Combinatorial Mutagenesis and Selection of Improved Signal Sequences and Their Application for High-Level Production of Translocated Heterologous Proteins in Escherichia coli

Abstract

bWe previously designed the consensus signal peptide (CSP) and demonstrated that it can be used to strongly stimulate heterologous protein production in Escherichia coli. A comparative study using CSP and two bacterial signal sequences, pelB and ompA, showed that the effect of signal sequences on both expression level and translocation efficiency can be highly protein specific. We report here the generation of CSP mutant libraries by a combinatorial mutagenesis approach. Degenerated CSP oligonucleotides were cloned in frame with the 5= end of the bla gene, encoding the mature periplasmic ␤-lactamase released from its native signal sequence. This novel design allows for a direct selection of improved signal sequences that positively affect the expression level and/or translocation efficiency of ␤-lactamase, based on the ampicillin tolerance level of the E. coli host cells. By using this strategy, 61 different CSP mutants with up to 8-fold-increased ampicillin tolerance level and up to 5.5-fold-increased ␤-lactamase expression level were isolated and characterized genetically. A subset of the CSP mutants was then tested with the alternative reporter gene phoA, encoding periplasmic alkaline phosphatase (AP), resulting in an up to 8-fold-increased production level of active AP protein in E. coli. Moreover, it was demonstrated that the CSP mutants can improve the production of the medically important human interferon ␣2b under high-cell-density cultivations. Our results show that there is a clear potential for improving bacterial signal sequences by using combinatorial mutagenesis, and bioinformatics analyses indicated that the beneficial mutations could not be rationally predicted.

In Vitro Prediction of the Evolution of GES-1 β-Lactamase Hydrolytic Activity

“…The A6T substitution in GES-1 systematically conferred a 3-to 8-fold increase in the MICs of CTX, ATM, and IPM (Tables 2 to 4). In accordance with our observation, a study including experimental mutagenesis of a consensus signal sequence fused to a ␤-lactamase gene increased the ampicillin tolerance level of the host cell up to 8-fold (21). To the best of our knowledge, it has never been clearly assessed that mutations in the signal sequence of any ␤-lactamase may confer increased resistance to ␤-lactams.…”

5′-coding sequence of thenasAgene ofAzotobacter vinelandiiis required for efficient expression

“…Future mechanistic studies could begin with ribosome‐binding assays, an approach that would tell whether the nasA 5′‐coding sequence directly promotes the formation of translational complexes. The findings from this study suggest the possibility of improving protein production in A. vinelandii and potentially other Pseudomonas species by optimizing 5′‐coding sequences or fusing a 5′ heterologous partner sequence, a strategy that has been tested successfully in E. coli (Stenstrom et al ., ; Heggeset et al ., ; Kucharova et al ., ). Engineering a 5′‐coding sequence to improve gene expression is straightforward and has potential applications in both basic research and industrial protein production.…”

Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

“…Both artificial operons harbour the phosphoglucomutase encoding sequence, celB , as the first gene; a spacer region; and the β-lactamase encoding sequence, bla , as the second gene (Figure 1). The celB gene was chosen as it can be efficiently transcribed and translated, hence would not introduce any undesired restrictions 5 ; and the bla gene was chosen as host’s resistance to ampicillin correlates with the produced amounts of β-lactamase, simplifying the identification of clones with the desired phenotype 18, 29 . As for the spacer region, it ensures that the translation of bla only occurs through de novo initiation as opposed to translational read-through 5 .…”