Rational design of a new variant of Reteplase with optimized physicochemical profile and large-scale production in Escherichia coli

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

Download Full-text

Efficient Production of l-Ribose with a Recombinant Escherichia coli Biocatalyst

Applied and Environmental Microbiology ◽

10.1128/aem.02768-07 ◽

2008 ◽

Vol 74 (10) ◽

pp. 2967-2975 ◽

Cited By ~ 30

Author(s):

Ryan D. Woodyer ◽

Nathan J. Wymer ◽

F. Michael Racine ◽

Shama N. Khan ◽

Badal C. Saha

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Active Form ◽

Apium Graveolens ◽

Efficient Production ◽

Scale Production ◽

Gene Encoding ◽

Large Scale Production ◽

One Step ◽

Rare Sugars

ABSTRACT A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.

Download Full-text

Large scale production of biologically active Escherichia coli glutamyl-tRNA reductase from inclusion bodies

Protein Expression and Purification ◽

10.1016/s1046-5928(03)00184-0 ◽

2003 ◽

Vol 31 (2) ◽

pp. 271-275 ◽

Cited By ~ 17

Author(s):

Stefan Schauer ◽

Corinna Lüer ◽

Jürgen Moser

Keyword(s):

Escherichia Coli ◽

Inclusion Bodies ◽

Large Scale ◽

Biologically Active ◽

Scale Production ◽

Large Scale Production

Download Full-text

Glycosyltransferase engineering for carbohydrate synthesis

Biochemical Society Transactions ◽

10.1042/bst20150200 ◽

2016 ◽

Vol 44 (1) ◽

pp. 129-142 ◽

Cited By ~ 30

Author(s):

John B. McArthur ◽

Xi Chen

Keyword(s):

Directed Evolution ◽

Rational Design ◽

Large Scale ◽

Enzyme Engineering ◽

Lessons Learned ◽

Scale Production ◽

Amino Acid Residues ◽

Large Scale Production ◽

Donor And Acceptor ◽

Substrate Binding Sites

Glycosyltransferases (GTs) are powerful tools for the synthesis of complex and biologically-important carbohydrates. Wild-type GTs may not have all the properties and functions that are desired for large-scale production of carbohydrates that exist in nature and those with non-natural modifications. With the increasing availability of crystal structures of GTs, especially those in the presence of donor and acceptor analogues, crystal structure-guided rational design has been quite successful in obtaining mutants with desired functionalities. With current limited understanding of the structure–activity relationship of GTs, directed evolution continues to be a useful approach for generating additional mutants with functionality that can be screened for in a high-throughput format. Mutating the amino acid residues constituting or close to the substrate-binding sites of GTs by structure-guided directed evolution (SGDE) further explores the biotechnological potential of GTs that can only be realized through enzyme engineering. This mini-review discusses the progress made towards GT engineering and the lessons learned for future engineering efforts and assay development.

Download Full-text