Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin.

Brazzein is a 54-amino-acid sweet-tasting protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. Brazzein, as isolated from the fruit, is 500 times sweeter than sucrose on a weight basis (9500 times sweeter on a per-molecule basis). A minor component of brazzein from fruit, des-pGlu1-brazzein, has 53 amino acid residues and has twice the sweetness of the parent protein. We have designed a gene for des-pGlu1- brazzein that incorporates codons that are optimal for protein production in Escherichia coli. Production of brazzein from the chemically synthesized gene resulted in recombinant protein with sweetness similar to that of brazzein isolated from the original source. The best yields were achieved by producing brazzein as a fusion with staphylococcal nuclease with a designed cyanogen bromide cleavage site. Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. This efficient protein production system for brazzein will facilitate such investigations.

Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein.

Brazzein, originally isolated from the fruit of the African plant Pentadiplandra brazzeana Baillon, is the smallest, most heat-stable and pH-stable member of the set of proteins known to have intrinsic sweetness. These properties make brazzein an ideal system for investigating the chemical and structural requirements of a sweet-tasting protein. We have used the three-dimensional structure of the protein (J. E. Caldwell et al. (1998) Nat. Struct. Biol. 5, 427-431) as a guide in designing 15 synthetic genes in expression constructs aimed at delineating the sweetness determinants of brazzein. Protein was produced heterologously in Escherichia coli, isolated, and purified as described in the companion paper (Assadi-Porter, F. M., Aceti, D., Cheng, H., and Markley, J. L., this issue). Analysis by one-dimensional (1)H NMR spectroscopy indicated that all but one of these variants had folded properly under the conditions used. A taste panel compared the gustatory properties of solutions of these proteins to those of sucrose and brazzein isolated from fruit. Of the 14 mutations in the des-pGlu1-brazzein background, four exhibited almost no sweetness, six had significantly reduced sweetness, two had taste properties equivalent to des-pGlu1-brazzein (two times as sweet as the major form of brazzein isolated from fruit which contains pGlu1), and two were about twice as sweet as des-pGlu1-brazzein. Overall, the results suggest that two regions of the protein are critical for the sweetness of brazzein: a region that includes the N- and C-termini of the protein, which are located close to one another, and a region that includes the flexible loop around Arg43.

Transcriptional regulation of Streptomyces coelicolor pathway-specific antibiotic regulators by the absA and absB loci.

The four antibiotics produced by Streptomyces coelicolor are all affected by mutations in the absA and absB loci. The absA locus encodes a putative two-component signal transduction system, and the absB locus encodes a homolog of Escherichia coli RNase III. We assessed whether these loci control synthesis of the antibiotics actinorhodin and undecylprodigiosin by regulating transcript abundance from the biosynthetic and regulatory genes specific for each antibiotic. Strains that were Abs- (for antibiotic synthesis deficient) due to mutations in absA or absB were examined. In the Abs- absA mutant strain, transcripts for the actinorhodin biosynthetic genes actVI-ORF1 and actI, and for the pathway-specific regulatory gene actII-ORF4, were substantially lower in abundance than in the parent strain. The level of the transcript for the undecylprodigiosin pathway-specific regulatory gene redD was similarly reduced in this mutant. Additionally, a strain that exhibits precocious hyperproduction of antibiotics (Pha phenotype) due to disruption of the absA locus contained elevated levels of the actVI-ORF1, actII-ORF4, and redD transcripts. In the absB mutant strain, actVI-ORF1, actI, actII-ORF4, and redD transcript levels were also substantially lower than in the parent strain. These results establish that the abs genes affect production of antibiotics through regulation of expression of the antibiotic-specific regulatory genes in S. coelicolor.

Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis.

Acetate kinase was purified 102-fold to a specific activity of 656 mumol of ADP formed/min/mg of protein from acetate-grown Methanosarcina thermophila. The enzyme was not intrinsically membrane bound. The native enzyme (Mr 94,000) was an alpha 2 homodimer with a subunit Mr of 53,000. The activity was optimum between pH 7.0 and 7.4. A pI of 4.7 was determined. The enzyme was stable to O2 and stable to heating at 70 degrees C for 15 min but was rapidly inactivated at higher temperatures. The apparent Km for acetate was 22 mM and for ATP was 2.8 mM. The enzyme phosphorylated propionate at 60% of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. The enzyme required one of several divalent cations for activity; the maximum rate was obtained with Mn2+. Western blots of cell extract proteins showed that acetate grown cells synthesized higher quantities of the acetate kinase than did methanol grown cells.

EPR properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide dehydrogenase activity from acetate-grown Methanosarcina thermophila. Evidence that acetyl-CoA is a physiological substrate.

The carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila was further studied by EPR spectroscopy. The as purified enzyme exhibited no paramagnetic species at 113 K; however, enzyme reduced with CO exhibited a complex EPR spectrum comprised of two paramagnetic species with g values of g1 = 2.089, g2 = 2.078, and g3 = 2.030 (signal I) and g1 = 2.057, g2 = 2.049, and g3 = 2.027 (signal II). Isotopic substitution with 61Ni, 57Fe, or 13CO resulted in broadening of the EPR spectra indicating a Ni-Fe-C spin-coupled complex. Pure signal II was obtained following treatment of the CO-reduced enzyme with acetyl-CoA but not by addition of acetyl phosphate or CoASH. Acetate-grown cells were highly enriched in acetate kinase (EC 2.7.2.1) and CoASH-dependent phosphotransacetylase (EC 2.3.1.8) activities. These results suggest acetyl-CoA is a physiological substrate for the carbon monoxide dehydrogenase complex synthesized in acetate-grown cells of M. thermophila.