Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate

Anda, R.; Lara, Alvaro R.; Hernández, Vanessa; Hernández-Montalvo, Verónica; Gosset, Guillermo; Bolívar, Francisco; Ramı́rez, Octavio T.

doi:10.1016/j.ymben.2006.01.002

Cited by 120 publications

(99 citation statements)

References 41 publications

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“…Transcriptome analysis in these fast-growing strains indicated that the expressions of gene galP (coding for galactose permease), glk (coding for glucose kinase), and pgi (coding for phosphoglucose isomerase) were increased as compared to the wild-type strain, suggesting that these genes were important in enhancing glucose uptake Flores et al 2002). Hence, it was reported that the overexpression of galP and glk enhanced the growth rate of microbes in glucose medium Lu et al 2012) and improved the yields of valuable products such as aromatics (Yi et al 2003), recombinant protein (De Anda et al 2006), and ethanol (Hernandez-Montalvo et al 2003). Combinatorial modulation of galP and glk gene expressions has been used to improve cell growth rate (Lu et al 2012), but it is unclear if such an approach will increase the production of isoprenoids.…”

Section: Introductionmentioning

confidence: 99%

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Zhang

Zou

Chen

et al. 2015

Appl Microbiol Biotechnol

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Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways.These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.

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Section: Introductionmentioning

confidence: 99%

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Zhang

Zou

Chen

et al. 2015

Appl Microbiol Biotechnol

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“…These mutants have a reduced growth rate in glucose minimal medium and exhibit a strongly reduced rate of acetate formation (45) even in complex media, where the growth rate is unaffected (4). Lower acetate production is an advantage for industrial applications, and hence PTS mutants, as well as strains with additional manipulations, such as the upregulation of the mgl operon, galP (3,7,24,33), and/or the glucokinase (24), have already been analyzed. Nevertheless, a systematic approach to studying the impact of all systems capable of translocating glucose has not been attempted.…”

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confidence: 99%

Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems

Steinsiek

Bettenbrock

2012

J Bacteriol

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In Escherichia coli, several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit have not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose-limited continuous cultivations. Deletion of EIIBC disturbs glucose transport severely in batch cultures; cyclic AMP (cAMP)-cAMP receptor protein (CRP) levels rise, and induction of the mgl operon occurs. Nevertheless, Mgl activity is not essential for growth of these mutants, since deletion of this transporter did not affect the growth rate; the activities of the remaining transporters seem to be sufficient. Under conditions of glucose limitation, mgl is upregulated 23-fold compared to levels for growth under glucose excess. Despite the strong induction of mgl upon glucose limitation, deletion of this transport system did not lead to further changes. Although the galactose transporters are often regarded as important for glucose uptake at micromolar concentrations, the glucose as well as mannose PTS might be sufficient for growth at this relatively low dilution rate.T he transport of carbon sources, especially glucose, is an important field of research that has been previously investigated in Escherichia coli. The main uptake system under conditions of glucose excess is the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). It plays an important role not only in the transport and phosphorylation of carbon sources but also in the regulation of carbon metabolism and chemotaxis (for an overview, see reviews in references 11a, 47a, 50, and 50a). Glucose transport into the cytoplasm by EIICB Glc (encoded by ptsG) is coupled to its phosphorylation. The phosphate group is derived from phosphoenolpyruvate (PEP) and is transferred via a cascade of proteins, enzyme I (EI), HPr, EIIA, and EIIB. EI and HPr are general PTS proteins, while the EII proteins are carbohydrate specific. Like the glucose PTS, the mannose PTS (encoded by manXYZ) has been shown to play a role in transportation and phosphorylation of glucose under aerobic conditions with glucose as the sole carbon source (26), although PtsM has a lower affinity for glucose than PtsG (K m PtsM , 1.3 mM [9]; K m PtsG , 10 to 20 M [26]). Besides the phosphotransferase systems, the galactose transport systems, the galact...

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“…By deleting one of the phosphotransferase system genes, e.g. ptsG, ptsH or ptsI, the uptake through the major glucose transporter is several impeded, resulting in a reduced glycolytic flux and reduced acetate pathway (Chou et al, 1994;Siguenza et al, 1999;De Anda et al, 2006;Wong et al, 2008). To restore the strong reduction in growth rate as consequence of hampering the main glucose transporter De Anda et al (2006) overexpressed the alternative glucose transporter gene galP (coding for a galactose permease) and exploited the native glucose kinase (Glk) transporter.…”

Section: An Alternative Approach To Reduce Acetate Production and Impmentioning

confidence: 99%

Increasing Recombinant Protein Production in E. coli by an Alternative Method to Reduce Acetate

Waegeman¹,

Mey²

2012

Advances in Applied Biotechnology

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Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate

Cited by 120 publications

References 41 publications

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems

Increasing Recombinant Protein Production in E. coli by an Alternative Method to Reduce Acetate

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