Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.

Comparison of two bacterial azoreductases acquired during adaptation to growth on azo dyes.

Selection for utilization of carboxy-Orange I [1-(4'-carboxyphenylazo)-4-naphthol] in the chemostat yielded Pseudomonas strain K24 which was unable to grow on carboxy-Orange II [1-(4'-carboxyphenylazo)-2-naphthol] while selection for growth on carboxy-Orange II had previously led to strain KF46 which did not utilize carboxy-Orange I. Orange I azoreductase of strain K24, the key enzyme of dye degradation, was purified 80-fold with 17% yield to electrophoretic homogeneity and compared to the previously purified Orange II azoreductase of strain KF46. Common properties of the two enzymes were their monomeric structure, their specificity for NADPH and NADH as cosubstrates, the range of their Km values for substrates and cosubstrates as well as their reactivity towards a series of substrate analogs. They differed from each other with respect to molecular weight (21,000 and 30,000) and in the absolute requirement of Orange I azoreductase for a hydroxy group in the 4'position of the naphthol ring of the substrate molecule as compared to the requirement for substrates with a 2-naphthol moiety by Orange II azoreductase. The pure enzymes did not exhibit immunological cross-reaction with each other. Crude extracts of strains K24 and KF46 and of azoreductase-negative strains isolated at different stages of the adaptation experiments, however, contained material which cross-reacted (CRM) with both anti Orange I azoreductase serum and anti Orange II azoreductase serum. The CRM may represent a common precursor protein of the azoreductases in strains K24 and KF46.

Regulatory citrate lyase mutants of Salmonella typhimurium.

Citrate lyase, the key enzyme of anaerobic citrate catabolism, could not be deleted from Salmonella typhimurium. The only class of mutants found had a mode of covalent regulation that strongly resembled the Escherichia coli system: citrate lyase was only active, i.e., acetylated, when a cosubstrate was present.

Properties of purified Orange II azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46.

Orange II azoreductase [NAD(P)H: 1-(4'-sulfophenylazo)-2-naphthol oxidoreductase], an enzyme catalyzing the reductive cleavage of the azo bridge of Orange II and related dyes, was purified to electrophoretic homogeneity from Pseudomonas species, strain KF46. This organism utilized carboxy-Orange II [1-(4'-carboxyphenylazo)-2-naphthol] but not Orange II as the sole source of carbon, energy, and nitrogen. Orange II azoreductase was induced 80-fold by both Orange II and carboxy-Orange II. With two successive runs of affinity chromatography using two chromatographic media with different triazinyl dyes as ligands, the enzyme was purified 120-fold with 43% yield. The purified enzyme is a monomer with a molecular weight of 30,000. Its Km values were 1.5 microM for both Orange II and carboxy-Orange II, 5 microM for NADPH, and 180 microM for NADH. A survey of the efficiency of various Orange dyes as substrates for Orange II azoreductase showed that: (a) a hydroxy group in the 2-position of the naphthol ring is required; (b) charged groups in proximity to the azo group hinder the reaction; (c) a second polar substituent on the dye molecule impedes the reaction; (d) electron-withdrawing groups on the phenyl ring accelerate the reaction.

Energy-dependent inactivation of citrate lyase in Enterobacter aerogenes.

Enterobacter aerogenes was grown in continous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation.

H2-dependent anaerobic growth of Escherichia coli on L-malate: succinate formation.

Escherichia coli grew anaerobically on L-malate only in the presence of H2; 91% of the L-malate utilized was converted to succinate. Anaerobically isolated membrane vesicles catalyzed the reduction of fumarate with H2 and contained a b-type cytochrome. Cytochrome c552 was present in the "periplasmic space."