ligation independent cloning serial cloner

These enzymes transfer carbohydrate residues from activated sugar donors to a multitude of acceptor molecules, including proteins, saccharides, lipids and small molecules. In nature, glycosylation reactions are primarily catalysed by nucleotide-sugar-dependent glycosyltransferases. Glycosylated vitamin C and anthocyanidins are more stable than their non-glycosylated precursors, alteration of the sugar residues of quercetin affects pharmacological properties, and transfer of sugars to the diterpenol steviol results in sweet tasting steviosides. Volatile terpenols become non-volatile by glycosylation, allowing them to be applied as slow release aroma chemicals and the water solubility of resveratrol increases by a factor of 1700 when sugars are attached, which results in improved bioavailability. The transfer of sugars to small molecules makes them non-volatile, more water-soluble and improves their stability, bioavailability and functionality. Recently, the glycosylation reaction has attracted much attention because volatility, poor water solubility and instability limit the application of many small molecules in consumer products. Glycosylation plays a central role in cellular communication, transport and storage of metabolites in organisms. coli, which offers the potential for large-scale and cost-effective production. For the moment, the most promising method for whole-cell biocatalysis seems to be the use of engineered E.

When using whole-cell biocatalysts for biocatalysis, substrate and product transmembrane transport and cofactor regeneration capacity of the used strain must similarly be considered. by using tightly regulated promoters or by attaching solubility-enhancing fusion tags. Strategies for overcoming this disadvantage of heterologous expression are the optimization of the expression system, e.g. However, the production of misfolded and inactive aggregates such as inclusion bodies is problematic when recombinant proteins are over-expressed in bacteria. coli as whole-cell biocatalyst offers additional benefits such as cofactor regeneration, high catalytic efficiency and mild and simple reaction conditions. Escherichia coli is the host of choice if post-translational modifications of the enzyme are not required for its activity. The production of catalytically active proteins in heterologous microbial and fungal hosts leads to lower costs and high yields, which is crucial for the economy of an industrial process. Traditionally, enzymes are used by food and feed industry, but are increasingly implemented in emerging markets such as pharmaceutical and fine chemicals industries. The selection of a small solubility-enhancing protein tag and exploitation of the T7 polymerase-induction system allowed the formation of increased levels of functional recombinant protein, thereby improving the performance of the whole-cell biocatalyst.Įnzymes offer tremendous opportunities for the chemical industry as they can efficiently and economically convert molecules and replace well-known and established industrial processes with more sustainable ones.

The product concentration was improved 8.6-fold compared with E. coli BL21(DE3)/pET-SUMO_VvGT14ao yielded the highest titres. coli expression strains, resulting in 18 strains that were tested for glycosylation efficiency with terpenols and a phenol. Seven expression plasmids differing in the resistance gene, origin of replication, promoter sequence, and fusion protein tag were generated and transformed into four different E. coli expressing VvGT14ao, a glycosyltransferase gene from grape ( Vitis vinifera). The aim of this work was to increase the titre of terpenyl glucosides in biotransformation assays with E. However, there is still a need to optimize the biocatalysts. Glycosides are becoming increasingly more relevant for various industries as low-cost whole-cell-biocatalysts are now available for the manufacture of glycosides.

Ligation independent cloning serial cloner