RESUMEN
L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.
Asunto(s)
Escherichia coli/genética , Ingeniería Genética/métodos , Glucosa/metabolismo , Serina/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , L-Serina Deshidratasa/metabolismo , Mutagénesis Sitio-Dirigida , Fosfoglicerato-Deshidrogenasa/metabolismoRESUMEN
Extracellular serine protease SFP2 from Streptomyces fradiae var. k11 with high feather-degrading activity was purified. The partial amino acid sequences of internal peptide of purified SFP2 were determined, and the partial gene encoding SFP2 was cloned by PCR using the degenerate primers designed according to the amino acid sequences. Complete sfp2 gene was cloned by screening the genomic DNA library of Streptomyces fradiae var. k11. The Open Reading Frame of sfp2 including pre- pro-enzyme is 924bp long (EMBL Accession number: AJ784940). The signal peptide sequence is as long as 114bp, the precursor sequence is 810bp and the mature enzyme is 576bp long, encoding 191 amino acid resides with the putative molecular weight of 19.112kD. In E. coli and Bacillus subtilis, the two sequences encoding SFP2 pro-enzyme and mature enzyme were both expressed successfully. The pro-enzyme expressed had normal biological function and its mature product had normal enzymatic activity.