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Riboflavin is widely used as a food additive. Here, multiple strategies were used to increase riboflavin production in Escherichia coli LS31T. First, purR deletion and co-overexpression of fbp, purF, prs, gmk, and ndk genes resulted in an increase of 18.6% in riboflavin titer (reaching 729.7 mg/L). Second, optimization of reduced nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide ratio and respiratory chain activity in LS31T increased the titer up to 1020.2 mg/L. Third, the expression level of the guaC gene in LS31T was downregulated by ribosome binding site replacement, and the riboflavin production was increased by 10.6% to 658.5 mg/L. Then, all the favorable modifications were integrated together, and the resulting strain LS72T produced 1339 mg/L of riboflavin. Moreover, the riboflavin titer of LS72T reached 21 g/L in fed-batch cultivation, with a yield of 110 mg riboflavin/g glucose. To our knowledge, both the riboflavin titer and yield obtained in fed-batch fermentation are the highest ones among all the rationally engineered strains.

The plasmid-driven T7 system (PDT7) is a flexible approach to trigger protein overexpression; however, most of the reported PDT7 rely on many auxiliary elements or inducible systems to attenuate the toxicity from the orthogonality of the T7 system, which limits its application as the one-step cloning and protein expression system. In this study, we developed a stable and robust PDT7 via tailoring the genetic elements. By error-prone mutagenesis, a mutated T7RNAP with TTTT insertion conferred a trace but enough amount of T7RNAP for stable and efficient PDT7, denoted as PDT7m. The replication origin was kept at the same level, while the ribosome binding site (RBS) of the T7 promoter was the most contributing factor, thus enhancing the protein expression twofold using PDT7m. For application as a host-independent screening platform, both constitutive and IPTG-inducible PDT7m were constructed. It was found that each strain harnessed different IPTG inducibilities for tailor-made strain selection. Constitutive PDT7m was successfully used to express the homologous protein (i.e., lysine decarboxylase) or heterologous protein (i.e., carbonic anhydrase, CA) as a one-step cloning and protein expression tool to select the best strain for cadaverine (DAP) or CA production, respectively. Additionally, PDT7m is compatible with the pET system for coproduction of DAP and CA simultaneously. Finally, PDT7m was used for in vivo high-end chemical production of aminolevulinic acid (ALA), in which addition of the T7 terminator successfully enhanced 340% ALA titer, thus paving the way to rapidly and effectively screening the superior strain as a cell factory.

In the described experiments, we identify and clone a novel serine palmitoyltransferase (SPT) isolated from the gram-negative obligatory aerobic bacteria Sphingobium chungbukense strain KCTC2955. SPT catalyzes the first rate-limiting reaction in the de novo biosynthesis of sphingolipids, in S. chungbukense. The DNA sequence of the cloned SPT gene (1791 bp) has an open reading frame encoding 405 amino acids with a predicted molecular mass of 41 kDa; the sequence contains the consensus PLP-binding motif (GTFSKSXXXXGG) which is conserved among serine palmitoyltransferase. The ribosomal binding site GGGA lays 6 bp upstream of the ORF of 1218 bp, which is initiated by an ATG codon and terminated by TGA. The cloned SPT gene succeeded in overproducing the SPT protein under the T7 promoter of the expression vector pET 21a in Escherichia coli BL21(DE3), in which the product amounted to about 20–30% of the total protein isolated from the cell extract. Sphingobium SPT showed about a 68.4–89.3% homology with other enzymes in the serine palmitoyltransferase family, and amino acid residues proposed to be involved in catalysis were conserved. The activity of purified recombinant-SPT using Ni-NTA metal-affinity chromatography was measured via the radioactivity of the [3H]-3-ketodihydrosphingosone (KDS) formation using l-[3H]-serine. We have identified a novel SPT gene from S. chungbukense KCTC2955 and overexpressed the recombinant SPT in E. coli. This study is a first step in understanding the reaction mechanism of SPT in bacteria as well as the first focused on the genus Sphingobium.

Lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides (HMOs), has attracted substantial attention for its nutraceutical potentials and applications in the production of complex HMOs. In this study, Escherichia coli was metabolically engineered to efficiently produce LNT II using glycerol as a carbon source and lactose as a substrate. The UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway was strengthened, and β-1,3-N-acetylglucosaminyltransferase (LgtA) was introduced to construct an LNT II-producing base strain. To increase the titer and yield of LNT II, combinatorial optimization of the copy number and the ribosomal binding site sequence was performed to tune the gene expression strength and translation rates of the pathway enzymes. Next, multipoint mutations were introduced to glucosamine-6-phosphatesynthase (GlmS) to relieve the feedback inhibition. Then, a series of engineered strains were constructed by blocking the futile pathways by the deletion of the relevant genes. Finally, the culture conditions were optimized. LNT II titer was improved step-by-step from 0.53 to 5.52 g/L in shake-flask cultivations. Fed-batch culture of the final engineered strain produced 46.2 g/L of LNT II, with an LNT II productivity and content of 0.77 g/(L·h) and 0.95 g/g dry cell weight, respectively.

