Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell Biotransformation Efficiency

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.

Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications

“…Heterologous enzymes expressed within E. coli may aggregate or be unstable, however, being bound to a stable molecule such as DNA may improve their stability and reduce levels of aggregation. More recently, Park et al was involved in the stable expression of immobilized enzymes within E. coli using the plasmid display method ( Park et al, 2020 ). Once again the Oct-1 DBD was fused to two fucosyltransferases, and soluble expression and functional activity was improved in the fused constructs ( Park et al, 2020 ).…”

“…More recently, Park et al was involved in the stable expression of immobilized enzymes within E. coli using the plasmid display method ( Park et al, 2020 ). Once again the Oct-1 DBD was fused to two fucosyltransferases, and soluble expression and functional activity was improved in the fused constructs ( Park et al, 2020 ). Further research into this technology tackling the issue of inclusion bodies is needed to optimize and provide further proof of concept.…”

Catalytic Kinetics Considerations and Molecular Tools for the Design of Multienzymatic Cascade Nanoreactors

“…Only the complex between the plasmid and the 1,2fucosylltransferase was purified, but the expression of both enzymes and the scaffold increased the concentration of products in whole cells of E. coli when compared with cells without scaffold. [117] Different DNA binding domains, with zinc finger motifs have been fused to different enzymes allowing the assemble of enzymatic cascades for the production of lycopene using as scaffold a plasmid carrying the specific sequences that are recognized for the zinc fingers. [118] Similarly, a cascade for detection of inorganic pyrophosphate (PPi), composed of the enzymes orthophosphate dikinase (PDKK) and luciferase (FLuc) were fused to zinc finger motifs and assembled in short DNA fragments showing improved efficiency by shorting the distances between the target DNA sequences.…”

Directed Evolution of Soluble α-1,2-Fucosyltransferase Using Kanamycin Resistance Protein as a Phenotypic Reporter for Efficient Production of 2'-Fucosyllactose

“…The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes. Keywords: 2′-fucosyllactose, α-1,2-fucosyltransferase, directed evolution, solubility biosensor, kanamycin resistance, Escherichia coli insoluble and unstable nature of α-1,2-fucosyltransferase under process conditions [11,12].…”

“…In engineered Escherichia coli [ 7 , 8 ] and Saccharomyces cerevisiae [ 9 , 10 ] capable of producing 2'-FL, lactose is fucosylated by a heterologous α-1,2-fucosyltransferase using guanosine 5'‐diphosphate (GDP)-L-fucose as a fucose donor. However, despite several attempts to construct high-performance strains, the 2'-FL titers are often reported to be low because of the insoluble and unstable nature of α-1,2-fucosyltransferase under process conditions [ 11 , 12 ].…”