Synonymous Codon Changes Research Articles

In silico framework for genome analysis

Genomes hold the complete genetic information of an organism. Examining and analyzing genomic data plays a critical role in properly understanding an organism, particularly the main characteristics, functionalities, and evolving nature of harmful viruses. However, the rapid increase in genomic data poses new challenges and demands for extracting meaningful and valuable insights from large and complex genomic datasets. In this paper, a novel Framework for Genome Data Analysis (F4GDA), is developed that offers various methods for the analysis of viral genomic data in various forms. The framework’s methods can not only analyze the changes in genomes but also various genome contents. As a case study, the genomes of five SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) VoC (variants of concern), which are divided into three types/groups on the basis of geographical locations, are analyzed using this framework to investigate (1) the nucleotides, amino acids and synonymous codon changes in the whole genomes of VoC as well as in the Spike (S) protein, (2) whether different environments affect the rate of changes in genomes, (3) the variations in nucleotide bases, amino acids, and codon base compositions in VoC genomes, and (4) to compare VoC genomes with the reference genome sequence of SARS-CoV-2.

Synthetic genomes unveil the effects of synonymous recoding.

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1-4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement-recoding-is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic Escherichia coli genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organism's genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

Suppression of the dwarf phenotype of an Arabidopsis mutant defective in thermospermine biosynthesis by a synonymous codon change in the SAC51 uORF.

Thermospermine plays a critical role in negatively regulating xylem development in angiosperms. A mutant of Arabidopsis thaliana that is defective in thermospermine biosynthesis, acaulis5 (acl5), exhibits a dwarf phenotype with excessive xylem formation. Mechanistically thermospermine acts in attenuating the inhibitory effect of an evolutionarily conserved upstream open reading frame (uORF) on the main ORF of SAC51, which encodes a basic helix-loop-helix protein involved in xylem repression. Here, we revealed that a semidominant suppressor of acl5, sac503, which partially restores the acl5 phenotype, has a point mutation in the conserved uORF of SAC51 with no amino acid substitution in the deduced peptide sequence. In transgenic lines carrying the β-glucuronidase (GUS) reporter gene fused with the SAC51 5' region containing the uORF, the mutant construct was shown to confer higher GUS activity than does the wild-type SAC51 construct. We confirmed that sac503 mRNA was more stable than SAC51 mRNA in acl5. These results suggest that the single-base change in sac503 positively affects the translation of its main ORF instead of thermospermine. We further found that the uORF-GUS fusion protein could be synthesized in planta from the wild-type and sac503 translational fusion constructs.

Codon-Restrained Method for Both Eliminating and Creating Intragenic Bacterial Promoters.

Future applications of synthetic biology will require refactored genetic sequences devoid of internal regulatory elements within coding sequences. These regulatory elements include cryptic and intragenic promoters, which may constitute up to a third of the predicted Escherichia coli promoters. The promoter activity is dependent on the structural interaction of core bases with a σ factor. Rational engineering can be used to alter key promoter element nucleotides interacting with σ factors and eliminate downstream transcriptional activity. In this paper, we present codon-restrained promoter silencing (CORPSE), a system for removing intragenic promoters. CORPSE exploits the DNA-σ factor structural relationship to disrupt σ70 promoters embedded within gene coding sequences with a minimum of synonymous codon changes. Additionally, we present an inverted CORPSE system, iCORPSE, which can create highly active promoters within a gene sequence while not perturbing the function of the modified gene.

Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites.

Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. Here we show that the accessibility of translation initiation sites modelled using the mRNA base-unpairing across the Boltzmann's ensemble significantly outperforms alternative features. This approach accurately predicts the successes or failures of expression experiments, which utilised Escherichia coli cells to express 11,430 recombinant proteins from over 189 diverse species. On this basis, we develop TIsigner that uses simulated annealing to modify up to the first nine codons of mRNAs with synonymous substitutions. We show that accessibility captures the key propensity beyond the target region (initiation sites in this case), as a modest number of synonymous changes is sufficient to tune the recombinant protein expression levels. We build a stochastic simulation model and show that higher accessibility leads to higher protein production and slower cell growth, supporting the idea of protein cost, where cell growth is constrained by protein circuits during overexpression.