Escherichia coli is a widely used platform for metabolic engineering due to its fast growth and well-established engineering techniques. However, there has been a demand for faster-growing E. coli for higher production of desired substances. Here, to increase the growth of E. coli cells, we optimized the expression level of Hfq protein, which plays an essential role in stress responses. Six variants of the hfq gene with a different ribosome binding site sequence and thereby a different expression level were constructed. When the Hfq expression level was optimized in DH5α, its growth rate was increased by 12.1% and its cell density was also increased by 4.5%. RNA-seq and network analyses revealed the upregulation of stress response genes and metabolic genes, which increases the tolerance against pH changes. When the same strategy was applied to five other E. coli strains (BL21 (DE3), JM109, TOP10, W3110, and MG1655), all their growth rates were increased by 18–94% but not all their densities were increased (− 12 − + 32%). In conclusion, the Hfq expression optimization can increase cell growth rate and probably their cell densities as well. Since the hfq gene is highly conserved across bacterial species, the same strategy could be applied to other bacterial species to construct faster-growing strains.

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells., Synthetic biologists often co-opt heterologous parts to affect new functions in living cells, yet such an approach has rarely been extended to structural components of the ribosome. Here, the authors describe generalizable methods to express ribosomes from divergent microbes in E. coli and maximize their function.

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBSs (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, we optimized the expression of three homologous genes responsible for BC production, pgm, galU, and ndp, and thereby greatly increased it under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/l, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.

Intracellular pathogens are responsible for an enormous amount of worldwide morbidity and mortality, and each has evolved specialized strategies to establish and maintain their replicative niche. Listeria monocytogenes is a facultative intracellular pathogen that secretes a pore-forming cytolysin called listeriolysin O (LLO), which disrupts the phagosomal membrane and, thereby, allows the bacteria access to their replicative niche in the cytosol. Nonsynonymous and synonymous mutations in a PEST-like domain near the LLO N terminus cause enhanced LLO translation during intracellular growth, leading to host cell death and loss of virulence. Here, we explore the mechanism of translational control and show that there is extensive codon restriction within the PEST-encoding region of the LLO messenger RNA (mRNA) (hly). This region has considerable complementarity with the 5' UTR and is predicted to form an extensive secondary structure that overlaps the ribosome binding site. Analysis of both 5' UTR and synonymous mutations in the PEST-like domain that are predicted to disrupt the secondary structure resulted in up to a 10,000-fold drop in virulence during mouse infection, while compensatory double mutants restored virulence to WT levels. We showed by dynamic protein radiolabeling that LLO synthesis was growth phase-dependent. These data provide a mechanism to explain how the bacteria regulate translation of LLO to promote translation during starvation in a phagosome while repressing it during growth in the cytosol. These studies also provide a molecular explanation for codon bias at the 5' end of this essential determinant of pathogenesis.

Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro. Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.

Untranslated regions (UTRs) of the transcripts play significant roles in translation regulation and continue to raise many intriguing questions in our understanding of cellular stress physiology. Internal ribosome entry site (IRES) mediated alternative translation initiations are emerging as unique mechanisms. Present study is aimed to indentify a functional short 92 base pair length putative sequence located at the 5' untranslated region of bovine heat shock protein 90 AA1 (Hsp90AA1) may interact with ribosomal as well as eukaryotic initiation factor binding site. Here we have predicted both the two and three dimensional structures of bovine Hsp90AA1 IRES (MF400854) element with their respective free energy. Molecular interactions between bovine RPS5 and IRES have been determined after the preparation of docking complex of IRES bound RPS5. Structure of bovine ribosomal translational initiation factor (TIF) has also been determined and docked with IRES. Molecular interaction between bovine TIF and IRES was analyzed from the complex structure. We further detected the relative expression efficiency of the viral (original) in relation with Hsp90AA1 IRES-driven GFP expression, which revealed that efficiency under the control of identified bovine Hsp90AA1 IRES was slightly lower than viral origin. It was also noted that identified bovine HSP90 IRES may increase the expression level of GFP under in vitro heat stressed condition.

The fast-growing Vibrio natriegens is an attractive robust chassis for diverse synthetic biology applications. However, V. natriegens lacks the suitable constitutive regulatory parts for precisely tuning the gene expression and, thus, recapitulating physiologically relevant changes in gene expression levels. In this study, we designed, constructed, and screened the synthetic regulatory parts by varying the promoter region and ribosome binding site element for V. natriegens with different transcriptional or translational strengths, respectively. The fluorescence intensities of the cells with different synthetic regulatory parts could distribute evenly over a wide range of 5 orders of magnitude. The selected synthetic regulatory parts had good stability in both nutrient-rich and minimal media. The precise combinatorial modulation of galP (GalP = galactose permease) and glk (Glk = glucokinase) from Escherichia coli by using three synthetic regulatory parts with different strengths was confirmed in a phosphoenolpyruvate:carbohydrate phosphotransferase system with inactive V. natriegens strain to alter the glucose transport. This work provides the simple, efficient, and standardized constitutive regulatory parts for V. natriegens synthetic biology.

R-(-)-linalool is widely used in pharmaceutical, agrochemical, and fragrance industries. However, plant extraction furnishes only limited and unstable R-(-)-linalool yields that do not satisfy market demand. Therefore, a sustainable yet efficient and productive method is urgently needed. To induce the R-(-)-linalool biosynthesis pathway in Escherichia coli, we expressed several heterologous (3R)-linalool synthases (LISs) and then chose a suitable LIS from Streptomyces clavuligerus (bLIS) for further study. The bLIS expression was markedly elevated by using optimized ribosomal binding sites and protein fusion tags. To increase the geranyl diphosphate content, we tested various alterations in prenyltransferases and their mutants. The final strain accumulated 100.1 and 1027.3 mg L-1 R-(-)-linalool under shake flask and fed-batch fermentation conditions, respectively. The latter is the highest reported R-(-)-linalool yield to date. This work could lay theoretical and empirical foundations for engineering terpenoid pathways and optimizing other metabolic pathways.

