Ribosome-binding Sequence Research Articles

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.

Exploring the mechanisms behind the E. coli pH-responsive riboswitch.

Riboswitches are 5’-untranslated regions of mRNA that change their conformation in response to ligand binding, dictating whether the downstream gene is transcribed and/or translated into protein. A “pH-responsive element” (PRE) riboswitch discovered in E. coli, however, does not require a ligand binding event in a traditional sense to rearrange its structure and instead senses the environmental H+ concentration. Specifically, at neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome binding sequence (RBS), whereas at alkaline pH, the PRE adopts a translationally active structure with an exposed RBS. The precise mechanism that controls pH-responsive riboswitching has remained unclear, with two proposed models that are yet to be distinguished; pH either regulates the transcription rate of RNA polymerase (RNAP) or acts on the RNA itself. Previous work suggested that alkaline pH enhances RNAP pause longevity during PRE synthesis to drive folding of structural motifs in the active conformer. I investigated this proposal by performing the first rigorous kinetic study of E. coli RNAP pausing at key sites for PRE folding under different pH conditions. I find that pH influences RNAP pausing, but not in the proposed manner; instead, my experiments uncovered that alkaline pH either decreases or has no effect on RNAP pause longevity. These results indicate that modulation of RNAP pausing is not the sole mechanism by which pH affects PRE folding and invite the possibility that the riboswitch RNA is actively involved in sensing the cytosolic pH (Stephen and Mishanina, J. Biol. Chem. 2022, 298:102302). I am currently conducting chemical probing and cryo-EM studies of PRE structure to explore the pH-induced RNA structural changes in rigorous detail, which will ultimately illuminate the mechanism for how pH-dependent riboswitching occurs.

Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5’-Untranslated Region of the bac Operon

Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.

Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5'-Untranslated Region of the bac Operon.

Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains ofB. subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on thebacABCDEFoperon. Examination of the sequence surrounding the upstream of thebacoperon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of thebacoperon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of thebacoperon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.

Light-Dependent Control of Bacterial Expression at the mRNA Level.

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down- and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several 10-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

Alkaline pH has an unexpected effect on transcriptional pausing during synthesis of the Escherichia coli pH-responsive riboswitch

Riboswitches are 5′-untranslated regions of mRNA that change their conformation in response to ligand binding, allowing post-transcriptional gene regulation. This ligand-based model of riboswitch function has been expanded with the discovery of a “pH-responsive element” (PRE) riboswitch in Escherichia coli. At neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome-binding sequence, whereas at alkaline pH, the PRE adopts a translationally active structure. This unique riboswitch does not rely on ligand binding in a traditional sense to modulate its alternative folding outcomes. Rather, pH controls riboswitch folding by two possible modes that are yet to be distinguished; pH either regulates the transcription rate of RNA polymerase (RNAP) or acts on the RNA itself. Previous work suggested that RNAP pausing is prolonged by alkaline pH at two sites, stimulating PRE folding into the active structure. To date, there has been no rigorous exploration into how pH influences RNAP pausing kinetics during PRE synthesis. To provide that understanding and distinguish between pH acting on RNAP versus RNA, we investigated RNAP pausing kinetics at key sites for PRE folding under different pH conditions. We find that pH influences RNAP pausing but not in the manner proposed previously. Rather, alkaline pH either decreases or has no effect on RNAP pause longevity, suggesting that the modulation of RNAP pausing is not the sole mechanism by which pH affects PRE folding. These findings invite the possibility that the RNA itself actively participates in the sensing of pH.

Ribosome-binding Sequences (RBS) Engineering of Key Genes in Escherichia coli for High Production of Fatty Alcohols

Ribosome-binding Sequences (RBS) Engineering of Key Genes in Escherichia coli for High Production of Fatty Alcohols

Structural and mechanistic study of a pH-responsive riboswitch

Riboswitches are structured, 5'-untranslated pieces of a messenger RNA that control whether the downstream transcript gets translated into protein. Recently, a “pH-responsive element” (PRE) riboswitch was uncovered at the alx gene locus in E. coli. At neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome binding sequence, while at alkaline pH, the PRE adopts a translationally active structure. In this unique riboswitch system, pH governs riboswitch folding by regulating the transcription rate of RNA polymerase (RNAP).

