Metabolic engineering of microbes for oligosaccharide and polysaccharide synthesis

Abstract

Metabolic engineering has recently been embraced as an effective tool for developing whole-cell biocatalysts for oligosaccharide and polysaccharide synthesis. Microbial catalysts now provide a practical means to derive many valuable oligosaccharides, previously inaccessible through other methods, in sufficient quantities to support research and clinical applications. The synthesis process based upon these microbes is scalable as it avoids expensive starting materials. Most impressive is the high product concentrations (up to 188 g/L) achieved through microbe-catalyzed synthesis. The overall cost for selected molecules has been brought to a reasonable range (estimated $30-50/g). Microbial synthesis of oligosaccharides and polysaccharides is a carbon-intensive and energy-intensive process, presenting some unique challenges in metabolic engineering. Unlike nicotinamide cofactors, the required sugar nucleotides are products of multiple interacting pathways, adding significant complexity to the metabolic engineering effort. Besides the challenge of providing the necessary mammalian-originated glycosyltransferases in active form, an adequate uptake of sugar acceptors can be an issue when another sugar is necessary as a carbon and energy source. These challenges are analyzed, and various strategies used to overcome these difficulties are reviewed in this article. Despite the impressive success of the microbial coupling strategy, there is a need to develop a single strain that can achieve at least the same efficiency. Host selection and the manner with which the synthesis interacts with the central metabolism are two important factors in the design of microbial catalysts. Additionally, unlike in vitro enzymatic synthesis, product degradation and byproduct formation are challenges of whole-cell systems that require additional engineering. A systematic approach that accounts for various and often conflicting requirements of the synthesis holds the key to deriving an efficient catalyst. Metabolic engineering strategies applied to selected polysaccharides (hyaluronan, alginate, and exopolysaccharides for food use) are reviewed in this article to highlight the recent progress in this area and similarity to challenges in oligosaccharide synthesis. Many naturally occurring microbes possess highly efficient mechanisms for polysaccharide synthesis. These mechanisms could potentially be engineered into a microbe for oligosaccharide and polysaccharide synthesis with enhanced efficiency.

Figure 1

Metabolic pathway for UDP-galactose synthesis using sucrose synthase [9]. Enzymes – 1: sucrose synthase (SusA), 2: UDP-glucose 4'-epimerase (GalE), 3: β1,4-galactosyltransferase (β1,4GalT), 4: α1,4-galactosyltransferase (α1,4GalT).

Figure 2

Metabolic pathway for UDP-galactose synthesis using galactokinase and glucose-1-phosphate uridyltransferase [8]. Enzymes – 1: glucose-1-phosphate uridyltransferase (GalU), 2: pyrophosphatase (ppa), 3: galactokinase (GalK), 4: galactose-1-phosphate uridyltransferase (GalT), 5: α1,4-galactosyltransferase (lgtC).

Figure 3

Metabolic pathway for UDP-galactose synthesis using endogenous UDP-glucose synthesis pathway [11]. Enzymes – 1: phosphoglucose isomerase, 2: phosphoglucomutase, 3: UDP-glucose pyrophosphorylase, 4: UDP-glucose 4'-epimerase.

Figure 4

Metabolic pathway for CMP-NeuAc synthesis using CMP-NeuAc synthase [13]. Enzymes – 1: sialic acid permease (NanT), 2: sialic acid aldolase (NanA), 3: CMP-NeuAc synthase, 4: α2,3-sialyltransferase, 5: β-galactoside permease (LacY).

Figure 5

Metabolic pathway for CMP-NeuAc synthesis from NeuAc and orotic acid [14]. Enzymes – 1: CMP-NeuAc synthetase (NeuA), 2: α2,3-sialyltransferase, 3: CTP synthetase (PyrG).

Figure 6

Metabolic pathway for GDP-fucose synthesis using natural GDP-fucose synthesis pathway [15]. Enzymes – 1: phosphomannose isomerase (ManA), 2: phosphomannomutase (ManB), 3: mannose-1-phosphate guanylyltransferase (ManC), 4: GDP-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (Gmd, WcaG), 5: putative UDP-glucose lipid carrier transferase (WcaJ).

Figure 7

Metabolic pathway for GDP-fucose synthesis using bacterial coupling [16]. Enzymes – 1: glucokinase (Glk), 2: phosphomannomutase (ManB), 3: mannose-1-phosphate guanylyltransferase (ManC), 4: GDP-mannose-4,6-dehydratase (Gmd), 5: GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (WcaG), 6: α1,3-fucosyltransferase (FucT).

Figure 8

Metabolic pathway for GDP-fucose synthesis in Saccharomyces cerevisiae [17]. Enzymes – 1: GDP-mannose-4,6-dehydratase (Gmd), 2: GDP-4-keto-6-deoxy-mannose-3,5-epimerase (GmeR), 3: 4-reductase (GmeR), 4: GDP-fucose pyrophosphorylase, 5: fucose kinase.