Evaluation of the production of alginate-like exopolysaccharides (ALE) and tryptophan in aerobic granular sludge systems.

The engineering and microbiological aspects involved in the production of alginate-like exopolysaccharides (ALE) and tryptophan (TRY) in aerobic granular sludge systems were evaluated. The inclusion of short anoxic phase (A/O/A cycle-anaerobic, oxic, and anoxic phase) and the control of sludge retention time (SRT ≈ 10 days) proved to be an important strategy to increase the content of these bioproducts in granules. The substrate concentration also has a relevant impact on the production of ALE and TRY. The results of the microbiological analysis showed that slow-growing heterotrophic microbial groups (i.e., PAOs and GAOs) might be associated with the production of ALE, and the EPS-producing fermentative bacteria might be associated with the TRY production. The preliminary economic evaluation indicated the potential of ALE recovery in AGS systems in decreasing the OPEX (operational expenditure) of the treatment, especially for larger sewage treatment plants or industrial wastewaters with a high organic load.

Removing the by-products acetic acid and NH4 + from the L-tryptophan broth by vacuum thin film evaporation during L-tryptophan production

Background: During L-tryptophan production by Escherichia coli, the by-products, acetic acid and NH4 +, accumulate in the fermentation broth, resulting in inhibited cell growth and activity and decreased L-tryptophan production. To improve the L-tryptophan yield and glucose conversion rate, acetic acid and NH4 + were removed under low-temperature vacuum conditions by vacuum scraper concentrator evaporation; the fermentation broth after evaporation was pressed into another fermenter to continue fermentation. To increase the volatilisation rate of acetic acid and NH4 + and reduce damage to bacteria during evaporation, different vacuum evaporation conditions were studied. Results: The optimum operating conditions were as follows: vacuum degree, 720 mm Hg; concentration ratio, 10%; temperature, 60°C; and feeding rate, 300 mL/min. The biomass yield of the control fermentation (CF) and fermentation by vacuum evaporation (VEF) broths was 55.1 g/L and 58.3 g/L at 38 h, respectively, (an increase of 5.8%); the living biomass yield increased from 8.9 (CF) to 10.2 pF (VEF; an increase of 14.6%). L-tryptophan production increased from 50.2 g/L (CF) to 60.2 g/L (VEF) (an increase of 19.9%), and glucose conversion increased from 18.2% (CF) to 19.5% (VEF; an increase of 7.1%). The acetic acid concentrations were 2.74 g/L and 6.70 g/L, and the NH4 + concentrations were 85.3 mmol/L and 130.9 mmol/L in VEF and CF broths, respectively. Conclusions: The acetic acid and NH4 + in the fermentation broth were quickly removed using the vacuum scraper concentrator, which reduced bacterial inhibition, enhanced bacterial activity, and improved the production of L-tryptophan and glucose conversion rate.

Anthranilate synthase subunit organization in Chromobacterium violaceum.

Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

Evolution of bacterial trp operons and their regulation.

Survival and replication of most bacteria require the ability to synthesize the amino acid L-tryptophan whenever it is not available from the environment. In this article we describe the genes, operons, proteins, and reactions involved in tryptophan biosynthesis in bacteria, and the mechanisms they use in regulating tryptophan formation. We show that although the reactions of tryptophan biosynthesis are essentially identical, gene organization varies among species--from whole-pathway operons to completely dispersed genes. We also show that the regulatory mechanisms used for these genes vary greatly. We address the question--what are some potential advantages of the gene organization and regulation variation associated with this conserved, important pathway?

New insights into regulation of the tryptophan biosynthetic operon in Gram-positive bacteria.

The tryptophan operon of Bacillus subtilis serves as an excellent model for investigating transcription regulation in Gram-positive bacteria. In this article, we extend this knowledge by analyzing the predicted regulatory regions in the trp operons of other fully sequenced Gram-positive bacteria. Interestingly, it appears that in eight of the organisms examined, transcription of the trp operon appears to be regulated by tandem T-box elements. These regulatory elements have recently been described in the trp operons of two bacterial species. Single T-box elements are commonly found in Gram-positive bacteria in operons encoding aminoacyl tRNA synthetases and proteins performing other functions. Different regulatory mechanisms appear to be associated with variations of trp gene organization within the trp operon.

Genetic analysis of violacein biosynthesis by Chromobacterium violaceum.

Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule.

Genetic analysis of violacein biosynthesis by Chromobacterium violaceum

Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule

Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator.

The nucleotide sequence of a 2-4 kb BamHI-SalI fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.