A Versatile Toolbox for the Control of Protein Levels Using N^ε-Acetyl-l-lysine Dependent Amber Suppression

Abstract

The analysis of the function of essential genes in vivo depends on the ability to experimentally modulate levels of their protein products. Current methods to address this are based on transcriptional or post-transcriptional regulation of mRNAs, but approaches based on the exploitation of translation regulation have so far been neglected. Here we describe a toolbox, based on amber suppression in the presence of N-acetyl-l-lysine (AcK), for translational tuning of protein output. We chose the highly sensitive luminescence system LuxCDABE as a reporter and incorporated a UAG stop codon into the gene for the reductase subunit LuxC. The system was used to measure and compare the effects of AcK- and N-(tert-butoxycarbonyl)-l-lysine (BocK) dependent amber suppression in Escherichia coli. We also demonstrate here that, in combination with transcriptional regulation, the system allows protein production to be either totally repressed or gradually adjusted. To identify sequence motifs that provide improved translational regulation, we varied the sequence context of the amber codon and found that insertion of two preceding prolines drastically decreases luminescence. In addition, using LacZ as a reporter, we demonstrated that a strain encoding a variant with a Pro-Pro amber motif can only grow on lactose when AcK is supplied, thus confirming the tight translational regulation of protein output. In parallel, we constructed an E. coli strain that carries an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible version of the AcK-tRNA synthetase (AcKRS) gene on the chromosome, thus preventing mischarging of noncognate substrates. Subsequently, a diaminopimelic acid auxotrophic mutant (ΔdapA) was generated demonstrating the potential of this strain in regulating essential gene products. Furthermore, we assembled a set of vectors based on the broad-host-range pBBR ori that enable the AcK-dependent amber suppression system to control protein output not only in E. coli, but also in Salmonella enterica and Vibrio cholerae.

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

“…The expression of genes with amber stop codon insertions requires ncAAs, and their expression levels are presumed ncAA-dependent. At the same time, tunable translation control tools using site-specific ncAA incorporation have been studied 29 , 32 – 34 . Therefore, by inserting amber stop codons into genes that are important for controlling cell growth, such as genes involved in central carbon metabolic pathways, should be regulated by ncAA abundances.…”

Transcriptional regulation of theNε-fructoselysine metabolism inEscherichia coliby global and substrate-specific cues

“…We further noticed a sequence matching the CRP/CAP (cAMP Response Protein/Catabolite Activator Protein) binding site centered around position −41.5 and thus being in perfect distance to constitute a class II promoter (Lawson et al , 2004). To test our hypotheses on transcriptional regulation of the P frlABCD promoter we initially fused 294bp (−219/+75) 5’ of frlABCD with the lux-operon luxCDABE of Photorhabdus luminescens (Volkwein et al , 2017) and measured the light output over a time course of 24 h in E. coli BW25113 wild-type cells grown in Lysogeny broth (LB/Miller) (Bertani, 2004). With this, we reached a maximal luminescence per OD 600 of about 1×10 6 RLU showing that this region comprises a fully functional promoter (Fig.…”

Expanding the Genetic Code of Lactococcus lactis and Escherichia coli to Incorporate Non-canonical Amino Acids for Production of Modified Lantibiotics

“…This unwanted mutation can in our experiments be excluded by the obtained mass spectrometry data. Additionally, with the first documented OTS shuttle vector system for E. coli , Salmonella enterica , and Vibrio cholerae , new developments also for Gram-positive species are expected in the long run ( Volkwein et al, 2017 ).…”