The aim of this study was to construct an expression vector mediated by the dual promoter that can simultaneously drive the recombinant protein production in eukaryotic and prokaryotic cells. The prokaryotic T7 promoter and ribosome binding site (RBS) was cloned downstream of CMV promoter in the eukaryotic expression vector pIRES-neo, and T7 termination sequence was inserted upstream of neomycin phosphotransferase gene to generate the dual promoter vector. The enhanced green fluorescent protein (eGFP) gene was used as reporter gene. Then, the resultant vector was transfected into Chinese hamster ovary (CHO) cells and transformed into Escherichia coli (E. coli) BL21, and the eGFP expression levels were analyzed by fluorescence microscopy, flow cytometry and Western blot, respectively. Fluorescence microscopy revealed that the eGFP was expressed in both CHO cells and E. coli BL21. Flow cytometry showed that the eGFP expression level had no significant difference between the dual promoter vector and control vector in transfected CHO cells. Western blot analysis indicated the eGFP expressed in transformed E. coli. In conclusion, a prokaryotic-eukaryotic double expression vector was successfully constructed, which has potential applications in rapid cloning and expression of recombinant proteins in both prokaryotic and eukaryotic expression systems.

Ricin toxin A subunit (RTA) removes an adenine from the universally conserved sarcin/ricin loop (SRL) on eukaryotic ribosomes, thereby inhibiting protein synthesis. No high affinity and selective small molecule therapeutic antidotes have been reported against ricin toxicity. RTA binds to the ribosomal P stalk to access the SRL. The interaction anchors RTA to the P protein C-termini at a well-defined hydrophobic pocket, which is on the opposite face relative to the active site. The RTA ribosome binding site has not been previously targeted by small molecule inhibitors. We used fragment screening with surface plasmon resonance to identify small molecular weight lead compounds that bind RTA and defined their interactions by crystallography. We identified five fragments, which bound RTA with mid-micromolar affinity. Three chemically distinct binding fragments were cocrystallized with RTA, and crystal structures were solved. Two fragments bound at the P stalk binding site, and the third bound to helix D, a motif distinct from the P stalk binding site. All fragments bound RTA remote from the catalytic site and caused little change in catalytic site geometry. Two fragments uniquely bound at the hydrophobic pocket with affinity sufficient to inhibit the catalytic activity on eukaryotic ribosomes in the low micromolar range. The binding mode of these inhibitors mimicked the interaction of the P stalk peptide, establishing that small molecule inhibitors can inhibit RTA binding to the ribosome with the potential for therapeutic intervention.

Background Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes. Results We changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173–2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined. Conclusions Our findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.

The 5'-untranslated region (5'-UTR) of prokaryotic mRNAs plays an essential role in post-transcriptional regulation. Bacillus species, such as Bacillus subtilis and Bacillus licheniformis, have gained considerable attention as microbial cell factories for the production of various valuable chemicals and industrial proteins. In this work, we developed a portable 5'-UTR sequence for enhanced protein output in the industrial strain B. licheniformis DW2. This sequence contains only ∼30 nt and forms a hairpin structure located right before the open reading frame. The optimized Shine-Dalgarno (SD) sequence was presented as a single strand on the loop of the hairpin for better ribosome recognition and recruitment. By optimizing the free energy of folding, this 5'-element could effectively enhance the expression of eGFP by ∼50-fold and showed good adaptability for other target proteins, including RFP, nattokinase, and keratinase. This 5'-UTR could promote the accessibility of both the SD sequence and start codon, leading to improved efficiency of translation initiation. Furthermore, the hairpin structure protected mRNA against 5'-exonucleases, resulting in enhanced mRNA stability. It is well-known that the stable structure at a ribosome binding site (RBS) impedes initiation in Escherichia coli. In this study, we presented a unique structure at a RBS that can effectively enhance protein production, which is an exception of this prevailing concept. By adjusting a single thermodynamic parameter and holding the other factors affecting protein output constant, a series of 5'-UTR elements with different expression strengths could be rationally designed for wide use in Bacillus sp.