Genome-Scale Analysis of Acetobacterium woodii Identifies Translational Regulation of Acetogenesis.

ABSTRACTAcetogens synthesize acetyl-CoA via the CO2-fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5′ untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity.IMPORTANCE Acetogens are capable of reducing CO2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5′-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5′-untranslated-region bioparts.

Identification and characterization of a hemagglutinin gene from Prevotella intermedia

In this study, we describe the cloning of a gene encoding hemagglutinin from Prevotella intermedia to characterize hemagglutinin function and elucidate its possible role in the infectious process mediated by the organism. We screened genomic libraries created in Escherichia coli JM109, using antibodies against P . intermedia , and the positive clones were subsequently tested for their hemagglutinating activity, using rabbit red blood cells. We isolated a clone of strain 0543 in which, a 3.6-kb P. intermedia chromosomal DNA fragment was inserted into the pUC18 plasmid. Our deletion analysis-based subcloning procedures identified a hemagglutinin gene―designated phg. The open-reading frame of phg encoded a 35-kDa protein, and the expression of a recombinant protein of the predicted size was observed in a fusion protein expression system. While the DNA analysis of phg revealed no homology to known DNA sequences, the putative promoter, ribosome binding, and transcriptional termination sequence were found in the flanking DNA regions. In the N-terminus of the deduced amino acid sequence, we found a consensus leader peptide sequence for secretory proteins. These findings indicate that hemagglutinin might be expressed on the surface of P. intermedia to mediate its function.

A novel RNA aptamer-modified riboswitch as chemical sensor

In this study, a novel label- and immobilization-free RNA aptamer-modified riboswitch-based biosensor was developed by using RNA aptamer modified secondary-structural scaffolds to control the identity of the ribosomal binding sequence (RBS). In the developed sensor, the duplex RNA aptamers-modified cis-repressor sequence is introduced upstream to the RBS of the indicating gene (gfp gene), leading to formatting an RNA bubble due to the none-complementary state of the RNA aptamers in the hairpin structure of the cis-repressor sequence. Without the presence of the target molecule, the ribosome cannot identify the RBS of the indicating gene as the RBS is hidden by the introduced cis-repressor, consequently, the indicating gene in the sensor would not be expressed, demonstrating the absence of the target. On the contrary, with the presence of the target molecule, the binding of aptamer with the target would induce the enlargement of the RNA bubble, leading to the separation of the cis-repressor sequence and RBS. Hence, the indicating gene would be expressed, manifesting the existence of the target. In addition, the developed sensor can quantitatively report the target concentrations by measuring the gfp gene-encoded GFP (green fluorescent protein) concentration. The approach proposed in this study can be used to construct sensors for detecting various chemicals by introducing the corresponding aptamers, therefore, this strategy can potentially provide a new set of analytical tools in the field of analytical chemistry.

Occlusion of the Ribosome Binding Site Connects the Translational Initiation Frequency, mRNA Stability and Premature Transcription Termination

