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

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.Trehalose (1-␣-glucopyranosyl-1-␣-glucopyranoside) is a nonreducing, particularly stable disaccharide formed by two glucose moieties (37). As a compatible osmolite (22) and protein stabilizer (39), trehalose shows a wide range of potential applications in biotechnology (increased stress tolerance of important crops, stability of recombinant proteins, etc.), as well as in the food industry (37).In the past, trehalose was produced by using Saccharomyces cerevisiae (32). The high cost of this system and the promising applications of the disaccharide led to the development of a new trehalose production process based on the enzymatic biotransformation of maltodextrins (25,26). The success of the enzymatic process has limited the interest in using microorganisms as alternative sources for trehalose synthesis. However, the recent development of metabolic engineering tools, allowing the rational design of microorganisms for metabolite production (27), prompted us to evaluate an alternative process for trehalose overproduction in the gram-positive bacterium Corynebacterium glutamicum (23). C. glutamicum was chosen for three major reasons: (i) it produces, and excretes, trehalose (15); (ii) it has a metabolic control architecture simpler than that of other microorganisms, maybe as a result of its comparatively small 3,500-kb genome size (10); and (iii) it is widely used in industrial biotechnological processes (10).Three pathways for trehalose synthesis have been characterized in C. glutamicum (45), similarly to that found in Mycobacterium species (7) (Fig. 1). The first is the TreS pathway, in which trehalose is formed by maltose isomerization (7, 31).The second is the TreY-TreZ pathway, le...

Enhanced Trehalose Production Improves Growth of Escherichia coli under Osmotic Stress

“…Trehalose is a nonreducing sugar in which the [1O1] glucosyl bond conceals the reactive ends of both glucose monomers (8,17,33,35). The inertness of this disaccharide is presumed to allow the accumulation of high intracellular concentrations without disturbing biochemical processes.…”

“…The trehalose biosynthetic operon (otsBA) from E. coli has been used to genetically engineer increased stress tolerance in plants (15) and in mammalian cells (18,40). This operon has recently been overexpressed in Corynebacterium glutamicum for the production of extracellular trehalose (33).…”

Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway

“…Tryptone soybean broth (TSB) medium was used for the cultivation of C. glutamicum. The culture media defined for C. glutamicum for shake flask experiments (DMCG I) and for batch bioreactor cultivation experiments (DMCG II) were prepared as described previously (7,22,23). Antibiotics were added to obtain final concentrations of 50 g of ampicillin and 20 g of chloramphenicol/ ml, while IPTG (isopropyl-␤-D-thiogalactopyranoside) was added to obtain a final concentration of 1.0 mM.…”

“…The latter vector was previously digested with the restriction enzyme XbaI upstream in the treZ gene and filled with DNA polymerase I (Klenow fragment). For the synthetic operon galU/treY, the galU gene was excised from pLPIgalU00 plasmid (22,23) with the restriction enzyme SmaI and ligated in the pJCtreY vector previously digested with the restriction enzyme XbaI upstream in the treY gene and filled with DNA polymerase I (Klenow fragment). Finally, for the synthetic operon galU/treYZ, the treZ fragment was excised from the TOPOtreZ construct and ligated in the pJCgalUtreY vectorpreviously digested with the restriction enzyme BsaI downstream in the galU and treY genes and filled with DNA polymerase I (Klenow fragment).…”

“…In previous studies (22,23), expression of the otsBA operon in C. glutamicum with coexpression of the galU gene-encoded Escherichia coli UDP-glucose pyrophosphorylase produced a significant rise in trehalose synthesis. Increased glycogen supplied during the single expression of galU suggests that the higher trehalose was synthesized through the TreYZ pathway (23).…”