Genetic manipulation of a primary metabolic pathway for l-ornithine production in Escherichia coli

Abstract

Metabolic engineering has been used to improve L-ornithine biosynthesis in Escherichia coli W3110. L-Ornithine production increased from 0.3 to 3.2 mg/g (dry cell weight) when the primary L-ornithine biosynthetic pathway was optimized by disrupting the pathway transcription repressor, thereby increasing the expression of the genes involved in the pathway, and by preventing conversion of L-ornithine into citrulline. When a feedback-resistant N-acetylglutamate synthetase gene (argA214) was placed under the control of the arabinose-inducible promoter, either in the chromosome or on a multicopy plasmid in the cell, the combination of overexpression of argA214 with an argF argI argR triple knockout mutation had an additive effect on L-ornithine production but only when exogenous glutamate was present. When speF (which encodes ornithine decarboxylase) and proB (which encodes gamma-glutamyl kinase) were inactivated to prevent the conversion of L-ornithine to putrescine and to block the biosynthesis of a side branch of L-ornithine, respectively, L-ornithine production was further enhanced approx. 140% from 5.5 to 13.2 mg/g (dry cell weight).

Optimization of Fermentation Conditions of the Engineered Corynebacterium Glutamicum to Enhance L-Ornithine Production by Response Surface Methodology

“…It may also be used to enhance the flux to L-ornithine. Only three papers reported the results of enhance of L-ornithine production by metabolic engineering so far [10][11][12]. Lee and Cho [10] reported that an engineered E. coli W3110 ( argFargIargRproBspeFP araB -arg214) produced L-ornithine of 13.2 mg/gDCW.…”

“…Only three papers reported the results of enhance of L-ornithine production by metabolic engineering so far [10][11][12]. Lee and Cho [10] reported that an engineered E. coli W3110 ( argFargIargRproBspeFP araB -arg214) produced L-ornithine of 13.2 mg/gDCW. Hwang et al [11] also reported that cooverexpression in a triple gene knockout Corynebacterium glutamicum (argFargR proB) resulted in L-ornithine production of 16.49 mg/g DCW, L-ornithine concentration of 179.14 mg/L.…”

Interaction of Transcriptional Repressor ArgR with Transcriptional Regulator FarR at the argB Promoter Region in Corynebacterium glutamicum

“…Table 1 lists the bacterial strains and plasmids used in this study. Wild-type strain C. glutamicum ATCC 13032 (American Type Culture Collection, Manassas, VA) and mutant strain C. glutamicum SJC 8074 (provided by Sangji University, South Korea) (10) were grown at 30°C in Luria-Bertani medium (29) and, for the production of ornithine and arginine, in mineral medium containing yeast extract (MMY) ) (17). In a 250-ml shake flask, a 1-ml preculture was inoculated into 50 ml of MMY medium.…”

Production of L-ornithine from sucrose and molasses by recombinant Corynebacterium glutamicum

“…Many papers have reported that L-ornithine can be produced from glucose by a citrulline-or arginine-requiring mutant of Brevibacterium ketoglutamicum (Choi et al 1996;Lee et al 2000), Corynebacterium glutamicum (Zhang et al 2009), or engineered C. glutamicum (Lee and Cho 2006;Hwang et al 2008;Hwang and Cho 2010, 2014Lee et al 2010b;Lu et al 2011Lu et al , 2012Jiang et al 2013a, b). However, glucose is commonly obtained from starch hydrolysis by a high-temperature saccharification process combined with α-amylase and glucoamylase (Khaw et al 2007).…”