Protein production is controlled by ribosome binding to the messenger RNA (mRNA), quantified in part by the binding affinity between the ribosome and the ribosome binding sequence on the mRNA. Using the E. coli lac operon as model, Ringquist et al. (1992) found a more than 1,000-fold difference in protein yield when varying the Shine-Dalgarno sequence and its distance to the translation start site. Their proposed model accounted for this large variation by only a variation in the binding affinity and the subsequent initiation rate. Here we demonstrate that the decrease in protein yield with weaker ribosome binding sites in addition is caused by a decreased mRNA stability, and by an increased rate of premature transcription termination. Using different ribosome binding site sequences of the E. coli lacZ gene, we found that an approximately 100-fold span in protein expression could be subdivided into three mechanisms that each affected expression 3- to 6-fold. Our experiments is consistent with a two-step ribosome initiation model, in which occlusion of the initial part of the mRNA by a ribosome simultaneously protects the mRNA from both premature transcription termination and degradation: The premature termination we suggest is coupled to the absence of occlusion that allows binding of transcription termination factor, possibly Rho. The mRNA stability is explained by occlusion that prevents binding of the degrading enzymes. In our proposed scenario, a mRNA with lower translation initiation rate would at the same time be “hit” by an increased premature termination and a shorter life-time. Our model further suggests that the transcription from most if not all natural promoters is substantially influenced by premature termination.

Mutually exclusive RNA secondary structures regulate translation initiation of DinQ in Escherichia coli.

Protein translation can be affected by changes in the secondary structure of mRNA. The dinQ gene in Escherichia coli encodes a primary transcript (+1) that is inert to translation. Ribonucleolytic removal of the 44 first nucleotides converts the +1 transcript into a translationally active form, but the mechanism behind this structural change is unknown. Here we present experimental evidence for a mechanism where alternative RNA secondary structures in the two dinQ mRNA variants affect translation initiation by mediating opening or closing of the ribosome binding sequence. This structural switch is determined by alternative interactions of four sequence elements within the dinQ mRNA and also by the agrB antisense RNA. Additionally, the structural conformation of +1 dinQ suggests a locking mechanism comprised of an RNA stem that both stabilizes and prevents translation initiation from the full-length dinQ transcript. BLAST search and multiple sequence alignments define a new family of dinQ-like genes widespread in Enterobacteriaceae with close RNA sequence similarities in their 5′ untranslated regions. Thus, it appears that a whole new family of genes is regulated by the same mechanism of alternative secondary RNA structures.

Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression.

Until recently, engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or one of several methods to knock down wasteful genes. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. Here, we report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. This development is important because the most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme. Our design includes a cis-repressor at the 5' end of the mRNA that forms a stem-loop helix, occluding the ribosomal binding sequence and blocking translation. A trans-expressed activating-RNA frees the ribosomal-binding sequence, which turns on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability. We describe here a cis-repressor that can completely shut off translation of antibiotic-resistance reporters and a trans-activator that restores translation. We have established that it is possible to use these riboregulators to achieve translational control of gene expression over a wide dynamic range. We have also found that a targeting sequence can be modified to develop riboregulators that can, in principle, independently regulate translation of many genes. In a selection experiment, we demonstrated that by subtly altering the sequence of the trans-activator it is possible to alter the ratio of the repressed and activated states and to achieve intermediate translational control.

The sensitivity and significance analysis of parameters in the model of pH regulation on lactic acid production by Lactobacillus bulgaricus.

BackgroundThe excessive production of lactic acid by L. bulgaricus during yogurt storage is a phenomenon we are always tried to prevent. The methods used in industry either control the post-acidification inefficiently or kill the probiotics in yogurt. Genetic methods of changing the activity of one enzyme related to lactic acid metabolism make the bacteria short of energy to growth, although they are efficient ways in controlling lactic acid production.ResultsA model of pH-induced promoter regulation on the production of lactic acid by L. bulgaricus was built. The modelled lactic acid metabolism without pH-induced promoter regulation fitted well with wild type L. bulgaricus (R2LAC = 0.943, R2LA = 0.942). Both the local sensitivity analysis and Sobol sensitivity analysis indicated parameters Tmax, GR, KLR, S, V0, V1 and dLR were sensitive. In order to guide the future biology experiments, three adjustable parameters, KLR, V0 and V1, were chosen for further simulations. V0 had little effect on lactic acid production if the pH-induced promoter could be well induced when pH decreased to its threshold. KLR and V1 both exhibited great influence on the producing of lactic acid.ConclusionsThe proposed method of introducing a pH-induced promoter to regulate a repressor gene could restrain the synthesis of lactic acid if an appropriate strength of promoter and/or an appropriate strength of ribosome binding sequence (RBS) in lacR gene has been designed.

Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis.

BackgroundProkaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.ResultsSequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.ConclusionThe results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-684) contains supplementary material, which is available to authorized users.

Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 104 positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.

Kinetics of Allosteric Transitions in S-adenosylmethionine Riboswitch Are Accurately Predicted from the Folding Landscape

Riboswitches are RNA elements that allosterically regulate gene expression by binding cellular metabolites. The SAM-III riboswitch, one of several classes that binds S-adenosylmethionine (SAM), represses translation upon binding SAM (OFF state) by encrypting the ribosome binding sequence. We have carried out simulations of the RNA by applying mechanical force (f) to the ends of SAM-III, with and without SAM, to get quantitative insights into the f-dependent structural changes. Force-extension (z) curves (FECs) for the apo (ON) state, obtained in simulations in which f is increased at a constant loading rate, show three intermediates, with the first one being the rupture of SAM binding region, which is greatly stabilized in the OFF state. Force-dependent free energy profiles, G(z,f), as a function of z, obtained in equilibrium constant force simulations, reveal the intermediates observed in FECs. The predicted stability difference between the ON and OFF states using G(z,f) is in excellent agreement with experiments. Remarkably, using G(z,f)s and estimate of an effective diffusion constant at a single value of f allows us to predict the f-dependent transition rates using theory of first passage times for both the apo and holo states. To resolve the kinetics of assembly of SAM-III riboswitch in structural terms, we use force stretch-quench pulse sequences in which the force on RNA is maintained at a low (fq) value starting from a high value for a time period tq. Variation of tq over a wide range results in resolution of elusive states involved in the SAM binding pocket and leads to accurate determination of folding times down to fq = 0. Quantitative measure of the folding kinetics, obtained from the folding landscape, allows us to propose that, in contrast to riboswitches regulating transcription, SAM-III functions under thermodynamic control provided the basal concentration of SAM exceeds a small critical value. All of the predictions are amenable to tests in single molecule pulling experiments.

Nonomuraea sp. ID06-A0189 inulin fructotransferase (DFA III-forming): gene cloning, characterization and conservation among other Nonomuraea species

The inulin fructotransferase (DFA III-forming)(EC 4.2.2.18) gene in Nonomuraea sp. ID06-A0189 was amplified from genomic DNA, sequenced and expressed in Escherichia coli. The 1326-bp gene, designated as Nsp-ift, encodes a protein composed of a putative 37-amino-acid signal peptide and 404-amino-acid mature protein. A putative ribosomal binding sequence was identified 12 bases upstream from the start codon. However, a typical bacterial promoter could not be found by in silico analysis. The deduced amino-acid sequence of the enzyme was most similar to that of inulin fructotransferase (DFA I-forming) in Frankia sp. EAN1pec. Phylogenetic analysis of deduced amino-acid sequences indicated that Nonomuraea sp. ID06-A0189 and Frankia sp. EAN1pec inulin fructotransferases formed a distinct clade from those from Arthrobacter sp. H65-7, A. globiformis and Bacillus sp. snu-7 that showed 57, 56 and 56% identity to that of Nsp-ift, respectively. The Nsp-ift without a putative signal peptide was successfully expressed in E. coli and partially purified using His-tag affinity chromatography. The recombinant enzyme displayed optimum temperature between 65 and 70 °C, optimum pH between 5.5 and 6.0 and remained stable up to 70 °C. The properties were identical to those of the original enzyme. Of 10 Nonomuraea species tested by Southern hybridization, enzyme activity measurements and PCR, only Nonomuraea sp. ID06-A0189 has the Nsp-ift gene, suggesting that Nsp-ift is not highly conserved in this genus.

Functional reconstruction of bovine P450scc steroidogenic system in <i>Escherichia coli</i>

Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.