Abstract
Recombinant whole-cell biocatalysts are widely used for biotransformation of valuable products. However, some key enzymes involved in biotransformation processes are unstable and cannot be easily expressed in the functional form. In this study, we describe a versatile platform for enzyme stabilization inside the cell: Intracellularly Immobilized Enzyme System (IIES). A 1,2-fucosyltransferase from Pedobactor saltans (PsFL) and a 1,3-fucosyltransferase from Helicobacter pylori (HpFL), chosen as model proteins, were fused with Oct-1 DNA-binding domain, which mediated the formation of a plasmid–protein complex. Oct-1 fusion enabled both soluble and stable expression of recombinant proteins in the cytoplasm because the fusion proteins were stabilized on the plasmid like immobilized enzymes bound to solid surface. As a result, Oct-1-fusion proteins exhibited significantly greater product titer and yield than non-fusion proteins. Use of fusion proteins PsFL-Oct-1 with C-terminal Oct-1 and Oct-1-PsFL with N-terminal Oct-1 resulted in ~3- and ~2-fold higher 2′-fucosyllactose titers, respectively, than with the use of PsFL alone. When Oct-1 was fused to HpFL, which requires dimerization through heptad repeats, almost two times more 3-fucosyllactose was produced. Fucosyllactose has been used as a food additive because it has various beneficial effects on human health. We anticipate that IIES using Oct-1 fusion protein developed in this study can be applied to stabilize other unstable enzymes.
Highlights
Escherichia coli is generally the first choice of host for the production of proteins and chemicals, because of its fast growth rate, a well-established expression system, high production yield, and low manufacturing cost
Schistosoma japonicum glutathione S-transferase (GST), E. coli maltose-binding protein (MBP), E. coli N-utilization substance A (NusA), and E. coli thioredoxin are commonly used as fusion partners to prevent inclusion body formation (Esposito and Chatterjee, 2006)
Oct-1 DNAbinding domain (DBD) itself exhibited both high expression level and high solubility (Park et al, 2013). These results suggest that Oct-1 DBD is a good fusion tag to demonstrate intracellularly immobilized enzyme system (IIES) among other DBDs with their relatively large size, or complex structures that are difficult to produce in the cytoplasm of E. coli (Yesudhas et al, 2017)
Summary
Escherichia coli is generally the first choice of host for the production of proteins and chemicals, because of its fast growth rate, a well-established expression system, high production yield, and low manufacturing cost. In E. coli cytoplasm, recombinant proteins tend to interact with non-specific hydrophobic patches, leading to the formation of inclusion bodies (Baneyx and Mujacic, 2004). Schistosoma japonicum glutathione S-transferase (GST), E. coli maltose-binding protein (MBP), E. coli N-utilization substance A (NusA), and E. coli thioredoxin are commonly used as fusion partners to prevent inclusion body formation (Esposito and Chatterjee, 2006). Fusion partners provide a general protective effect against insoluble aggregation, some soluble fusions lack detectable activities (Sachdev and Chirgwin, 1999). These soluble aggregates are formed by agglomeration of misfolded proteins of interest, while precipitation is prevented by the presence of the soluble fusion partners (Nomine et al, 2001). An alternative fusion system should be developed to increase soluble expression levels and simultaneously block agglomeration formation between misfolded target proteins
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