The transcriptional landscape of a rewritten bacterial genome reveals control elements and genome design principles

Sequence rewriting enables low-cost genome synthesis and the design of biological systems with orthogonal genetic codes. The error-free, robust rewriting of nucleotide sequences can be achieved with a complete annotation of gene regulatory elements. Here, we compare transcription in Caulobacter crescentus to transcription from plasmid-borne segments of the synthesized genome of C. ethensis 2.0. This rewritten derivative contains an extensive amount of supposedly neutral mutations, including 123’562 synonymous codon changes. The transcriptional landscape refines 60 promoter annotations, exposes 18 termination elements and links extensive transcription throughout the synthesized genome to the unintentional introduction of sigma factor binding motifs. We reveal translational regulation for 20 CDS and uncover an essential translational regulatory element for the expression of ribosomal protein RplS. The annotation of gene regulatory elements allowed us to formulate design principles that improve design schemes for synthesized DNA, en route to a bright future of iteration-free programming of biological systems.

Nonoptimal Codon Usage Is Critical for Protein Structure and Function of the Master General Amino Acid Control Regulator CPC-1.

Under amino acid starvation conditions, eukaryotic organisms activate a general amino acid control response. In Neurospora crassa, Cross Pathway Control Protein 1 (CPC-1), the ortholog of the Saccharomyces cerevisiae bZIP transcription factor GCN4, functions as the master regulator of the general amino acid control response. Codon usage biases are a universal feature of eukaryotic genomes and are critical for regulation of gene expression. Although codon usage has also been implicated in the regulation of protein structure and function, genetic evidence supporting this conclusion is very limited. Here, we show that Neurospora cpc-1 has a nonoptimal NNU-rich codon usage profile that contrasts with the strong NNC codon preference in the genome. Although substitution of the cpc-1 NNU codons with synonymous NNC codons elevated CPC-1 expression in Neurospora, it altered the CPC-1 degradation rate and abolished its amino acid starvation-induced protein stabilization. The codon-manipulated CPC-1 protein also exhibited different sensitivity to limited protease digestion. Furthermore, CPC-1 functions in rescuing the cell growth of the cpc-1 deletion mutant and activation of the expression of its target genes were impaired by the synonymous codon changes. Together, these results reveal the critical role of codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein folding.IMPORTANCE The general amino acid control response is critical for adaptation of organisms to amino acid starvation conditions. The preference to use certain synonymous codons is a universal feature of all genomes. Synonymous codon changes were previously thought to be silent mutations. In this study, we showed that the Neurospora cpc-1 gene has an unusual codon usage profile compared to other genes in the genome. We found that codon optimization of the cpc-1 gene without changing its amino acid sequence resulted in elevated CPC-1 expression, an altered protein degradation rate, and impaired protein functions due to changes in protein structure. Together, these results reveal the critical role of synonymous codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein structure.

Codon optimization: a mathematical programing approach.

Synthesizing proteins in heterologous hosts is an important tool in biotechnology. However, the genetic code is degenerate and the codon usage is biased in many organisms. Synonymous codon changes that are customized for each host organism may have a significant effect on the level of protein expression. This effect can be measured by using metrics, such as codon adaptation index, codon pair bias, relative codon bias and relative codon pair bias. Codon optimization is designing codons that improve one or more of these objectives. Currently available algorithms and software solutions either rely on heuristics without providing optimality guarantees or are very rigid in modeling different objective functions and restrictions. We develop an effective mixed integer linear programing (MILP) formulation, which considers multiple objectives. Our numerical study shows that this formulation can be effectively used to generate (Pareto) optimal codon designs even for very long amino acid sequences using a standard commercial solver. We also show that one can obtain designs in the efficient frontier in reasonable solution times and incorporate other complex objectives, such as mRNA secondary structures in codon design using MILP formulations. http://alpersen.bilkent.edu.tr/codonoptimization/CodonOptimization.zip.

Coding Region Mutation Screening in Optineurin in Chinese Normal-Tension Glaucoma Patients.

