Exploring the Nonconserved Sequence Space of Synthetic Expression Modules in Bacillus subtilis

Abstract

Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a "brute-force" approach by designing and synthesizing a large combinatorial library comprising ∼12 000 unique synthetic expression modules (SEMs) for Bacillus subtilis. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned 5 orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong veg expression module. Analyses of the synthetic modules indicated that sequences at the 5'-end of the mRNA were the most important contributing factor to the differences in expression levels, presumably by preventing formation of strong secondary mRNA structures that affect translation initiation. When the gfp coding region was replaced by the coding region of the xynA gene, encoding the industrially relevant B. subtilis xylanase enzyme, only a 3-fold improvement in xylanase production was observed. Moreover, the correlation between GFP and xylanase expression levels was weak. This suggests that the differences in expression levels between the gfp and xynA constructs were due to differences in 5'-end mRNA folding and consequential differences in the rates of translation initiation. Our data show that the use of large libraries of SEMs, in combination with high-throughput technologies, is a powerful approach to improve the production of a specific protein, but that the outcome cannot necessarily be extrapolated to other proteins.

Fine-tuning biosensor dynamic range based on rational design of cross-ribosome-binding sites in bacteria

“…2c, d ), suggesting that the maximum dynamic range can be achieved at optimal TIR. However, TIR higher than the optimal TIR could cause low biosensor dynamic range, which could be due to the rapid expression of sfGFP resulting in misfolding or unfolding, thus affecting the natural folding of sfGFP 22, 24 . Therefore, we hypothesized that the RBS could affect protein folding by regulating the TIR of protein.…”

“…E. coli BL21 (DE3) cells containing the plasmid libraries were cultured to saturation, and then incubated at a concentration of 1% into 250-mL flasks containing LB medium at 250 rpm and 37 °C. After 2 h, inducers were added to the desired final concentration, and incubation was resumed for 12 h. The induced cultures were diluted into cold PBS and kept on ice until evaluation with a BD FACS AriaII cell sorter (Becton Dickinson) 24 . At least 100,000 events were captured for each sample.…”

Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors of Streptococcus Species

“…The promoters that we tested here exhibit moderate to high levels of gene expression, but the platforms that we have developed could be used to screen for weaker or stronger promoters in a more comprehensive approach. It is important to note that optimal expression for one protein (e.g., GFP) might not correlate with that for another because of differences in 5=-end mRNA folding affecting mRNA stability and translation initiation (33). This is an especially important consideration here, as we did not use the same RBS for each promoter-sfgfp fusion.…”

Biotransformation and chiral resolution of d,l‐alanine into pyruvate and d‐alanine with a whole‐cell biocatalyst expressing l‐amino acid deaminase

“…N-terminal coding sequences can influence the protein expression levels by impacting ribosome extension at the initial stage of translation and the efficiency of ribosome binding to mRNA [33][34][35][36]. Therefore, we selected ten NCSs from highly expressed proteins in B. subtilis [25] and introduced them into the 5 -end of the pm1 gene (Fig.…”