Metabolic engineering of Escherichia coli for the production of l -valine based on transcriptome analysis and in silico gene knockout simulation

Abstract

The L-valine production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on transcriptome analysis and gene knockout simulation of the in silico genome-scale metabolic network. Feedback inhibition of acetohydroxy acid synthase isoenzyme III by L-valine was removed by site-directed mutagenesis, and the native promoter containing the transcriptional attenuator leader regions of the ilvGMEDA and ilvBN operon was replaced with the tac promoter. The ilvA, leuA, and panB genes were deleted to make more precursors available for L-valine biosynthesis. This engineered Val strain harboring a plasmid overexpressing the ilvBN genes produced 1.31 g/liter L-valine. Comparative transcriptome profiling was performed during batch fermentation of the engineered and control strains. Among the down-regulated genes, the lrp and ygaZH genes, which encode a global regulator Lrp and L-valine exporter, respectively, were overexpressed. Amplification of the lrp, ygaZH, and lrp-ygaZH genes led to the enhanced production of L-valine by 21.6%, 47.1%, and 113%, respectively. Further improvement was achieved by using in silico gene knockout simulation, which identified the aceF, mdh, and pfkA genes as knockout targets. The VAMF strain (Val ⌬aceF ⌬mdh ⌬pfkA) overexpressing the ilvBN, ilvCED, ygaZH, and lrp genes was able to produce 7.55 g/liter L-valine from 20 g/liter glucose in batch culture, resulting in a high yield of 0.378 g of L-valine per gram of glucose. These results suggest that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification, transcriptome profiling, and systems-level in silico analysis.systems biology ͉ global regulator ͉ exporter ͉ in silico prediction M ost amino acid-producing bacterial strains have been constructed by random mutagenesis. A significant disadvantage of this approach is the possibility that the random distribution of mutations in regions not directly related to amino acid biosynthesis can cause unwanted changes in physiology and growth retardation. Rational metabolic engineering by specific targeted modifications can overcome this disadvantage. The recent development of omics technology, combined with computational analysis, now provides a new avenue for strain improvement (1-4) by providing new information extracted from a large number of data, which is termed ''systems biotechnology'' (5).L-valine, an essential hydrophobic and branched-chain amino acid, is used as a component of cosmetics and pharmaceuticals as well as animal feed additives. L-valine has been produced by employing bacteria belonging to the genera Brevibacterium, Corynebacterium, and Serratia, which have been improved by random mutation and selection (6, 7). A recent report describes the production of L-valine by rationally constructed Corynebacterium glutamicum, in which a feedback inhibition-resistant small subunit of acetohydroxy acid synthase (AHAS; encoded by ilvN) was generated by site-directed mutagen...

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

“…At the same time, endogenous pathways can be shut down or reduced [80] to optimize the balance between heterologous and endogenous biochemical activities [79]. Side effects or roadblocks encountered can be ameliorated or removed by multiple rounds of engineering [79,81,82].…”

The growing scope of applications of genome-scale metabolic reconstructions using Escherichia coli

“…Applications of the E. coli GEM range from pragmatic to theoretical studies, and can be classified into five general categories ( Fig. 3): 1) metabolic engineering [20][21][22][23][24][25][26][27][28][29][30] ; 2) biological discovery [31][32][33][34][35][36][37] ; 3) assessment of phenotypic behavior 19, ; 4) biological network analysis [64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79] ; and 5) studies of bacterial evolution [80][81][82] . The in silico methods used to probe the E. coli GEM in each study are summarized in Fig.…”

“…Through the application of computational methods that incorporate linear, mixed integer linear, and non-linear programming, it has been demonstrated that model-directed strain design can lead to increased metabolite production [20][21][22][23][24][25][26][27][28][29][30] . In these studies, the E. coli GEM is principally used to analyze the metabolite production potential of E. coli and identify metabolic interventions needed to enable the production of the product of interest.…”

“…In these studies, the E. coli GEM is principally used to analyze the metabolite production potential of E. coli and identify metabolic interventions needed to enable the production of the product of interest. Thus, E. coli strains have been systematically designed through in silico analysis to overproduce target metabolites such as lycopene 23,24 , lactic acid 25 , ethanol 26 , succinic acid 27,28 , Lvaline 29 , L-threonine 30 , additional amino acids 21 , as well as diverse products from hydrogen to vanillin 22 . Select exemplary metabolic engineering applications will be described in more detail.…”

“…Two studies producing the amino acids L-valine 29 and L-threonine 30 have demonstrated the broad usage of GEM aided computation for strain design. In the first study, GEM aided modeling was employed in three different areas to increase the production of L-threonine to industrial titers 30 .…”