Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

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.Trehalose (␣-glucopyranosyl-␣-glucopyranoside) is a stable, odor-free, nonreducing disaccharide that is widespread in nature (34). Its protein-stabilizing properties have suggested a wide range of commercial applications, ranging from cosmetics to the agricultural food sector (18). In the 1990s, the high cost of this disaccharide triggered the development of methods based on immobilized enzymes to obtain trehalose from maltodextrins (20,21). This achievement drastically reduced the commercial price of trehalose (34). A microbiological alternative for large-scale production of trehalose relying on Corynebacterium glutamicum (19), a gram-positive bacterium able to synthesize and excrete this sugar (37, 41), was recently proposed (27).Trehalose synthesis in C. glutamicum during growth on glucose proceeds through two major pathways ( Fig. 1): (i) from UDP-glucose and glucose-6-phosphate (OtsAB pathway) and (ii) from malto-oligosaccharides or ␣-1,4 glucans (TreYZ pathway) (36,42). Recently, a sixfold increase in OtsBA pathway activity in C. glutamicum (27) was achieved through heterologous expression of the Escherichia coli otsBA operon encoding the trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) (17). However, this approach resulted in only a twofold increase in the amount of excreted trehalose (27). These results suggested that further improvements in trehalose synthesis were hampered by the low activity of UDP-glucose pyrophosphorylase, which is responsible for UDP-glucose synthesis (17) according to the following reaction: glucose-1-P ϩ UTP 3 UDP-glucose ϩ pyrophosphate. This enzyme is critical in Lactococcus lactis and Streptococcus thermophilus during exopolysaccharide accumulation (3, ...

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.…”

“…Increased glycogen supplied during the single expression of galU suggests that the higher trehalose was synthesized through the TreYZ pathway (23). Rather than trying to establish the interrelationship in the two pathways' mechanisms, the aim of the present study was to seek higher trehalose production, making use of the simultaneous expression of C. glutamicum treY and treZ genes in the TreYZ pathway and the E. coli galU gene (11,23). …”

The glgX gene product of Corynebacterium glutamicum is required for glycogen degradation and for fast adaptation to hyperosmotic stress

“…Previous studies have revealed a link between glycogen and trehalose synthesis in C. glutamicum, suggesting a role of glycogen metabolism for fast adaptation to hyperosmotic growth conditions (Padilla et al, 2004;Seibold et al, 2007;Tzvetkov et al, 2003;Wolf et al, 2003). We therefore examined the effects of salt addition in the exponentialgrowth phase on growth, substrate consumption, glycogen level and intracellular trehalose formation of C. glutamicum WT, using minimal medium with 1.5 % glucose as a carbon source.…”

“…before complete consumption of the sugar substrate (Seibold et al, 2007). Therefore, it seems unlikely that glycogen in this organism serves as a long-term carbon storage compound, and in fact, glycogen metabolism in C. glutamicum has previously been suggested to be important for trehalose synthesis (Padilla et al, 2004;Seibold et al, 2007;Tzvetkov et al, 2003;Wolf et al, 2003). It has been shown that linear maltodextrins (formed as intermediates during glycogen synthesis and degradation) serve as substrates for the two-step trehalose biosynthetic pathway via maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) (Carpinelli et al, 2006;De Smet et al, 2000).…”

Functional metagenomics reveals novel salt tolerance loci from the human gut microbiome

“…Padilla et al (2004) increased the UDP glucose supply and consequently the levels of accumulated trehalose by expressing the E. coli galU gene in Corynebacterium glutamicum. In our study, the expression of both the galE(3) and galE(25) genes in the osmosensitive strain E. coli MKH13 resulted in a statistically significant increased salt (NaCl and KCl) tolerance phenotype (Figures 2b and c).…”