16S rRNA Promoter Research Articles

Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coli.

Thermophilic cellulases can play a crucial part in the efficient breakdown of cellulose-a major component of lignocellulosic plant biomass, however, their commercial production needs simple and robust biomanufacturing biosystems. In this study, two cellulases (β-glucosidase and endoglucanase) were heterologously expressed in Escherichia coli under a chloroplast-derived constitutive promoter and expression-enhancing terminator. The genes encoding the cellulases were sourced from a thermophilic bacterium Thermotoga maritima to exploit their industrially needed thermotolerance potential. The codon-optimized gene sequences were synthesized and placed under a tobacco chloroplast 16S rRNA promoter (Prrn), along with the 5' UTR (untranslated region) from gene 10 of phage T7 (T7g10). A six-residue long histidine tag (His6-tag) was attached to the N-terminus for protein detection. A high-level of expression of β-glucosidase and endoglucanase in E. coli was recorded from the chloroplast promoter and terminator. Furthermore, the activity assays confirmed that the recombinant enzymes maintained their activity at elevated temperatures. Thermostability analysis showed that recombinant enzymes retained their thermotolerance even after being expressed in a non-native host. Where, β-glucosidase and endoglucanase showed their optimum activities at 90 °C and 100 °C, respectively. Examination of the 3D structures of T. maritima cellulases revealed differential ionic interactions contributing to this high degree of thermotolerance. The study highlights the feasibility of producing thermostable versions of recombinant enzymes in E. coli at high levels. Our finding underscores the potential of this approach to meet industrial demands for efficient enzyme production employing E. coli as a robust biomanufacturing platform.

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts

BackgroundWe investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N-terminally fused to the coding region of cel6A, an endoglucanase from Thermobifida fusca. We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5′UTR, previously reported to enhance protein expression, to regulate the expression of the TetC-cel6A gene. We further investigated the accumulation of TetC-Cel6A under N-deplete growth conditions.ResultsBoth of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII-cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N-deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N-replete medium.ConclusionsThe DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Investigating the Production of Foreign Membrane Proteins in Tobacco Chloroplasts: Expression of an Algal Plastid Terminal Oxidase

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5′UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.

Cloning and expression of β-glucosidases from Bifidobacterium lactis AD011

For the conversion of β-glycosides, 3 genes (BLA_0039, BLA_0141, and BLA_0893) annotated as β-glucosidase (E.C 3.2.1.21) from the genome of Bifidobacterium lactis AD011 were cloned and expressed both in Escherichia coli DH5α and Bifidobacterium bifidum BGN4 using previously established bifidobacterial expression system under the control of 16S rRNA promoter. Deduced amino acid sequence analysis revealed that BLA_0141 is highly similar to β-d-glucosidase from Bifidobacterium breve clb and is included in glycosyl hydrolase family 1 (GHF1) while BLA_0893 belongs to glycosyl hydrolase family 3 (GHF3). Recombinant BLA_0893 showed β-glucosidase activity and converted ginsenoside Rb1 to Rd. Recombinant BLA_0141 showed broad range of activities including β-glucosidase, β-galactosidase, β-xylosidase, cellobiosidase, and α-arabinopyranosidase and converted ginsenoside Rb1 and Rb2 to Rd. They also hydrolized cellobiose into glucose. The recombinant enzyme activities of both BLA_0141 and BLA_0893 were stable around pH 4.5–7.5 and below 50°C.

Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts

Chloroplast transformation offers an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants. An important advantage for the isolation of proteins produced in the chloroplast would be the use of affinity tags for rapid purification by affinity chromatography. To date, only His-tags have been used. In this study, we have tested the feasibility of expressing two additional affinity tags: glutathione-S-transferase (GST) and a His-tagged derivative of the maltose-binding protein (His₆-MBP). By using the chloroplast 16S rRNA promoter and 5' untranslated region of phage T7 gene 10, GST and His₆-MBP were expressed in homoplastomic tobacco plants at approximately 7% and 37% of total soluble protein, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His₆-MBP by using a twin-affinity purification procedure involving first immobilised nickel followed by binding to amylose. Interestingly, expression of GST led to cytoplasmic male sterility. Overall, our work expands the tools available for purifying recombinant proteins from the chloroplast.

Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5′ untranslated region optimization

Microalgae have the potential to be a valuable biotechnological platform for the production of recombinant proteins. However, because of the complex regulatory network that tightly controls chloroplast gene expression, heterologous protein accumulation in a wild-type, photosynthetic-competent algal chloroplast remains low. High levels of heterologous protein accumulation have been achieved using the psbA promoter/5' untranslated region (UTR), but only in a psbA-deficient genetic background, because of psbA/D1-dependent auto-attenuation. Here, we examine the effect of fusing the strong 16S rRNA promoter to the 5' UTR of the psbA and atpA genes on transgene expression in the chloroplast of Chlamydomonas reinhardtii. We show that fusion of the 16S promoter had little impact on protein accumulation from the psbA 5' UTR in a psbA-deficient genetic background. Furthermore, the 16S/psbA promoter/UTR fusion was silenced in the presence of wild-type levels of D1 protein, confirming that the psbA 5' UTR is the primary target for D1-dependent auto-repression. However, fusion of the 16S promoter to the atpA 5' UTR significantly boosts mRNA levels and supports high levels of heterologous protein accumulation in photosynthetic-competent cells. The 16S/atpA promoter/UTR drove LUXCT protein accumulation to levels close to that of psbA in a psbA- background, and drove expression of a human therapeutic protein to levels only twofold lower than the psbA 5' UTR. The 16S/atpA promoter/UTR combination should have utility for heterologous protein production when expression from a photosynthetic-competent microalgal strain is required.

Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO(3) supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l alpha-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.

A genomic-library based discovery of a novel, possibly synthetic, acid-tolerance mechanism in Clostridium acetobutylicum involving non-coding RNAs and ribosomal RNA processing

We generated a genomic library from sheared Clostridium acetobutylicum ATCC 824 DNA, whereby inserts can be expressed in both directions from the thiolase promoter, P thl . Serial transfer of library-bearing C. acetobutylicum cultures exposed to increasing butyrate concentrations enriched for inserts containing fragments of rRNA genetic loci. The selected library inserts were placed so that antisense (to the rRNAs) non-coding RNAs (ncRNAs) would be transcribed from P thl . Different enriched inserts imparted similar butyrate-tolerance characteristics. A minimal tolerance fragment (RDNA7) was identified as the 16S-rRNA promoter region. Expressed on plasmid pRD7 off P thl , RDNA7 can produce putative ncRNAs termed ncRNA RD7. C. acetobutylicum 824(pRD7) showed superior resistance to butyrate and other carboxylic acids. Transcriptional analysis of butyrate stress identified 120 differentially expressed genes between 824(pRD7) and 824(pSOS95del). The few upregulated genes included the ffh gene of the putative signal recognition particle (SRP) system. Northern analysis of ncRNA RD7 and corresponding antisense RNAs demonstrated multiple ncRNA RD7 molecules in 824(pRD7). Several corresponding antisense RNA molecules were identified both in 824(pRD7) and 824(pSOS95del), but at much higher levels in 824(pRD7). Northern analysis of 16S rRNA expression suggested complex RDNA7-dependent rRNA processing. Our data suggest that by hybridizing against unprocessed rRNA precursors, ncRNA RD7 alters rRNA processing, and these alterations result in acid tolerance, possibly through a mechanism involving the Ffh protein.

Comparison of Bacteroides thetaiotaomicron and Escherichia coli 16S rRNA gene expression signals

There are barriers to cross-expression of genes between Bacteroides spp. and Escherichia coli. In this study, a lux-based reporter system was developed for Bacteroides and used to compare the promoter structure and function of a Bacteroides thetaiotaomicron 4001 (BT4001) 16S rRNA promoter with those of E. coli in vivo. Analysis of the BT4001 sequences upstream of the 16S rRNA gene revealed the same overall structure known for E. coli 16S rRNA promoters in that there were two promoters separated by approximately 150 bp. However, the BT4001 16S rRNA promoter contains the proposed Bacteroides -7 and -33 consensus sequences instead of the E. coli -10 and -35 consensus sequences. The biological activity of various configurations of the BT4001 16S rRNA promoter was analysed. Experiments pairing the BT4001 16S rRNA promoter with an E. coli RBS, and vice-versa, confirmed that gene expression between the two species is restricted at the level of transcription. In Bacteroides, a difference in translation initiation also appears to limit expression of foreign genes.

