Palythine-threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata.

Using a high-resolution reverse-phase liquid chromatography method we found that the tissues of the hermatypic coral Pocillopora capitata (collected in Santiago Bay, Mexico) contain a high diversity of primary and secondary mycosporine-like amino acids (MAAs) typical of some reef-building coral species: mycosporine-glycine, shinorine, porphyra-334, mycosporine-methylamine-serine, mycosporine-methylamine-threonine, palythine-serine, palythine and one additional novel predominant MAA, with an absorbance maximum of 320 nm. Here we document the isolation and characterization of this novel MAA from the coral P. capitata. Using low multi-stage mass analyses of deuterated and non deuterated compounds, high-resolution mass analyses (Time of Flight, TOF) and other techniques, this novel compound was characterized as palythine-threonine. Palythine-threonine was also present in high concentrations in the corals Pocillopora eydouxi and Stylophora pistillata indicating a wider distribution of this MAA among reef-building corals. From structural considerations we suggest that palythine-threonine is formed by decarboxylation of porphyra-334 followed by demethylation of mycosporine-methylamine-threonine.

Are high-lysine cereal crops still a challenge?

The essential amino acids lysine and threonine are synthesized in higher plants via a pathway starting with aspartate that also leads to the formation of methionine and isoleucine. Lysine is one of most limiting amino acids in plants consumed by humans and livestock. Recent genetic, molecular, and biochemical evidence suggests that lysine synthesis and catabolism are regulated by complex mechanisms. Early kinetic studies utilizing mutants and transgenic plants that over-accumulate lysine have indicated that the major step for the regulation of lysine biosynthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, recent strong evidence indicates that lysine catabolism is also subject to control, particularly in cereal seeds. The challenge of producing crops with a high-lysine concentration in the seeds appeared to be in sight a few years ago. However, apart from the quality protein maize lines currently commercially available, the release of high-lysine crops has not yet occurred. We are left with the question, is the production of high-lysine crops still a challenge?

Are high-lysine cereal crops still a challenge?

The essential amino acids lysine and threonine are synthesized in higher plants via a pathway starting with aspartate that also leads to the formation of methionine and isoleucine. Lysine is one of most limiting amino acids in plants consumed by humans and livestock. Recent genetic, molecular, and biochemical evidence suggests that lysine synthesis and catabolism are regulated by complex mechanisms. Early kinetic studies utilizing mutants and transgenic plants that over-accumulate lysine have indicated that the major step for the regulation of lysine biosynthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, recent strong evidence indicates that lysine catabolism is also subject to control, particularly in cereal seeds. The challenge of producing crops with a high-lysine concentration in the seeds appeared to be in sight a few years ago. However, apart from the quality protein maize lines currently commercially available, the release of high-lysine crops has not yet occurred. We are left with the question, is the production of high-lysine crops still a challenge?.

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production.

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987). Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability. A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of AprTcr clones after cultivation in non selective media). We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.

[Selection of bacterial strains for the production of threonine]. / Selección de cepas bacterianas para la producción de treonina.

The production of L-threonine in submerged culture was studied in the following bacterial strains; Brevibacterium flavum ATCC 21269, Corynebacterium acetoacidophilum ATCC 21270, Escherichia coli ATCC 21149, E. coli NRRL 12098, E. coli NRRL 12099 and E. coli NRRL 12100. Erlenmeyer flasks with different volumetric relations liquid/recipient, were used to study the influence of the volumetric oxygen transfer rate. B. flavum reached levels of threonine of 0.72 g/l at 96 hours of culture with a volumetric relation liquid/recipient of 1:5. With a relation 1:25 the maximal level was reached at 48 hours (0.60 g/l of threonine) (Table 1). In addition to threonine this strain accumulated in the culture media glutamic acid (+/- 2 g/l), alanine or glycine and proline. With E. coli ATCC 21149 the aeration favored the production of threonine reaching levels of 0.38 g/l in six day cultures with valine and alanine at levels approximate to 2 g/l. Excepting C. acetoacidophilum, all the strains produced threonine at levels of 0.30 to 3.55 g/l (Table 5). E. coli NRRL 12098 and E. coli NRRL 12100 produced only threonine, the culture medium being free from other aminoacids. With E. coli NRRL 12098 levels of 2 g/l were attained but the production was restricted to the presence of yeast extract in the media (Table 2). E. coli NRRL 12100 was the best strain and the inoculum media influenced the production (Table 3) and the best threonine levels were reached in the best aeration conditions assayed (Table 4) with 3.55 g/l for volumetric relations 1:10 and 1.25 g/l for 1:5.

Localized hydroxylamine mutagenesis, and cotransduction of threonine and lysine genes, in Streptomyces venezuelae.

A lysate of the generalized transducing phage SV1, grown on the prototrophic type strain 10712 of Streptomyces venezuelae, was mutagenized with hydroxylamine and used to transduce a lysineless auxotroph to lysine independence on supplemented minimal agar. A complex threonine mutant, strain VS95, was isolated from among the transductants and was shown to be carrying at least two different thr mutations. These were about 50% cotransducible with alleles of four independently isolated lysA mutations, as were two other independently isolated threonine mutations, thr-1 and hom-5. The location of thr genes close to lysA occurs in at least three other streptomycetes, but apparently not in Streptomyces coelicolor A3(2), in which the lysA and thr loci are at diametrically opposite locations on the linkage map. This first observation of cotransduction between loci governing the biosynthesis of different amino acids in the genus Streptomyces demonstrates the feasibility of fine-structure genetic analysis by transduction in these antibiotic-producing bacteria.