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We focused on the production of docosahexaenoic acid (DHA)-containing microbial lipids by Aurantiochytriumsp. using of defatted soybean (DS) as a nitrogen source. Defatted soybean is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytriumsp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytriumsp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytriumsp. 6-2 showed 15.8 ± 3.4 g/L DHA productivity (in 54.8 ± 12.1 g/L total fatty acid production) in 1 L of the FDS medium. Therefore, DHA produced by Aurantiochytriumsp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.Graphical AbstractFor the development of novel fish feed, docosahexaenoic acid (DHA) produced by a novel isolated strain Aurantiochytriumsp. 6-2 was applied as an alternative source of DHA by using defatted soybean treated with koji.

α-Tomatine is a steroidal glycoalkaloid in tomato plants and degrades with ripening. The aglycone form, tomatidine, is reported to have beneficial effects. In this study, the ability of food-related microorganisms to produce tomatidine from α-tomatine was evaluated. A total of 11 strains of Aspergillusspecies belonging to the section Nigriexhibited tomatinase activity, and Aspergillus luchuensisJCM 22302 was selected for optimization due to its high activity in its mycelia, conidia, and non-mycotoxin-producing property. Next, using A. luchuensisJCM22302 conidia, the highest yield was obtained in a 24-h reaction with 50 m mof acetic acid–sodium acetate buffer (pH 5.5) at 37 °C. Similar to the tomato pathogen Fusarium oxysporum f. lyceopersici, the time course analysis suggested that A. luchuensisJCM 22302 removed the entire sugar moiety in a single step. Future research will focus on utilizing conidia for large-scale tomatidine production because of their high tolerance and manageability.Graphical AbstractBlack koji was found to contain tomatinase activity. It was implied that A. luchuensisJCM22302 removed the whole sugar moiety of α-tomatine.

Rhizobium radiobacterC58 was found to convert 4-hydroxyisoleucine (HIL) and 2-amino-3-methyl-4-ketopentanoate (AMKP), bioactive oxidative derivatives of l-isoleucine, in both cases producing 2-aminobutyrate. Three native enzymes involved in these metabolisms were purified by column chromatography and successfully identified. In this strain, HIL was converted to acetaldehyde and 2-aminobutyrate by coupling action of the transaminase rrIlvE and the aldolase HkpA. AMKP was also converted to acetate and 2-aminobutyrate by coupling action of rrIlvE and a hydrolase DkhA. In the multi-enzymatic reactions, HkpA catalyzes the retro-aldol reaction of 4-hydroxy-3-methyl-2-ketopentanoate into acetaldehyde and 2-ketobutyrate, and DkhA catalyzes hydrolytic cleavage of the carbon-carbon bond of 2,4-diketo-3-methylpentanoate into acetate and 2-ketobutyrate. rrIlvE catalyzes reversible transamination between HIL and 4-hydroxy-3-methyl-2-ketopentanoate, AMKP and 2,4-diketo-3-methylpentanoate, and 2-ketobutyrate and 2-aminobutyrate. The results suggested that the conversion activity of Rhizobiumbacteria plays an important role in the complex biological metabolic networks associated with HIL and AMKP.Graphical AbstractHIL and AMKP are metabolized to 2-amimobutyrate by Rhizobiumbacteria in a multi-step reaction.

In natural indigo dyeing, the water-insoluble indigo included in the composted indigo leaves called sukumois converted to water-soluble leuco-indigo through the reduction activities of microorganisms under alkaline conditions. To understand the relationship between indigo reduction and microorganisms in indigo-fermentation suspensions, we isolated and identified the microorganisms that reduce indigo and analyzed the microbiota in indigo-fermentation suspensions. Indigo-reducing microorganisms, which were not isolated by means of a conventional indigo carmine-reduction assay method, were isolated by using indigo as a direct substrate and further identified and characterized. We succeeded in isolating bacteria closely related to Corynebacterium glutamicum, Chryseomicrobium aureum, and Enterococcussp. for the first time. Anthraquinone was found to be an effective mediator that facilitated the indigo-reduction activity of the isolated strains. On analysis of the microbiota in indigo-fermentation suspensions, the ratio of indigo-reducing bacteria and others was found to be important for maintaining the indigo-reduction activity.Graphical AbstractThe new indigo-reducing bacteria were isolated by the novel method from the indigo fermentation suspensions. The dyeing activities were explained by the analysis of microbiota.

ABSTRACTCyclooxygenases are responsible for the production of prostaglandin H2(PGH2) from arachidonic acid. PGH2can be converted into some bioactive prostaglandins, including prostaglandin F2α(PGF2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF2αhas been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF2αcan be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF2αwas accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF2αwere accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.

2′-O-Methylribonucleosides (2′-OMe-NRs) are promising raw materials for nucleic acid drugs because of their high thermal stability and nuclease tolerance. In the course of microbial screening for metabolic activity toward 2′-OMe-NRs, Lactobacillus buchneriLBK78 was found to decompose 2′-O-methyluridine (2′-OMe-UR). The enzyme responsible was partially purified from L. buchneriLBK78 cells by a four-step purification procedure, and identified as a novel nucleoside hydrolase. This enzyme, LbNH, belongs to the nucleoside hydrolase superfamily, and formed a homotetrameric structure composed of subunits with a molecular mass around 34 kDa. LbNH hydrolyzed 2′-OMe-UR to 2′-O-methylribose and uracil, and the kinetic constants were Kmof 0.040 mM, kcatof 0.49 s−1, and kcat/Kmof 12 mM−1 s−1. In a substrate specificity analysis, LbNH preferred ribonucleosides and 2′-OMe-NRs as its hydrolytic substrates, but reacted weakly with 2′-deoxyribonucleosides. In a phylogenetic analysis, LbNH showed a close relationship with purine-specific nucleoside hydrolases from trypanosomes.