Heterologous expression of cholesterol oxidase in Bifidobacterium longum under the control of 16S rRNA gene promoter of bifidobacteria

We have constructed a constitutive high-level-expression vector for the genus Bifidobacterium and used it to express cholesterol oxidase from Streptomyces coelicola. The promoter region of the 16S rRNA gene was amplified by inverse PCR and used for the construction of pBES16PR. The optimal ribosome-binding site (RBS) for Bifidobacterium was incorporated in pBES16PR. In order to test the efficacy of this expression vector, we constructed pBES16PR-CHOL with the structural gene for cholesterol oxidase under the control of the 16S rRNA promoter, and used it to transform Bifidobacterium longum. The gene was successfully expressed and high level of cholesterol oxidase activity was obtained in B. longum. This is the first report of an expression vector for the genus Bifidobacterium using a 16S rRNA gene promoter and successful expression of cholesterol oxidase.

Artificial control of transgene expression in Chlamydomonas reinhardtii chloroplast using the lac regulation system from Escherichia coli

Systems that can control the expression of a gene both temporally and spatially are important for the study of transgenic plants. Here, we describe an artificial, controllable gene expression system using the lac regulation system from Escherichia coli that we constructed in the Chlamydomonas reinhardtii chloroplast. This system consists of a controllable reporter gene expression cassette and the Lac repressor expression cassette. We created controller promoters by modifying two promoter sequences, rbcL and 16S rRNA, known to be highly active in the C. reinhardtii chloroplast. We inserted a synthetic lac operator sequence in different positions around these promoters, and both repression and induction of transcription were examined using appropriate repressor and inducer molecules. The effect of differing amounts of repressor protein on transcription was also investigated in stable chloroplast transformants. In the case of the modified rbcL promoter, although complete transcription repression was not achieved with the repressor, rapid, full induction was achieved within 1 h. In contrast, although the modified 16S rRNA promoter permitted almost complete repression, full transcription induction was not observed.

Development of Novel Types of Plastid Transformation Vectors and Evaluation of Factors Controlling Expression

Two new vector types for plastid transformation were developed and uidA reporter gene expression was compared to standard transformation vectors. The first vector type does not contain any plastid promoter, instead it relies on extension of existing plastid operons and was therefore named "operon-extension" vector. When a strongly expressed plastid operon like psbA was extended by the reporter gene with this vector type, the expression level was superior to that of a standard vector under control of the 16S rRNA promoter. Different insertion sites, promoters and 5'-UTRs were analysed for their effect on reporter gene expression with standard and operon-extension vectors. The 5'-UTR of phage 7 gene 10 in combination with a modified N-terminus was found to yield the highest expression levels. Expression levels were also strongly dependent on external factors like plant or leaf age or light intensity. In the second vector type, named "split" plastid transformation vector, modules of the expression cassette were distributed on two separate vectors. Upon co-transformation of plastids with these vectors, the complete expression cassette became inserted into the plastome. This result can be explained by successive co-integration of the split vectors and final loop-out recombination of the duplicated sequences. The split vector concept was validated with different vector pairs.

Site-directed gene disruption in Xylella fastidiosa

As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, called pSP3, which replicates both in Escherichia coli and in Xylella. Vector pSP3 was constructed by ligating to the E. coli plasmid pBluescript, a kanamycin resistance gene under the control of the Xylella 16S rRNA promoter and the indigenous Xylella plasmid pXF1.3. Transformation experiments have shown that pSP3 replicates stably in Xylella. When a DNA fragment encompassing part of the Xylella xpsD gene was cloned into pSP3, specific integration of the construct into this gene was observed in 10% of the transformants, as early as after two passages of the culture. These results indicate that this vector can be used to generate site-specific gene disruption by homologous recombination in Xylella fastidiosa.

Site-directed gene disruption in Xylella fastidiosa.

As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, called pSP3, which replicates both in Escherichia coli and in Xylella. Vector pSP3 was constructed by ligating to the E. coli plasmid pBluescript, a kanamycin resistance gene under the control of the Xylella 16S rRNA promoter and the indigenous Xylella plasmid pXF1.3. Transformation experiments have shown that pSP3 replicates stably in Xylella. When a DNA fragment encompassing part of the Xylella xpsD gene was cloned into pSP3, specific integration of the construct into this gene was observed in 10% of the transformants, as early as after two passages of the culture. These results indicate that this vector can be used to generate site-specific gene disruption by homologous recombination in Xylella fastidiosa.

Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNAin vitro

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.