Purpose To study the roles of sequence alterations in the optineurin (OPTN) gene-coding region in normal-tension glaucoma (NTG) among Chinese patients. Methods Genomic DNA was extracted from 190 NTG patients and 201 control subjects. The thirteen exons of OPTN were amplified by polymerase chain reaction and analyzed by direct sequencing. Detected sequence changes were compared between NTG patients and control subjects. Results Seven sequence changes in OPTN were identified in both NTG patients and control subjects. Among them, c.464G>A (T34 T), c.509C>T (T49T), c.806G>A (V148V), and c.959T>C (P199P) were synonymous codon changes, whilst c.655T>A (M98K), c.1996G>A (R545Q), and c.1582T>C (I407T) were missense changes. Two previously reported heterozygous mutations, c.458G>A (E50K) in exon 4 and c.691_692insAG in exon 6, were not found in this study. Out of these seven OPTN sequence variants, c.464G>A (T34T) was significantly associated with NTG in both the allelic and genotypic association analyses (allelic association: p = 0.0001, OR = 2.20, 95% CI: 1.46-3.31; genotypic association: p = 0.0001), whereas the association of other variants with NTG did not reach statistical significance (p > 0.05). Variants c.1582 T>C (I407T) and c.806G>A (V148V) were identified in one and two NTG patients, respectively, but not in the control subjects. Conclusions This study confirmed the association of the OPTN T34T variant with NTG, suggesting that OPTN is a susceptibility gene for NTG in Chinese. Moreover, a variant with amino acid change (I407T) was identified in NTG but not in controls. Further studies are warranted to assess whether this variant is a causative mutation for NTG.

Synonymous codon changes at the 5′-end of the gene strongly impact the heterologous protein expression in Escherichia coli

This study describes the impact of 5′-end codon modulation on the expression of a heterologous gene, human granulocyte colony stimulating factor (GCSF), in Escherichia coli. Fourteen different constructs (pGCSF-01 to pGCSF-14) carrying single or multiple synonymous substitutions at +2, +3 and further down from +4 to +7 codons, were prepared and their expression was monitored in E. coli BL21 Codon-Plus (DE3) RIPL using a strong T7 lac-promoter based expression system. A single nucleotide change at +2 Thr codon (ACC→ACA) either alone or in combination with +3 Pro codon (CCC/CCT/CCA) resulted in the expression enhancement of an otherwise poorly expressed native-GCSF, to a level that corresponded to 45–50% of the total E. coli BL21 CodonPlus (DE3) RIPL cellular proteins. The differences in GCSF expression amongst different constructs could be attributed to the preferential or non-preferential codon usage, reduced number of G/C nucleotides and the stability of mRNA secondary structure formed near the 5′-end coding region. The expression of GCSF achieved was in the form of biologically inactive inclusion bodies that were solubilized using mild concentration of a non-ionic surfactant and refolded by a simplified, step-dialysis approach. Biological activity of the purified GCSF, assessed in induced neutropenic mice, was similar to the commercially available preparation of the GCSF analog (filgrastim).

Synonymous Codon Changes in Measles (HMV) and Canine Distemper (CDV) Viral Nucleic Acid Sequences Result in Gene‐Specific Changes in Levels of Viral Protein Expression

The purpose of this study was to characterize the effects of human and canine codon optimization on expression of viral proteins from HMV and CDV nucleic acid sequences. All the codon changes made were synonymous in that the amino acid coded for was unchanged. The human and canine CUB optimized HMV and CDV sequences had increased nucleic acid and codon identity as compared to the WT sequences. Protein expression from human and canine CUB optimized and sub‐optimized constructs of the N, F, M, H and P/C proteins was assessed in transfected human (293 HEK) and canine (A‐72) cells using fluorescence, Cellomic and, for proteins for which antibodies were available, western analysis.Results1) codon optimization increased protein expression of HMV and CDV N and F proteins in both cell lines; 2) synonymous codon changes did not affect expression of the HMV and CDV P and C and the HMV M proteins, but both optimization and sub‐optimization to canine CUB increased expression of the CDV M protein in human cells; 3) synonymous codon changes increased expression of the HMV H protein in both cell lines, but did not significantly affect CDV H protein expression. Overall both CUB optimization and sub‐optimization affected protein expression independent of viral infection in ways that varied with the gene and cell type, and differed between HMV and CDV. These results indicate that synonymous codon changes in viral nucleic acid sequences can alter protein expression in a variety of ways, some of which are likely to impact viral pathogenicity.Support or Funding InformationThis project was sponsored by the Department of Defense Threat Reduction Agency. The content of the information does not necessarily reflect the position or the policy of the federal government, and no official endorsement should be inferred.

Large-Effect Beneficial Synonymous Mutations Mediate Rapid and Parallel Adaptation in a Bacterium.

