activity of NADH:Fe(III)EDTA oxidoreductase in the formation of ethylene from KMBA in vivo is discussed.

Microbial production of C2-hydrocarbons, ethane, ethylene and acetylene

Article de synthese sur la production dans des conditions aerobies (champignons, levures, bacteries, actinomycetes)

A method of graphically analyzing substrate-inhibition kinetics.

A model of substrate inhibition for enzyme catalysis was extended to describe the kinetics of photosynthetic production of ethylene by a recombinant cyanobacterium, which exhibits light-inhibition behavior similar to the substrate-inhibition behavior in enzyme reactions. To check the validity of the model against the experimental data, the model equation, which contains three kinetic parameters, was transformed so that a linear plot of the data could be made. The plot yielded reasonable linearity, and the parameter values could be estimated from the plot. The linear-plot approach was then applied to other inhibition kinetics including substrate inhibition of enzyme reactions and inhibitory growth of bacteria, whose analyses would otherwise require nonlinear least-squares fits or data measured in constrained ranges. Plots for three totally different systems all showed reasonable linearity, which enabled visual validation of the assumed kinetics. Parameter values evaluated from the plots were compared with results of nonlinear least-squares fits. A normalized linear plot for all the results discussed in this work is also presented, where dimensionless rates as a function of dimensionless concentration lie in a straight line. The linear-plot approach is expected to be complementary to nonlinear least-squares fits and other currently used methods in analyses of substrate-inhibition kinetics. Copyright 1999 John Wiley & Sons, Inc.

Overexpression and in vitro reconstitution of the ethylene-forming enzyme from Pseudomonas syringae

Abstract By replacing a native promoter with lac and tac promoters, the gene encoding an ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 was overexpressed in Escherichia coli . The EFE protein expressed by a multicopy plasmid accounted for more than 30% of the total cellular protein, resulting in ethylene-forming activities higher than 10 μl of ethylene (mg cell) −1 h −1 in recombinant E. coli cells. However, most of the EFE protein accumulated as inactive inclusion bodies particularly at elevated temperatures (>30°C). We present an efficient procedure for reconstituting an active enzyme from inclusion bodies by solubilization with 8 M urea and dialysis. The reconstituted EFE has specific activity identical to that of the native enzyme from P. syringae , suggesting that the EFE protein has an intrinsic folding capability in vitro .