Salmonella enterica serovar Typhimurium invades the intestinal epithelium and induces inflammatory diarrhea using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Expression of the SPI1 T3SS is controlled by three AraC-like regulators, HilD, HilC and RtsA, which form a feed-forward regulatory loop that leads to activation of hilA, encoding the main transcriptional regulator of the T3SS structural genes. This complex system is affected by numerous regulatory proteins and environmental signals, many of which act at the level of hilD mRNA translation or HilD protein function. Here, we show that the sRNA MicC blocks translation of the hilD mRNA by base pairing near the ribosome binding site. MicC does not induce degradation of the hilD message. Our data indicate that micC is transcriptionally activated by SlyA, and SlyA feeds into the SPI1 regulatory network solely through MicC. Transcription of micC is negatively regulated by the OmpR/EnvZ two-component system, but this regulation is dependent on SlyA. OmpR/EnvZ control SPI1 expression partially through MicC, but also affect expression through other pathways, including an EnvZ-dependent, OmpR-independent mechanism. MicC-mediated regulation plays a role during infection, as evidenced by a SPI1 T3SS-dependent increase in Salmonella fitness in the intestine in the micC deletion mutant. These results further elucidate the complex regulatory network controlling SPI1 expression and add to the list of sRNAs that control this primary virulence factor. IMPORTANCE The Salmonella SPI1 T3SS is the primary virulence factor required for causing intestinal disease and initiating systemic infection. The system is regulated in response to a large variety of environmental and physiological factors such that the T3SS is expressed at only the appropriate time and place in the host during infection. Here we show how the sRNA MicC affects expression of the system. This work adds to our detailed mechanistic studies aimed at a complete understanding of the regulatory circuit.

[EN] Models of gene expression considering host-circuit interactions are relevant for understanding both the strategies and associated trade-offs that cell endogenous genes have evolved and for the efficient design of heterologous protein expression systems and synthetic genetic circuits. Here, we consider a small-size model of gene expression dynamics in bacterial cells accounting for host-circuit interactions due to limited cellular resources. We define the cellular resources recruitment strength as a key functional coefficient that explains the distribution of resources among the host and the genes of interest and the relationship between the usage of resources and cell growth. This functional coefficient explicitly takes into account lab-accessible gene expression characteristics, such as promoter and ribosome binding site (RBS) strengths, capturing their interplay with the growth-dependent flux of available free cell resources. Despite its simplicity, the model captures the differential role of promoter and RBS strengths in the distribution of protein mass fractions as a function of growth rate and the optimal protein synthesis rate with remarkable fit to the experimental data from the literature for Escherichia coli. This allows us to explain why endogenous genes have evolved different strategies in the expression space and also makes the model suitable for model-based design of exogenous synthetic gene expression systems with desired characteristics., This work was partially supported by grants MINECO/AEI, EU DPI2017-82896-C2-1-R, and MCIN/AEI/10.13039/501100011033 grant number PID2020-117271RB-C21. F.N.S.-N. is grateful to grant PAID-01-2017 (Universitat Politecnica de Valencia). The authors are very grateful to the anonymous reviewers for their comprehensive and in-depth reviews.

Bacterial small RNAs (sRNAs) are important post-transcriptional regulators in stress responses and virulence. They can be derived from an expanding list of genomic contexts, such as processing from parental transcripts by RNase E. The role of RNase III in sRNA biogenesis is less well understood despite its well-known roles in rRNA processing, RNA decay, and cleavage of sRNA-mRNA duplexes. Here, we show that RNase III processes a pair of cis-encoded sRNAs (CJnc190 and CJnc180) of the food-borne pathogen Campylobacter jejuni. While CJnc180 processing by RNase III requires CJnc190, RNase III processes CJnc190 independent of CJnc180 via cleavage of an intramolecular duplex. We also show that CJnc190 directly represses translation of the colonization factor PtmG by targeting a G-rich ribosome-binding site, and uncover that CJnc180 is a cis-acting antagonist of CJnc190, indirectly affecting ptmG regulation. Our study highlights a role for RNase III in sRNA biogenesis and adds cis-encoded RNAs to the expanding diversity of transcripts that can antagonize bacterial sRNAs.

Cyanobacteria, a group of diverse bacteria capable of oxygenic photosynthesis, are excellent models for investigating many important cellular processes, such as photosynthesis, nitrogen fixation, and prokaryotic cell differentiation. They also have great potential to become the next-generation cell factories for sustainable biosynthesis of valuable products. However, genetic manipulation in cyanobacteria is not as convenient as in other model bacteria. Particularly, handling essential genes in cyanobacteria has been difficult due to the lack of appropriate tools, limiting our understanding of many important cellular functions encoded by them. We recently develop a CRISPR-based method for constructing the conditional mutants of cyanobacterial essential genes by engineering the ribosome binding site to a theophylline-responsive riboswitch. Here, we provide the details of this method. The principle of this method could be used to construct conditional mutants in a wide range of bacterial species.

Organisms often harbor seemingly redundant proteins. In the bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium), the RNA chaperones CspC and CspE appear to play redundant virulence roles because a mutant lacking both chaperones is attenuated, whereas mutants lacking only one exhibit wild-type virulence. We now report that CspC—but not CspE—is necessary to activate the master virulence regulator PhoP when S. Typhimurium experiences mildly acidic pH, such as inside macrophages. This CspC-dependent PhoP activation is specific to mildly acidic pH because a cspC mutant behaves like wild-type S. Typhimurium under other PhoP-activating conditions. Moreover, it is mediated by ugtL, a virulence gene required for PhoP activation inside macrophages. Purified CspC promotes ugtL translation by disrupting a secondary structure in the ugtL mRNA that occludes ugtL’s ribosome binding site. Our findings demonstrate that proteins that are seemingly redundant actually confer distinct and critical functions to the lifestyle of an organism., Graphical Abstract Graphical AbstractRNA chaperone CspC promotes translation of virulence gene ugtL by disrupting secondary structure in ugtL mRNA leader, thereby advancing Salmonella virulence program directed by PhoP/PhoQ inside a macrophage phagosome.