A succinimide-assimilating bacterium, Pseudomonas putidas52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putidas52 as catalyst, 400 mMpyruvate was produced from 500 mMfumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mMof pyruvate was successfully produced from 1,000 mMfumarate in a 72-h reaction with washed cells from P. putida15160 as catalyst.

Linoleic acid isomerase in Lactobacillus plantarumwas found to be a novel multi-component enzyme system widespread in membrane and soluble fractions. The isomerization reaction involved a hydration step, 10-hydroxy-12-octadecenoic acid production from linoleic acid, as part of the reaction, and the hydration reaction was catalyzed by the membrane fraction. Both membrane and soluble fractions were required for the whole isomerization reaction, i.e., conjugated linoleic acid (CLA) production from linoleic acid, and for CLA production from 10-hydroxy-12-octadecenoic acid, a reaction intermediate. The multi-component enzyme system was inhibited by o-phenanthroline, and divalent metal ions such as Ni2+and Co2+restored activity. Metal oxides such as VO43+, MoO42+, and MnO42+enhanced activity. The multi-component enzyme systems required oxidoreduction cofactors such as NADH together with FAD or NADPH for total activity.

Arthrobacter simplexAKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, α-ketobutyrate, and L-glutamate in the presence of Escherichia coliharboring the branched chain amino acid transaminase gene (ilvE) from E. coliK12 substrain MG1655. By using resting cells of A. simplexAKU 626 and E. coliBL21(DE3)/pET-15b-ilvE, 3.2 mM4-hydroxyisoleucine was produced from 250 mMacetaldehyde, 75 mMα-ketobutyrate, and 100 mML-glutamate with a molar yield to α-ketobutyrate of 4.3% in 50 mMTris–HCl buffer (pH 7.5) containing 2 mMMnCl2·4H2O at 28 °C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and α-ketobutyrate was purified from A. simplexAKU 626. Mn2+and pyridoxal 5′-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coliwas investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coliconvert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate viaD-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM(121 g/l) fructose 1,6-diphosphate was produced from 1,111 mMglucose and 750 mMpotassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. colicells. With 178 mMenzymatically prepared fructose 1,6-diphosphate and 400 mMacetaldehyde as substrates, 246 mM(52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.

Bioprocesses, which involve biocatalysts for the production of useful compounds, are expected to become a leading player in green chemistry. The first step in bioprocess development is screening for useful biological reactions in the immense number of microorganisms with infinite diversity and versatility. This review introduces some examples of bioprocess development that started from process design stemming from the discovery of unique metabolic processes, reactions, and enzymes in microbial nucleic acid and lipid metabolisms.

An isomer of the conjugated linoleic acid (CLA) produced from linoleic acid by Lactobacillus plantarumwas identified as cis-9,trans-11-octadecadienoic acid by proton nuclear magnetic resonance spectroscopy. Together with earlier results, we concluded that the bacterium produces two CLA isomers, cis-9,trans-11- and trans-9,trans-11-octadecadienoic acid from linoleic acid. The addition of L-serine, glucose, AgNO3, or NaCl to the reaction mixture reduced production of the latter.

Ricinoleic acid (12-hydroxy-cis-9-octadecaenoic acid) was an effective substrate for conjugated linoleic acid (CLA) production by washed cells of Lactobacillus plantarumAKU 1009a. The CLA produced was a mixture of cis-9,trans-11- and trans-9,trans-11-octadecadienoic acids. Addition of α-linolenic acid to the culture medium increased the CLA productivity of the washed cells. In the presence of lipase, castor oil, in which the main fatty acid component is ricinoleic acid, also was a substrate for CLA.

Bilirubin dehydrogenase, a membrane-bound enzyme that catalyzes the one-step oxidation of ditaurobilirubin and bilirubin to ditaurobiliverdin and biliverdin, respectively, in the presence of an electron acceptor, was found in Aspergillus ochraceusIB-3, and purified from the membrane fraction through solubilization by Triton X-100. Phenazine and quinone derivatives acted as electron acceptors. Accumulation of ditaurobiliverdin and biliverdin by enzyme catalysis increased the absorbance at 660 nm, which is far from the range of wavelengths affected by serum ingredients. The enzyme selectively oxidized ditaurobilirubin at low pH, so changes in the reaction pH enable the enzyme to discriminate between the bilirubin fractions ditaurobilirubin (an example of conjugated bilirubin) and bilirubin (an example of unconjugated bilirubin). Using the enzyme, 2 to 80 μMof ditaurobilirubin were measured accurately by monitoring the changes in absorbance at 660 nm.

An NAD+-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fuscaAKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The Kmand Vmaxfor (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mMand 53 μmol/min/mg, and 0.33 mMand 130 μmol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The Kmand Vmaxfor 2-hexanone reduction were 2.5 mMand 260 μmol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH2-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolisand phenylacetaldehyde reductase from Corynebacteriumsp.