Contrary to previous understanding, recent evidence indicates that synonymous codon changes may sometimes face strong selection. However, it remains difficult to generalize the nature, strength, and mechanism(s) of such selection. Previously, we showed that synonymous variants of a key enzyme-coding gene (fae) of Methylobacterium extorquens AM1 decreased enzyme production and reduced fitness dramatically. We now show that during laboratory evolution, these variants rapidly regained fitness via parallel yet variant-specific, highly beneficial point mutations in the N-terminal region of fae. These mutations (including four synonymous mutations) had weak but consistently positive impacts on transcript levels, enzyme production, or enzyme activity. However, none of the proposed mechanisms (including internal ribosome pause sites or mRNA structure) predicted the fitness impact of evolved or additional, engineered point mutations. This study shows that synonymous mutations can be fixed through strong positive selection, but the mechanism for their benefit varies depending on the local sequence context.

A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator.

Synonymous mutations, such as I507-ATC→ATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), the most frequent disease-associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small-molecule drugs are being developed to correct ΔF508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on I507-ATT and I507-ATC ΔF508 CFTR biogenesis and function. C4 stabilized I507-ATT ΔF508 CFTR band B, but without considerable biochemical and functional correction. VX-809 biochemically corrected ∼10% of both of the variants, leading to stable, forskolin+3-isobutyl-1-methylxanthine (IBMX)-activated whole-cell currents in the presence of the corrector. Omitting VX-809 during whole-cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX-809. Treatment of cells with the C4+VX-809 combination resulted in enhanced rescue and 2-fold higher forskolin+IBMX-activated currents of both I507-ATT and I507-ATC ΔF508 CFTR, compared with VX-809 treatment alone. The lack of an effect of C4 on I507-ATC ΔF508 CFTR, but its additive effect in combination with VX-809, implies that C4 acted on VX-809-modified I507-ATC ΔF508 CFTR. Our results suggest that binding of C4 and VX-809 to ΔF508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.

Genetics of GNE myopathy in the non-Jewish Persian population.

GNE myopathy is an autosomal recessive adult-onset disorder characterized by progressive muscle atrophy and weakness, initially involving the distal muscles, while often sparing the quadriceps. It is caused by variants in the GNE gene that encodes a key bifunctional enzyme in the sialic acid biosynthetic pathway. We investigated the clinical and molecular characteristics of 18 non-Jewish Persian patients from 11 unrelated GNE myopathy families. In addition, we reviewed the previously reported cases and suggest genotype-phenotype correlations for the identified variants. Comprehensive clinical and laboratory evaluations were carried out. Sequencing of the GNE gene was performed using genomic DNA from the patients. Screening of the identified variants was performed in all relevant family members. Molecular analyses identified three causative homozygous GNE variants in 11 families: c.2228T>C (p. M743T) in 7, c.830G>A (p.R277Q) in 2, and one novel variation (c.804G>A) in 2 families that results in a synonymous codon change (p.L268=) and likely creates a novel splice site affecting the protein function. This study confirms that c.2228T>C (p.M743T) is the most prevalent disease-causing variant in the non-Jewish Persian population, but other GNE variants can cause GNE myopathy in this population. The patients with all three different variants had similar ages of onset. The youngest patient was an 18-year-old girl in whom the c.830G>A (p.R277Q) variant was identified, whereas the oldest onset age (31 years) was seen in a male patient with c.804G>A (p.L268=). The results of this investigation expand our knowledge about the genotype-phenotype correlations in GNE myopathy and aid in clinical management and therapeutic interventions.

Investigating the role of site specific synonymous variation in disease association studies

Synonymous codon changes may not always be neutral indicating their significance in disease association studies, which is almost always overlooked. Synonymous substitutions may affect protein-folding rates leading to protein misfolding and aggregation. Genome wide analysis of 2301 mitochondrial genomes is performed to evaluate the significance of synonymous codons in disease association studies. The analysis revealed usage of rare codons at several sites in mitochondrial genes with rare codon usage higher for hydrophobic amino acids. The analysis suggests that variation data in association studies should be analyzed using site-specific codon usage values to infer the potential phenotypic impact of synonymous changes.

Causes and Effects of N-Terminal Codon Bias in Bacterial Genes

Most amino acids are encoded by multiple codons, and codon choice has strong effects on protein expression. Rare codons are enriched at the N terminus of genes in most organisms, although the causes and effects of this bias are unclear. Here, we measure expression from >14,000 synthetic reporters in Escherichia coli and show that using N-terminal rare codons instead of common ones increases expression by ~14-fold (median 4-fold). We quantify how individual N-terminal codons affect expression and show that these effects shape the sequence of natural genes. Finally, we demonstrate that reduced RNA structure and not codon rarity itself is responsible for expression increases. Our observations resolve controversies over the roles of N-terminal codon bias and suggest a straightforward method for optimizing heterologous gene expression in bacteria.