BackgroundBrucella is an important pathogen causing Brucellosis. Vaccine strains obtained by a single knockout cannot combine low virulence and immunogenicity. Our study modified the SD sequence and spacer sequence of the RBS of Brucella to affect its protein expression. We altered the RBS of LPS-associated genes to reduce LPS-associated protein expression while retaining LPS integrity.ResultsWe first established an evaluation system based on the reporter gene red fluorescent protein mCherry. The mCherry expression could be changed by altering the Shine Dalgarno sequence and spacer sequence of RBS. After optimizing the Shine Dalgarno sequence, mCherry expression was increased 4-fold in E. coli and decreased by 1/4 in Brucella. The mCherry expression was increased 1.5-fold in E. coli and decreased to 1/2 in Brucella when the length of the spacer sequence was 0. When the spacer sequence was NA (N = 4, 8, 12nt) or NG (N = 4, 8, 12nt), mCherry expression was reduced in both E. coli and Brucella. Accordingly, two mutant strains were constructed in an attempt to decrease the expression of LptA and LpxO, Brucella LPS-related genes, by 1/4. Silver staining experiments of LPS SDS-PAGE revealed an alteration in the composition of LPS in the two mutant strains. Polymyxin B experiments revealed that both mutant strains were more sensitive to Polymyxin B resistance.Conclusion: In Brucella, the expression of the target gene could be affected by changing the length or the composition of the RBS sequence. The LPS gene remained unchanged while reducing the expression of its associated protein, achieving the original goal of reducing bacterial virulence while retaining immunogenicity. It is a promising strategy to improve the safety and efficacy of vaccines.

We expressed the putative cyclopropane synthase Cfs1 from the mushroom Coprinopsis cinerea and the native bacterial Cfa enzyme in Escherichia coli as fusion proteins with an Nterminal 10xHis-tag and a proteolytic Factor Xa site, using the commercial pET-16b vector with the T7lac promoter for expression. Proteomics detected both proteins with high Mascot scores via trypsin-digested peptides in total cytoplasmic protein samples from transformed BL21(DE3) bacteria, induced by IPTG (isopropyl β-D-1- thiogalactopyranoside) for T7 RNA polymerase production from theT7 phage gene 1 present in the host genome under control by the lacUV5 promoter. In lower amounts, Cfs1 and Cfa were also found in protein samples from non-induced cells based on the known transcriptional leakiness of the pET-system in expression of T7 RNA polymerase. As a novelty, the results on Cfs1 from proteomics suggest that the sequence at the pET-16b-cfs1 fusion point CTCGAGGAT CCTATG (BamHI cloning site in italic, native cfs1 ATG start codon in bold) serves in transcripts as a minor RBS (ribosome binding site) to inadvertently initiate protein synthesis on the native cfs1 start codon. The first peptide MPAHHHPSSSAPCVSFPSSSK of the native Cfs1 protein was found in E. coli which is only possible if the normal cfs1 ATG was recognized as the start codon in protein biosynthesis. The GAGG-stretch in the above DNA sequence with a 7-bases-aligned spacing to the native cfs1 start codon features a partial Shine-Dalgarno (SD) sequence.

The human pathogen Chlamydia trachomatis proceeds through a multi phenotypic developmental cycle with each cell form specialized for different roles in pathogenesis. Understanding the mechanisms regulating this complex cycle has historically been hampered by limited genetic tools. In an effort to address this issue, we developed a translational control system to regulate gene expression in Chlamydia using a synthetic riboswitch. Here we demonstrate that translational control via a riboswitch can be used in combination with a wide range of promoters in C. trachomatis. The synthetic riboswitch E, inducible with theophylline, was used to replace the ribosome binding site of the synthetic promoter T5-lac, the native chlamydial promoter of the pgp4 plasmid gene and an anhydrotetracycline responsive promoter. In all cases the riboswitch inhibited translation, and high levels of protein expression was induced with theophylline. Combining the Tet transcriptional inducible promoter with the translational control of the riboswitch resulted in strong repression and allowed for the cloning and expression of the potent chlamydial regulatory protein, HctB. The ability to control the timing and strength of gene expression independently from promoter specificity is a new and important tool for studying chlamydial regulatory and virulence genes.

BackgroundTranslation initiation is an essential step for fidelity of gene expression, in which the ribosome must bind to the translation initiation region (TIR) and position the initiator tRNA in the P-site (1). For this to occur correctly, the TIR encompassing the ribosome binding site (RBS) needs to be highly accessible (2-5). ΔGunfold is a metric for computing accessibility of the TIR, but there is no automated way to compute it manually with existing software/tools limiting throughput.ResultsΔGunfoldleaderless allows users to automate the ΔGunfold calculation to perform high-throughput analysis. Importantly, ΔGunfoldleaderless allows calculation of TIRs of both leadered mRNAs and leaderless mRNAs which lack a 5’ UTR and which are abundant in bacterial, archaeal, and mitochondrial transcriptomes (4, 6, 7). The ability to analyze leaderless mRNAs also allows one additional feature where users can computationally optimize leaderless mRNA TIRs to maximize their gene expression (8, 9).ConclusionsThe ΔGunfold leaderless package facilitates high-throughput calculations of TIR accessibility, is designed to calculate TIR accessibility for leadered and leaderless mRNA TIRs which are abundant in bacterial/archaeal/organellar transcriptomes and allows optimization of leaderless mRNA TIRs for biotechnology.

