Fermentaciones industriales

African Journal of Biotechnology Vol. 9(22), pp. 3308-3317, 31 May, 2010 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2010 Academic Journals

Full Length Research Paper

Preliminary study on metabolic regulation and control of L-valine fermentation in a newly screened L-valine producing Brevibacterium flavum strain
Liu Huanmin and Zhang Weiguo*
Key laboratory ofIndustrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122 Jiangsu Province, PR China.
Accepted 12 March, 2010

A L-valine hyper-producer Brevibacterium flavum XQ-6 (Leu , Ile , AHV , -AB , 2-TA ) was obtained, which could be resistant to high concentration of -amino-butyric acid ( -AB) and 2-thiazolealanaine (2TA). The metabolic network of XQ-6 was regulated with theaddition of amino acids, organic acids, vitamins, bases and other organic things. The carbon flux was directed to L-valine by manipulating the specific activity of -ketoglutarate dehydrogenase complex (KGDH). With a combinational regulation strategy, the highest L-valine concentration of 67.7 g/L was achieved. Key words: L-valine, Brevibacterium flavum, HPLC, metabolic analysis. INTRODUCTIONL-valine is an essential amino acid mainly used for pharmaceutical purposes and as precursor in chemical synthesis (e.g., anti-viral drugs). It has a market volume of approximately 500 tons per year with relatively high market prices (Magnus et al., 2006; Eggeling et al., 2001). In the metabolism of Brevibacterium flavum, L-valine is formed in four steps beginning with the condensation of two pyruvatemolecules to acetolactate by acetolactate synthase (Figure 1) (Leyval et al., 2003). The same enzymes catalyzing the formation of L-valine are also involved in the formation of the other branched chain amino acids, L-leucine and L-isoleucine. The direct precursor of L-valine is - ketoisovalerate, which is also used for L-leucine synthesis as well as for the formation of pantothenate (H ser et al.,2005). L-valine can be produced by chemical synthesis (D, Lvaline), fermentation and by extraction from animal raw materials. Nowadays, the way of fermentative production is getting more important due to a higher flexibility of produced amounts and the rising sensibility against risks from products based on animal origins. The advances in the amino acid fermentation industry are closely connectedwith screening or selection of suitable putative production organisms and subsequent improvement of the production strains. Our attempts at strain improvement have relied on classical mutagenesis and screening procedures, which focused not only on deleting competing pathways and eliminating feedback regulations in the biosynthetic pathways but also many other things which are important to thepractice on the large scale in the plant. Brevibacterium lactofermentum NO.487 which could be resistant to high concentration of 2-thiazolealanaine (2TA) was obtained by Tsuchida from B. lactofermentum NO.2256 by N-methyl-N’-nitro-soguanidine (NTG) treatment in 1975. And the mutant produced 31 mg/mL L-valine in the medium containing 10% of glucose (Kinoshita, 1975). Using Corynebacterium glutamicum as astarting strain for mutagenesis, a L-valine hyper-producer was obtained by Katsumada in 1993, which could produce 26 mg/ml of L-valine. And a strain which produced 39 mg/mL of Lvaline was obtained by Katsumada in 1996 (Katsumada, 1993). More recently, L-valine excretion by C. glutamicum

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*Corresponding author. E-mail: zhangwg168@126.com. Tel: +86 510 85329312. Fax:+86 510 85910799. Abbreviations: EMP, Embden-Meyerhof-Parnas pathway; HPLC, high performance liquid chromatogratography; NTG, Nmethyl-N’-nitro-soguanidine; HMP, hexose monophophate pathway; KGDH, -ketoglutarate dehydrogenase complex; AB, -amino-butyric acid; DES, diethylstilboestrol; 2-TA, 2thiazolealanaine.

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Figure 1. Biosynthesis of L-valine in Brevibacterium...