Synonymous codon changes in the oncogenes of the cottontail rabbit papillomavirus lead to increased oncogenicity and immunogenicity of the virus

Papillomaviruses use rare codons with respect to the host. The reasons for this are incompletely understood but among the hypotheses is the concept that rare codons result in low protein production and this allows the virus to escape immune surveillance. We changed rare codons in the oncogenes E6 and E7 of the cottontail rabbit papillomavirus to make them more mammalian-like and tested the mutant genomes in our in vivo animal model. While the amino acid sequences of the proteins remained unchanged, the oncogenic potential of some of the altered genomes increased dramatically. In addition, increased immunogenicity, as measured by spontaneous regression, was observed as the numbers of codon changes increased. This work suggests that codon usage may modify protein production in ways that influence disease outcome and that evaluation of synonymous codons should be included in the analysis of genetic variants of infectious agents and their association with disease.

Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production

In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication.

Polymorphisms in salivary‐gland transcripts of Russian wheat aphid biotypes 1 and 2

Abstract The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko) (Homoptera: Aphididae), is a major pest of small grains. As with plant‐feeding aphids in general, the interaction between RWA and host plants is governed, on the insect side, by proteins and enzymes in saliva. In this work, we examined sequence variations in transcripts encoding proteins and enzymes of RWA salivary glands. We conducted reverse transcription – polymerase chain reaction in RWA biotypes 1 and 2 using primers derived from pea aphid orthologs, and cloned regions of 17 putative salivary gland transcripts. For four of the transcripts, we observed no difference in sequences between the two biotypes. For the other 13 transcripts, for example, the transcripts encoding sucrase, trehalase and protein C002, large amount of variations, both within each biotype and between the two biotypes, were observed. Usually the two biotypes shared only one variant, which was typically the most common variant in both biotypes. Most of the transcripts had more non‐synonymous than synonymous codon changes among their variants. Our results offer possible molecular markers for distinguishing the two biotypes and insights into their evolution.

In this issue: Biotechnology Journal 6/2011

AbstractBirth, life and death of nascent polypeptide chainsJha and Komar, Biotechnol. J. 2011, 6, 623–640The journey of nascent polypeptides from synthesis at the ribosome (“birth”) to full function (“maturity”) involves multiple ways of tight regulation to ensure that proteins achieve their native, functional form. Unproductive co‐translational folding intermediates are subjects of co‐translational degradation (“death”). It has become clear that the kinetics of protein translation is predominantly modulated by synonymous codon usage along the mRNA, thereby providing an active mechanism for coordinating the synthesis, maturation and folding of nascent polypeptides. In this issue, Sujata Jha and Anton A. Komar from the Cleveland State University (OH, USA) provide an overview of the complex co‐translational events that accompany synthesis, maturation, folding and degradation of nascent polypeptide chainsImprint of codons on protein structureDeane and Saunders, Biotechnol. J. 2011, 6, 641–649The “central dogma” of biology is the highly specific translation of genetic code into amino acids and the impossibility of going from amino acid sequence to genetic code, because 18 of the 20 amino acids are encoded by more than one codon. However, synonymous codon changes have been shown to affect protein function without affecting protein expression levels. Evidence for co‐translational folding is growing rapidly, but the influence of codons on the protein structure attained is still highly contentious. It is theorised that the speed of codon translation modulates the time available for protein folding and hence the protein structure. In this issue, Charlotte M. Deane and Rhodri Saunders (Department of Statistics, Oxford University, UK) review past and present research regarding synonymous codons and codon translation speed.Membrane protein production in yeastAshe and Bill, Biotechnol. J. 2011, 6, 707–714Membrane proteins are key players in essential cellular processes and half of all prescription drugs produced from membrane proteins. To enable structural and functional studies membrane proteins have to be produced in recombinant hosts. However, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. Especially defined and rational optimization strategies are needed rather than relying on trial and error. Recent transcriptome and subsequent genetic analysis has identified genes implicated in high‐yielding yeast cells. These results have led to suggestions for cellular alterations to increase yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher production rates. In this issue, Mark P. Ashe and Roslyn M. Bill (Manchester and Birmingham, UK) highlight a potential bottleneck at the protein folding or translocation stage of protein production.