Heterologous expression of Escherichia coli ppsA (phosphoenolpyruvate synthetase) and galU (UDP-glucose pyrophosphorylase) genes in Corynebacterium glutamicum, and its impact on trehalose synthesis

ABSTRACT Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.

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Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.70.1.370-376.2004 ◽

2004 ◽

Vol 70 (1) ◽

pp. 370-376 ◽

Cited By ~ 39

Author(s):

Leandro Padilla ◽

Reinhard Krämer ◽

Gregory Stephanopoulos ◽

Eduardo Agosin

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Corynebacterium Glutamicum ◽

Metabolic Flux ◽

Krebs Cycle ◽

Exponential Growth Phase ◽

Fourfold Increase ◽

Potential Applications ◽

Defined Culture ◽

Phosphate Synthase

ABSTRACT Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.

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Heterologous expression and secretion of an antifungal Bacillus subtilis chitosanase (CSNV26) in Escherichia coli

Bioprocess and Biosystems Engineering ◽

10.1007/s00449-012-0834-x ◽

2012 ◽

Vol 36 (7) ◽

pp. 985-992 ◽

Cited By ~ 8

Author(s):

Olfa Kilani-Feki ◽

Fakher Frikha ◽

Imen Zouari ◽

Samir Jaoua

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Heterologous Expression

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Heterologous expression of maize chloroplast protein synthesis elongation factor (EF-Tu) enhances Escherichia coli viability under heat stress

Plant Science ◽

10.1016/s0168-9452(02)00273-x ◽

2002 ◽

Vol 163 (6) ◽

pp. 1075-1082 ◽

Cited By ~ 22

Author(s):

T Moriarty ◽

R West ◽

G Small ◽

D Rao ◽

Z Ristic

Keyword(s):

Escherichia Coli ◽

Protein Synthesis ◽

Heat Stress ◽

Heterologous Expression ◽

Elongation Factor ◽

Chloroplast Protein ◽

Maize Chloroplast ◽

Chloroplast Protein Synthesis

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Heterologous expression of mammalian cytochromes P450 as active forms in Escherichia coli,

Journal of Bioscience and Bioengineering ◽

10.1016/s1389-1723(00)90025-2 ◽

2000 ◽

Vol 89 (3) ◽

pp. 294

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Cytochromes P450

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Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

Applied and Environmental Microbiology ◽

10.1128/aem.01904-12 ◽

2012 ◽

Vol 78 (20) ◽

pp. 7407-7413 ◽

Cited By ~ 37

Author(s):

Qian Zhang ◽

Tao Yan

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Potassium Acetate ◽

Desiccation Stress ◽

Desiccation Resistance ◽

Content Type ◽

Soil Desiccation ◽

E Coli ◽

Organic Solute ◽

Trehalose Synthesis

ABSTRACTNaturalized soilEscherichia colipopulations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soilE. colistrains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day−1) than that of MG1655 (0.85 day−1). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among theE. colistrains. AllE. colistrains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman's ρ = −1.0;P= 0.02).De novotrehalose synthesis was further determined for 15E. colistrains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. MostE. colistrains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soilE. colistrains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).

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