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Site-directed mutagenesis was used to investigate the cloning, sequencing and overexpression in Escherichia coli cells of the low-specificity L-threonine aldolase (L-TA) gene from the Pseudomonas strain NCIMB 10558, purification and characterization of the recombinant enzyme and the identification of the active-site lysine residue of the enzyme. The amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei and other hypothetical proteins from other microorganisms.

A novel nucleoside oxidase was purified from Flavobacterium meningosepticum. The nucleoside oxidase catalyzes the oxidation of nucleosides to its carboxylic acid congener and forms hydrogen peroxide in the process. This enzyme is induced with the presence of nucleosides indicating that it is involved in nucleoside catabolism in F. meningosepticum. The physiological function of this enzyme is yet to be elucidated.

Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 [right arrow] Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 [right arrow] Asn substitution as well as Ser195 [right arrow] Aia substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-[NH.sub.2]) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

The gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereoscopically catalyzes the interconversion of L-allo-threonine and glycine was isolated. The active-site lysine residue of the novel enzyme was identified by site-directed mutagenesis. Two hypothetical proteins, GLY1 protein of Saccharomyces cerevisiae and GLY1-like protein of Caenorhabditis elegans that have substantial similarity to L-allo-TA in amino acid sequence were also identified.

A novel Mortierella alpina mutant (M209-7) derived from the delta12 desaturase-defective Mut48 strain produced greater amounts of 5,811-eicosatrienoic acid (mead acid) compared to its parent strain. The M209-7 strain was similar to Mut48 in morphology, glucose consumption, total fatty acid composition and growth. Furthermore, optimization of cultivation temperature and glucose feeding enhanced the synthesis of mead acid by the mutant Mortierella alpina M209-7 strain.

The cDNA encoding aldehyde reductase (ALR) in the red yeast Sporobolomyces salmonicolor has been cloned using oligonucleotide probes. The ALR gene contains an open reading frame of 323 residues with two introns in the noncoding sequence and four introns in the coding sequence. The encoded polypeptide has a molecular mass of 35,232 Da and its amino acid sequence is similar to that of other aldo-keto reductases, especially in the N-terminal region. An Escherichia coli strain expressing the ALR gene produces an enzyme structurally and catalytically similar to the yeast ALR.

A novel delta 5-desaturase defective mutant, S14, obtained from an arachidonic-acid generating fungus, Mortierella alpina, 1S-4, generates dihomo-gamma-linolenic acid (DGLA). Under favorable conditions, more than twograms of DGLA per liter of culture broth is generated by delta 5-desaturase-defective mutant. This is possible when it is developed for seven days in a 5-litre jar fermentor at 28 degrees Celsius under submerged batch culture conditions.

A delta 5-desaturase-defective mutant, termed Mut44, of the fungus Mortierella alpina 1S-4 was found to exhibit high mycelial dihomo-gamma-linolenic acid (DHGA) activity. The mutant grew in a simple medium with significant DHGA productivity. Various factors affecting the DHGA production were the glucose and yeast extract concentrations and growth temperature. There were no problems, however, with the inhibitors present in oils. Most of the DHGA was present in the triglyceride fraction among lipid classes.

Mortierella alpina 1S-4 is a soil fungus capable of producing high levels of polyunsaturated fatty acids (PUFA). To elucidate the pathway of PUFA biosynthesis in this fungus, fatty acid desaturation mutants were generated using N-methyl-N'-nitro-N-nitrosoguanidine. The mutagenesis yielded three mutants which were further characterized. The analysis showed that the mutants were defective in delta-5-, delta-6- and delta-12 desaturation, respectively. These mutants may be used for fermentative production of fatty acids, as well as sources of material for complementation analysis.

Abstract Background Reproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway. Results Using E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2′-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2′-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2′-deoxyinosine, the deaminated product of 2′-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process. Conclusion A critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary metabolites that accumulate at very low levels in the metabolic pool, will grow. The development of an efficient production method for these compounds will have a great impact in both fields of applied microbiology and industry and will also serve as a good example of a microbial platform for energy-requiring bioprocesses.