A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.
Metab Eng
; 25: 30-7, 2014 Sep.
Article
en En
| MEDLINE
| ID: mdl-24953302
ABSTRACT
Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Transducción de Señal
/
Gliceraldehído 3-Fosfato Deshidrogenasa (NADP/)
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Mejoramiento Genético
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Corynebacterium glutamicum
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Ingeniería Metabólica
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Lisina
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NADP
Idioma:
En
Revista:
Metab Eng
Asunto de la revista:
ENGENHARIA BIOMEDICA
/
METABOLISMO
Año:
2014
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
BE
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BELGICA
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BELGIQUE
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BELGIUM
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BÉLGICA