![]() In addition, advances in synthetic biology and metabolic engineering have led to a renaissance in whole-cell biocatalysis, which can enable the manufacture of value-added products from cheap feedstock. 21- 23 Whole-cell biocatalysts offer unique advantages, including endogenous cofactor regeneration, and are widely used for the efficient production of fine and bulk chemicals and active pharmaceutical ingredients. 18- 20 Enzyme activity can be improved through directed evolution coupled with an efficient screening system. 8 Poor transformation rates can be a result of low substrate availability, toxicity of the substrate or product, low enzyme concentration, or the use of inefficient enzymes. 17 However, some whole-cell biocatalysts show inefficient conversion rates. coli cells as whole-cell biocatalysts may provide an even more cost-effective solution. 8, 16 Nevertheless, the in vitro production of glycosides still requires purified enzymes and starting concentrations of UDP-glucose or UDP. The in vitro production of glycosides has been improved by adding additional enzymes for UDP-sugar regeneration. UGTs can be produced by, and purified from genetically modified Escherichia coli cells. 13 A number of plant UGTs have already been characterized and they produce economically interesting glycosides such as flavonoids, terpenols, and phenolic compounds. 11, 12 One of the largest groups of GTs are the UDP-glycosyltransferases (UGTs), which use uridine diphosphate (UDP)-sugars as a donor. Activated sugars are expensive fine chemicals and have to be recycled in order to establish an economical process. GTs transfer an activated sugar-moiety to an acceptor molecule. 8 Identification and characterization of new glycosyltransferases (GTs) provide more efficient enzymes for their production 9 and enables a growing variety of possible glycoside products. However, as biotechnological production offers a cost-effective alternative to classical chemical synthesis, low-cost glycosides are gaining importance for numerous applications. 7 Low-value glycosides are not available to industry. Protective group chemistry coupled with long reaction times and low yields results in high production costs. 6Ĭlassical chemical synthesis can only be used for the production of high-value glycosides because the synthesis requires multiple protection and deprotection steps. ![]() 4, 5 The use as industrial water treatment agent was also proposed. 2 Glycosides are currently used as sweeteners, 3 and as drugs. 1 Therefore, glycosylation modulates the effect of pharmaceuticals. The aglycone gains polarity due to the sugar moiety, resulting in higher water solubility, and altered bioavailability of the glycoside. Glycosides are conjugates of one or more sugars and a secondary molecule, which is referred to as the aglycone. Therefore, in addition to the quality of the mutant library, an efficient and stable expression system is crucial to achieve high concentrations of active enzyme and product, as formation of inclusion bodies and other inactive forms of the biocatalyst reduces productivity. An up to 60-fold increase in whole-cell product quantity was achieved. Detailed analysis revealed that the reason for the increase in product titer was mainly due to different expression effects of the mutant genes rather than improved enzyme kinetics. The screening of only 176 colonies yielded three putative candidates. We generated glycosyltransferase mutants through error-prone-PCR and screened the library using a small-scale whole-cell biotransformation system to identify highly productive strains. Directed evolution, coupled with a suitable screening method, can tackle this challenge. However, many production systems still suffer from low yields. Due to continuous improvement in production, glycosides can now be used in low-cost products by various industries. Biotechnological production of glycosides is an economically competitive manufacturing alternative to classical chemical synthesis.
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