Background The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine–Dalgarno (SD) sequence augmented by an upstream translational “enhancer” (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency. Methods and results Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call “CON_G”) that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh. Conclusions The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.

Salmonella enterica serovar Typhimurium invades the intestinal epithelium and induces inflammatory diarrhea using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Expression of the SPI1 T3SS is controlled by three AraC-like regulators, HilD, HilC and RtsA, which form a feed-forward regulatory loop that leads to activation of hilA, encoding the main transcriptional regulator of the T3SS structural genes. This complex system is affected by numerous regulatory proteins and environmental signals, many of which act at the level of hilD mRNA translation or HilD protein function. Here, we show that the sRNA MicC blocks translation of the hilD mRNA by base pairing near the ribosome binding site. This binding blocks translation but does not induce degradation of the hilD message. Our data indicate that micC is transcriptionally activated by SlyA, and SlyA feeds into the SPI1 regulatory network solely through MicC. Transcription of micC is negatively regulated by the OmpR/EnvZ two-component system, but this regulation is dependent on SlyA. OmpR/EnvZ control SPI1 expression partially through MicC, but also affect expression through other mechanisms. MicC-mediated regulation plays a role during infection, as evidenced by an increase in Salmonella fitness in the intestine in the micC deletion mutant that is dependent on the SPI1 T3SS. These results further elucidate the complex regulatory network controlling SPI1 expression and add to the list of sRNAs that control this primary virulence factor.IMPORTANCEThe Salmonella SPI1 T3SS is the primary virulence factor required for causing intestinal disease and initiating systemic infection. The system is regulated in response to a large variety of environmental and physiological factors such that the T3SS is expressed at only the appropriate time and place in the host during infection. Here we show how the sRNA MicC affects expression of the system. This work adds to our detailed mechanistic studies aimed at a complete understanding of the regulatory circuit.

Picobirnaviruses (PBVs) are bi-segmented dsRNA viruses that have been detected in various animal species including vertebrates and invertebrates. In this study, 17 complete or incomplete PBV segment-2 and one unsegmented PBV-like virus sequence were identified in fecal samples from different bird species using viral metagenomic approach. The bird PBV and PBV-like virus retained the conservative motifs that are conserved in dsRNA2 of common PBVs. The RdRp of these 17 PBVs shared the highest Amino acid (aa) identity of 45.90%∼94.19% with previous animal and human PBVs, while the RdRp of the unsegment PBV-like virus shared the highest aa sequence identity of 31.93% with one chicken PBV (GenBank No. MW837829). The unsegmented PBV-like virus unexpectedly used the yeast mitochondrial genetic code (transl_table=3) for all ORFs translation. In addition, the prokaryotic RBS sequence was not only detected upstream to ORF2 at position 360AGGAGG365 of this unsegmented PBV-like virus, but also found upstream to ORF of bird PBV dsRNA2. The presence of the prokaryotic ribosomal binding site in the bird PBV genomes, and the finding of one novel unsegmented PBV-like virus using the yeast mitochondrial genetic code for translation supported recent speculations that PBVs may actually infect prokaryotic or fungal host cells. This study enhanced our understanding of PBVs and provided data support for exploring the real host of PBVs.

Cell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

[EN] Optimal gene expression is central for the development of both bacterial expression systems for heterologous protein production, and microbial cell factories for industrial metabolite production. Our goal is to fulfill industry-level overproduction demands optimally, as measured by the following key performance metrics: titer, productivity rate, and yield (TRY). Here we use a multiscale model incorporating the dynamics of (i) the cell population in the bioreactor, (ii) the substrate uptake and (iii) the interaction between the cell host and expression of the protein of interest. Our model predicts cell growth rate and cell mass distribution between enzymes of interest and host enzymes as a function of substrate uptake and the following main lab-accessible gene expression-related characteristics: promoter strength, gene copy number and ribosome binding site strength. We evaluated the differential roles of gene transcription and translation in shaping TRY trade-offs for a wide range of expression levels and the sensitivity of the TRY space to variations in substrate availability. Our results show that, at low expression levels, gene transcription mainly defined TRY, and gene translation had a limited effect; whereas, at high expression levels, TRY depended on the product of both, in agreement with experiments in the literature., This research was partially supported by grants MINECO/AEI, EU DPI2017-82896-C21-R 662 and MICINN/AEI, EU PID2020-117271RB-C21. F.N.S.-N. thanks the UPV grant number PAID-01-2017.

