Overcoming expression bottlenecks in recombinant silk–elastin-like polypeptides via plasmid backbone and protein engineering approaches

Abstract

Silk–elastin-like polypeptides (SELPs) are sustainable recombinant biopolymers with applications in tissue engineering, drug delivery, or biosensing, to name a few. In contrast to the synthesis of traditional fossil-based polymers, SELPs are encoded by DNA, which leads to reproducible sequences and molecular weights. However, large-scale production of SELPs is hindered by the typical low yields attained in microbial expression systems. This is often attributed to (i) expression challenges caused by the repetitive nature of silk- and elastin-like tandem repeats in the SELP sequence, which can reduce ribosome processivity or cause mRNA instability through secondary structure formation; and (ii) cellular toxicity caused by SELP aggregation or inclusion body formation, particularly through irreversible aggregation of silk-like blocks. Here, we investigate strategies to prevent those limitations and enhance the heterologous expression of a positively charged SELP in Escherichia coli by systematically evaluating modifications at both the plasmid backbone and protein engineering levels. Plasmid backbone modifications encompass different antibiotic resistance markers (ampicillin vs. kanamycin), promoter switching (T7 vs. pBAD) and upregulation of glycine tRNA production. Protein engineering modifications included reducing molecular weight (from 57.5 to 38.5 kDa), varying the number of consecutive silk-like blocks (2, 4 or 8), and incorporating an N-terminal AKTK expression tag. Plasmid backbone modifications alone did not improve expression beyond its baseline yield (7.3 ± 3.1 mg L⁻¹). In contrast, adding an N-terminal AKTK tag alleviated translation bottlenecks and increased expression by 8.5-fold, independent of the number of consecutive silk-like blocks or the SELP molecular weight. Notably, alleviating this translation bottleneck enabled additional plasmid backbone optimisation strategies to further improve expression. As an example, the combined effect of adding an N-terminal AKTK tag plus upregulating glycine tRNA production resulted in a cumulative 17.0-fold improvement. Collectively, these findings highlight the critical role of N-terminal sequence engineering in unlocking efficient SELP translation and expression.