Follow Mary Nash Stoddard on Twitter

Wednesday, August 11, 2010

Aspartame Made from E-Coli Cultures

Proof of e-coli as a culture medium for amino acids in APM:

Optimization of d-hydantoinase production

d-hydantoinase was produced as an inducible enzyme in most of the microbial strains reported. Hydantoinase production in Pseudomonas species, Agrobacterium species, and Peptococcus anaerobius was observed to be induced by various substrates and substrate analogues. d-hydantoinase production was maximally induced by uracil in Pseudomonasputida19, and Agrobacterium sp.33. An unmetabolizable substrate analogue, 2,4-thiouracil, enhanced hydantoinase production up to five fold in Agrobacterium sp.41. In P. fluorescens and Bacillus stearothermophilus SD1, the enzyme was produced constitutively, and substrates or substrate analogues were unable to induce the enzyme production42,43.

Although production of hydantoinase has been reported in many organisms, only few reports about the medium optimization are available. Meat and beef extract, when used as nitrogen source, enhanced d-hydantoinase production inP. putida19, and Bacillus SD1 (ref. 43). This may be due to the high pyrimidine and purine content in these complex meat-derived nitrogen sources44. Yeast extract was used as the nitrogen source in Pseudomonas sp.42, and A. radiobacter45. Glucose or glycerol was used as a carbon source in most of the cases for the production of d-hydantoinase. In a recent study46, medium optimization studies were carried out for the production of d-hydantoinase in A. radiobacter NRRL B 1229, wherein with the use of an empirical modeling technique (response surface method) Achary et al. were able to achieve 35 U/ml of enzyme activity and 1.69 mg/ml of biomass in optimized complex medium. The optimized medium contained molasses as the carbon source, and ammonium nitrate as the nitrogen source. Mass production of d-hydantoinase from batch culture of recombinant E. coli in minimal medium with glycerol as the sole carbon source has been reported by Lee et al.47. They achieved 50 g dry cell weight per litre of broth, and an enzyme yield of 38000 U/g DCW in a 50 litre fermenter. They also reported that d-hydantoinase gene, which is expressed under its own promoter, is catabolically repressed by glucose in the recombinant strain.

Recently, genes from thermophilic Bacillus sp. have been cloned by various groups43,79. A gene for nonspecific hydantoinase from B. stearothermophilus from genomic DNA which encoded a 471 amino-acid-long peptide was also cloned79. The nucleotide sequence of this gene had a homology of 91% with the sequence of d-hydantoinase from a gram-positive organism Lu1220 (ref. 80). d-hydantoinase gene from B. stearothermophilus SD1 was cloned and overexpressed inE. coli43. The cloned enzyme was expressed as 20% of the total protein in E. coli. d-hydantoinase protein sequence decoded from the gene sequence of d-hydantoinase from B. stearothermophilus SD1 showed 20–28% homology with other hydantoinases, allantoinase and dihydroorotase protein sequences. This sequence exhibited more than 89% identity with hydantoinase from other thermophiles. Among the hydantoinases from thermophiles, C-terminal regions of enzymes were completely different, implying thereby that C-terminal region plays an important role in the biochemical properties of the enzymes81. Dihydropyrimidinase gene cloned by c-DNA cloning from rat liver and human liver showed 40% similarity with the
d-hydantoinase gene sequences from various microorganisms.

43 Lee, D. C., Lee, S. G., Hong, S. P., Sung, M. H. and Kim, H. S., Ann. N. Y. Acad. Sci., 1996, 799, 401–405.
Metabolic engineering of the E. colil-phenylalanine pathway for the production of d-phenylglycine (d-Phg).

Ulrike Müller, Friso van Assema, Michele Gunsior, Sonja Orf, Susanne Kremer, Dick Schipper, Anja Wagemans, Craig A Townsend, Theo Sonke, Roel Bovenberg, Marcel Wubbolts
d-phenylglycine (d-Phg) is an important side chain building block for semi-synthetic penicillins and cephalosporins such as ampicillin and cephalexin. To produce d-Phg ultimately from glucose, metabolic engineering was applied. Starting from phenylpyruvate, which is the direct precursor of l-phenylalanine, an artificial d-Phg biosynthesis pathway was created. This three-step route is composed of the enzymes hydroxymandelate synthase (HmaS), hydroxymandelate oxidase (Hmo), and the stereoinverting hydroxyphenylglycine aminotransferase (HpgAT). Together they catalyse the conversion of phenylpyruvate via mandelate and phenylglyoxylate to d-Phg. The corresponding genes were obtained from Amycolatopsis orientalis, Streptomyces coelicolor, and Pseudomonas putida. Combined expression of these activities in E. coli strains optimized for the production of l-phenylalanine resulted in the first completely fermentative production of d-Phg.

courtesy: Aspartame Consumer Safety Network