Background Computational approaches are often used to predict regulatory RNAs in bacteria, but their success is limited to RNAs that are highly conserved across phyla, in sequence and structure. The ANTAR regulatory system consists of a family of RNAs (the ANTAR-target RNAs) that selectively recruit ANTAR proteins. This protein-RNA complex together regulates genes at the level of translation or transcriptional elongation. Despite the widespread distribution of ANTAR proteins in bacteria, their target RNAs haven’t been identified in certain bacterial phyla such as actinobacteria. Results Here, by using a computational search model that is tuned to actinobacterial genomes, we comprehensively identify ANTAR-target RNAs in actinobacteria. These RNA motifs lie in select transcripts, often overlapping with the ribosome binding site or start codon, to regulate translation. Transcripts harboring ANTAR-target RNAs majorly encode proteins involved in the transport and metabolism of cellular metabolites like sugars, amino acids and ions; or encode transcription factors that in turn regulate diverse genes. Conclusion In this report, we substantially diversify and expand the family of ANTAR RNAs across bacteria. These findings now provide a starting point to investigate the actinobacterial processes that are regulated by ANTAR.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated globally and become a major threat to human life. The sequence type (ST) 11 CRKP is a dominant clone in Asia, especially China, but how this clone evolves in vivo, then adapts to host and facilitates dissemination remain largely unknown. We analyzed the genomic dynamics of 4 ST11-CRKP isolates sequencially isolated from the urine of a patient with initial fatal scrotal abscess and finally recovered without effective medication. Genomic differences were identified and their implications for pathogenesis and host adaptation were investigated. The related transcriptional pathways were further explored by RNA-Seq. Genomic analysis identified 4-24 mutations and 94%-100% were synonymous or intergenic. The mutation rate of ST11-CRKP was 2.1×10−6-1.7×10−5 substitutions/site/year over 47 days of antibiotics therapy. During this period, CRKP underwent several adaptive changes including tigecycline resistance and virulence attenuation. Tigecycline resistance was caused by ramR ribosomal binding site (RBS) deletion, which has been described by us previously. In this study, we demonstrated that mutations associated with acyltransferase (act) and ompK26 caused the virulence attenuation of ST11-CRKP. act deletion reduced the production of capsular polysaccharide and enhanced biofilm formation. RNA-Seq analysis revealed that act influenced the expression of ldhA, bglX, mtnK and metE which likely participate in capsular synthesis and biofilm formation. ompK26 affected the virulence by its overexpression caused by the deletion of upstream repressor binding site. Our finding suggested that the broad genomic diversity, high evolutionary capacity and rapid within-host adaptability of ST11-CRKP might contribute to the worldwide dissemination of this clone.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated worldwide and can cause life threatening infections, including pneumonia, bloodstream infections, urinary tract infections, intra-abdominal infection, liver abscess and meningitis. CRKP infection is the leading cause of high mortality in clinical. The sequence type (ST) 11 CRKP is a dominant clone and accounts for 60% of CRKP infections in China. Recently, the ST11-CRKP with high transmissibility are increasingly identified. Understanding how this clone evolved is crucial in controlling its further dissemination. The significance of our research is identifying the in vivo genomic dynamics of ST11-CRKP and the genetic basis for ST11-CRKP to facilitate persistence and dissemination, which will has broader biomedical impacts on understanding of ST11-CRKP dissemination. Furthermore, our study also highlights the importance of monitoring the development of variation in antibiotics susceptibility and virulence of bacteria in clinical practice, considering that pathogens can rapidly adapt to host during the treatment.

Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5′UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22°C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37°C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37°C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment., A.T.-A. was supported by the European Research Council under the European Union's Horizon 2020 research and innovation program [ERC-CoG-2014-646869]; and the Spanish Ministry of Science and Innovation [PID2019-105216GB-I00] grants. I.C. was supported by Centre National de la Recherche Scientifique (CNRS) through an International Project of Scientific Cooperation between CNRS and CSIC [PICS07507]; and by the framework of the labEx Net RNA, French National Research Agency [ANR-10-LABX-0036 and IMCBio ANR-17-EURE-0023]. Funding for open access charge: CSIC Open Access Publication Support Initiative, Unit of Information Resources for Research (URICI).

Bacterial small RNAs (sRNAs) are important post-transcriptional regulators in stress responses and virulence. They can be derived from an expanding list of genomic contexts, such as processing from parental transcripts by RNase E. The role of RNase III in sRNA biogenesis is less well understood despite its well-known roles in rRNA processing, RNA decay, and cleavage of sRNA-mRNA duplexes. Here, we show that RNase III processes a pair of cis-encoded sRNAs (CJnc190 and CJnc180) of the foodborne pathogen Campylobacter jejuni. While CJnc180 processing by RNase III requires CJnc190, In contrast, RNase III processes CJnc190 independent of CJnc180 via cleavage of an intramolecular duplex. We also show that CJnc190 directly represses translation of the colonization factor PtmG by targeting a G-rich ribosome binding site, and uncover that CJnc180 is a cis-acting antagonist of CJnc190, indirectly affecting ptmG regulation. Our study highlights a role for RNase III in sRNA biogenesis and adds cis-encoded RNAs to the expanding diversity of transcripts that antagonize bacterial sRNAs.

Gene overexpression causes a burden on ribosomes, which decreases the expression of other genes and growth rate. Here, we engineer a genetic ribosome actuator that increases gene expression rate and growth rate by over 150% and 80%, respectively. The actuator expresses the hydrolysis domain of the SpoT enzyme (SpoTH) in a bacterial strain with high basal levels of ppGpp, thus increasing ribosome availability. We use this actuator in a feedforward control arrangement, wherein SpoTH is co-expressed with a gene of interest (GOI). Without SpoTH co-expression, activation of the GOI decreases the expression of a constitutive gene and growth rate by over 80% and 40%, respectively. By contrast, with SpoTH co-expression, the ribosome binding site (RBS) of SpoTH can be tuned to achieve inappreciable change of either the expression of the constitutive gene or growth rate when the GOI is activated to the same level. We finally employ the actuator to keep growth rate constant while expressing dCas9 to a level that would otherwise decrease growth rate by more than 40% due to toxicity. This actuator will be relevant in applications where high cellular protein concentrations are desired without growth defects or global reduction of gene expression.

