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

Abstract

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra-and extracellular trehalose content.Trehalose contains two glucose molecules linked by an ␣-1,1 glycosidic bond. It is a stable, odor-free, and nonreducing disaccharide widely found in nature (27). Its properties as a protein and cell stabilizer make the molecule a promising biotechnological additive for tissue and organ preservation, protein technology, and several food applications.Trehalose is currently synthesized on an industrial scale by using the method patented by Hayashibara Biochemical Laboratories (17, 18), which is based on direct transformation from maltodextrin using two enzymes obtained from the culture of Rhizobium sp. strain M11 or Arthrobacter sp. strain Q36.Our approach to production is microbiological and makes use of the fact that Corynebacterium glutamicum a gram-positive bacterium (15), excretes significant amounts of trehalose during batch cultures (29, 32). C. glutamicum has been used extensively for the industrial production of vitamins and numerous L-amino acids for foodstuffs (16).C. glutamicum uses three pathways in trehalose synthesis (28, 33): the TreS pathway, directly converting maltose into trehalose catalyzed by trehalose synthase (TreS) (5, 21); the OtsAB pathway, wherein trehalose synthesis starts from UDPglucose and glucose-6-phosphate and is catalyzed in two steps by trehalose-6-phosphate synthase (OtsA), followed by trehalose-6-phosphate phosphatase (OtsB); and the TreYZ pathway, in which enzymatic conversion of the ␣-glucan polymer into trehalose is catalyzed in two steps by maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ), respectively. The regulatory mechanisms of each pathway remain unclear, and just how the three are interrelated is unknown. However, recent studies showed that under hyperosmotic conditions, osmoregulated trehalose synthesis in C. glutamicum is mediated by...

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

“…The same phenotype has been observed when glycogen synthesis is abolished by disruption of the glycogen synthase gene glgA in C. glutamicum DotsAB (Tzvetkov et al, 2003). Moreover, overexpression of the treYZ genes in C. glutamicum causes a decrease in the intracellular glycogen content (Carpinelli et al, 2006). All these findings indicate a link between trehalose formation through the TreYZ pathway and glycogen synthesis in the cells.…”

“…This corresponds to the same phenotype as C. glutamicum DtreY after an upshift of osmolality (Wolf et al, 2003). Since the glgX mutant contains glycogen, in principle, it should synthesize and contain all the intermediates of glycogen synthesis, including the linear chains of a-1,4-glycosidic-linked glucose residues, the products of glycogen synthase (Tzvetkov et al, 2003), and the substrate for TreZ (Carpinelli et al, 2006). The fact that the mutant contains no trehalose thus indicates that mainly the maltodextrins derived from the GlgX activity in the course of glycogen degradation are used for trehalose synthesis via the TreYZ pathway, and not the maltodextrins derived from de novo synthesis through the action of ADPglucose pyrophosphorylase and glycogen synthase.…”

“…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). Since trehalose in C. glutamicum acts as a cell-wall component (Tropis et al, 2005) and as a compatible solute under hyperosmotic and nitrogen-limitation conditions (Wolf et al, 2003), glycogen degradation and its control might be important for the cell-wall composition and for the response to osmotic stress in this organism.…”

Effect of trehalose on protein structure

“…24 It forms a part of the cell wall in different species of mycobacteria, including Mycobacterium tuberculosis and the phylogenetically related corynaebacteria (including Corynebacterium glutamicum). 12,23 Free trehalose is also found in the latter during hyperosmotic stress. In the animal kingdom, trehalose has been found in very high amounts in the adult roundworm (about 6% of the dry weight) and in the eggs of roundworm (about 9% of the dry weight).…”

“…[3][4][5][6][7][8][9][10][11] Similarly, articles reporting gene expression profiling relevant to trehalose as a bioprotector do not make a serious attempt to relate the protective action to physical and chemical properties of the molecule. [12][13][14][15][16][17] This review attempts to bridge this gap and to find out how data from physicists, chemists, and life scientists can be collected and collated so that an understanding of general interest to protein scientists may emerge. Trehalose is a white, odorless powder with relative sweetness 45% that of sucrose.…”