Landmines and other explosive remnants of war pose a global humanitarian problem that claims numerous casualties long after the conflict has ended. As there are no acceptable methodologies for the remote discovery of such devices, current detection practices still require the risky presence of personnel in the minefield. We have recently described bacterial sensor strains capable of reporting the existence of 2,4-dinitrotoluene (DNT) vapors in the soil above 2,4,6-trinitrotoluene (TNT)-based landmines, by generating a bioluminescent or a fluorescent signal. This may allow the identification of landmine location by remote imaging of an area over which the bacteria have been spread. In the study reported herein, we have improved the DNT-detection capabilities of these sensor strains by combining two DNT-responsive Escherichia coli gene promoters, yqjF and azoR, and subjecting them to three cycles of random mutagenesis by error-prone PCR, combined with segmentation and rearrangement (“DNA shuffling”). The activity of selected modified promoters was evaluated with the Aliivibrio fischeri and Photobacterium leiognathi luxCDABEG gene cassettes as the bioluminescent reporters, exhibiting a ten-fold background reduction that has led to a three-fold decrease in detection threshold. Signal intensity was further enhanced by modifying the ribosomal binding site of the yqjF gene promoter. The superior DNT detection capabilities on a solid matrix by the improved sensor strain were demonstrated. • Performance of microbial sensor strains for buried explosives was molecularly enhanced. • Manipulations included random mutagenesis, “DNA shuffling,” and RBS reprogramming. • The re-engineered constructs exhibited superior detection of trace explosives.

Picobirnaviruses (PBVs) are bisegmented double-stranded RNA viruses that have been detected in a wide variety of animal species including invertebrates and in environmental samples. Since PBVs are ubiquitous in feces/gut contents of humans and other animals with or without diarrhea, they were considered as opportunistic enteric pathogens of mammals and avian species. However, the virus remains to be propagated in animal cell cultures, or in gnotobiotic animals. Recently, the classically defined prokaryotic motif, the ribosomal binding site sequence, has been identified upstream of putative open reading frame/s in PBV and PBV-like sequences from humans, various animals, and environmental samples, suggesting that PBVs might be prokaryotic viruses. On the other hand, based on the detection of some novel PBV-like RNA-dependent RNA polymerase sequences that use the alternative mitochondrial genetic code (that of mold or invertebrates) for translation, and principal component analysis of codon usage bias for these sequences, it has been proposed that PBVs might be fungal viruses with a lifestyle reminiscent of mitoviruses. These contradicting observations warrant further studies to ascertain the true host/s of PBVs, which still remains controversial. In this minireview, we have focused on the various findings that have raised a debate on the true host/s of PBVs.

Simple Summary In synthetic biology, it is commonplace to design and insert gene expression constructs into cells for the production of useful proteins. In order to maximise production yield, it is useful to predict the performance of these “engineered cells” in advance of conducting experiments. This is typically a complex task, which in recent years has motivated the use of “whole-cell models” (WCMs) that act as computational tools for predicting different aspects of cell growth. Many useful WCMs exist, however a common problem is their over-simplification of ribosome movement on mRNA transcripts during translation. WCMs typically don’t consider that, for constructs with inefficient (“slow”) codons, ribosomes can stall and form “traffic jams”, thereby becoming unavailable for translation of other proteins. To more accurately address these scenarios, we have built a computational framework that combines whole-cell modelling with a detailed account of ribosome movement on mRNA. We show how our framework can be used to link the modular design of a gene expression construct (via its promoter, ribosome binding site and codon composition) to protein yield during continuous cell culture, with a particular focus on how the optimal design can change over time in the presence or absence of “slow” codons. Abstract The effect of gene expression burden on engineered cells has motivated the use of “whole-cell models” (WCMs) that use shared cellular resources to predict how unnatural gene expression affects cell growth. A common problem with many WCMs is their inability to capture translation in sufficient detail to consider the impact of ribosomal queue formation on mRNA transcripts. To address this, we have built a “stochastic cell calculator” (StoCellAtor) that combines a modified TASEP with a stochastic implementation of an existing WCM. We show how our framework can be used to link a synthetic construct’s modular design (promoter, ribosome binding site (RBS) and codon composition) to protein yield during continuous culture, with a particular focus on the effects of low-efficiency codons and their impact on ribosomal queues. Through our analysis, we recover design principles previously established in our work on burden-sensing strategies, namely that changing promoter strength is often a more efficient way to increase protein yield than RBS strength. Importantly, however, we show how these design implications can change depending on both the duration of protein expression, and on the presence of ribosomal queues.

A protocol for increasing soluble protein expression by fusing the chaperone GroEL apical domain with a gene of interest is described herein. GroEL apical domain, the minichaperone that functions independently of GroES and ATP in protein folding, is cloned downstream of the lambda CII ribosome binding site in the parent pRE vector. The pRE vector has tightly controlled transcription suitable for expressing toxic proteins. The GroEL minichaperone is fused to a glycine–serine rich linker followed by the enterokinase protease recognition sequence. A number of genes that are recalcitrant to protein production in the parent pRE vector 5were cloned into the pRE:GroEL fusion vector and successfully expressed as fusion proteins in Escherichia coli.