The Cyanobacterium Synechocystis sp. Strain PCC 6803 Expresses a DNA Methyltransferase Specific for the Recognition Sequence of the Restriction Endonuclease PvuI

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N⁶-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C⁵-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5′-CGATCG-3′, corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation SynMI (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.     

The NIaIV restriction and modification genes of Neisseria lactamica are flanked by leucine biosynthesis genes

The genes encoding the Neisseria lactamica restriction endonuclease IV (R.NIaIV) and its cognate DNA methyltransferase (M.NlaIV), both of which recognize the sequence GGNNCC, have been cloned in Escherichia coli and overexpressed using the T7 polymerase/promoter system. Analysis of a sequenced 3.58 kb fragment established the gene order, leuD-M.NlaIV-R.NlaIV-leuB. The predicted primary sequence of M.NlaIV (423 amino acids) shows the highest degree of identity to a pair of cytosine-specific methyltransferases, M.BanI (44.9%) and M.HgiCI (44.3%), which recognize the sequence GGYRCC (Y, pyrimidines; R, purines). In contrast, the R.NlaIV protein sequence (243 amino acids) is unique in the existing database, a situation that holds for most endonucleases. Flanking the NlaIV modification and restriction genes are homologues of the leuD and leuB genes of enteric bacteria, which code for enzymes in the leucine biosynthesis pathway. This gene context implies a possible new mode of gene regulation for the RM.NlaIV system, which would involve a mechanism similar to the recently discovered leucine/Lrp regulon in E. coli.     

Molecular Analysis of the Corynebacterium glutamicum Transketolase Gene

Transketolase is important in production of the aromatic amino acids in Corynebacterium glutamicum. The complete nucleotide sequence of the C. glutamicum transketolase gene has been identified. The DNA-derived protein sequence is highly similar to the transketolase of Mycobacterium tuberculosis, taxonomically related to C. glutamicum. The alignment of the N-terminus regions between both transketolases showed TTG to be the most probable start codon. Potential ribosomal binding and promoter regions were situated upstream from the TTG. The deduced amino acid sequence consists of 700 residues with a calculated molecular mass of 75 kDa, and contains all amino acid residues involved in cofactor and substrate binding in the well-characterized yeast transketolase sequence.     

DNA sequence of the araBAD-araC controlling region in Salmonella typhimurium LT2

The araB and araC genes of Salmonella typhimurium have been cloned onto the plasmid pBR322. Restriction analysis and subcloning of restriction fragments localized these genes to a 4.4 kb DNA fragment. Complementation analysis revealed that the cloned araB and araC genes from S. typhimurium complemented araB and araC mutant strains of Escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and araC mutant strains of S. typhimurium. The DNA sequences was determined for the S. typhimurium araB and araC controlling region and for the initially translated portions of these genes. The nucleotide sequence of the araB promoter was 87% homologous with the same region in E. coli and contained no deletions or insertions relative to the E. coli sequence. The presumed AUG codon corresponding to the amino terminus of the S. typhimurium araC protein was in the same location as in E. coli. There was, however, considerable divergence from the E. coli sequence preceding the translation start site. The nucleotide sequence of the initial 237 bp in the open reading frame of the S. typhimurium araC gene was 78% homologous with the same sequence in E. coli. By comparison, the amino acid sequence for this region was 91% conserved.     

A CRISPR/dCas9-assisted system to clone toxic genes in Escherichia coli

BackgroundThe cloning of toxic genes in E. coli requires strict regulation of the target genes' leaky expression. Many methods facilitating successful gene cloning of toxic genes are commonly exploited, but the applicability is severely limited.MethodsA CRISPR/dCas9-assisted system was used to clone toxic genes in E. coli. The plasmid-based and genome-integrated systems were designed in this study. And the green fluorescent protein characterization system was used to test the repression efficiency of the two systems.ResultsWe optimized the plasmid-based CRISPR/dCas9-assisted repression system via testing different sgRNAs targeting the Ptrc promoter and achieved inhibition efficiency up to 64.8%. The genome-integrated system represented 35.9% decreased GFP expression and was successfully employed to cloned four toxic genes from Corynebacterium glutamicum in E. coli.ConclusionsUsing this method, we successfully cloned four C. glutamicum-derived toxic genes that had been failed to clone in conventional ways. The CRISPR/dCas9-assisted gene cloning method was a promising tool to facilitate precise gene cloning of different origins in E. coli.General significanceThis system will be useful for cloning toxic genes from different origins in E. coli, and can accelerate the related research of gene characterization and heterologous expression in the metagenomic era.     

Synthetic promoter libraries for Corynebacterium glutamicum

The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7–59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.     

Identification and characterization of CbeI,a novel thermostable restriction enzyme from <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> DSM 6725 and a member of a new subfamily of HaeIII-like enzymes

Potent HaeIII-like DNA restriction activity was detected in cell-free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from Escherichia coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981-bp nucleic acid sequence, was optimally active at temperatures between 75°C and 85°C, and was stable for more than 1 week when stored at 35°C. The cleavage sequence was determined to be 5′-GG/CC-3′, indicating that CbeI is an isoschizomer of HaeIII. A search of the C. bescii genome sequence revealed the presence of both a HaeIII-like restriction endonuclease (Athe 2438) and DNA methyltransferase (Athe 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this restriction/modification activity is widespread in this genus. A phylogenetic analysis based on sequence alignment and conserved motif searches identified features of CbeI distinct from other members of this group and classified CbeI as a member of a novel subfamily of HaeIII-like enzymes.     

The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

Methylation at the 5-position of cytosine [m⁵C (5-methylcytidine)] occurs at three RNA nucleotides in Escherichia coli. All these modifications are at highly conserved nucleotides in the rRNAs, and each is catalyzed by its own m⁵C methyltransferase enzyme. Two of the enzymes, RsmB and RsmF, are already known and methylate 16S rRNA at nucleotides C967 and C1407, respectively. Here, we report the identity of the third E. coli m⁵C methyltransferase. Analysis of rRNAs by matrix-assisted laser desorption/ionization mass spectrometry showed that inactivation of the yccW gene leads to loss of m⁵C methylation at nucleotide 1962 in E. coli 23S rRNA. This methylation is restored by complementing the knockout strain with a plasmid-encoded copy of the yccW gene. Purified recombinant YccW protein retains its specificity for C1962 in vitro and methylates naked 23S rRNA isolated from the yccW knockout strain. However, YccW does not methylate assembled 50S subunits, and this is somewhat surprising as the published crystal structures show nucleotide C1962 to be fully accessible at the subunit interface. YccW-directed methylation at nucleotide C1962 is conserved in bacteria, and loss of this methylation in E. coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m⁵C methyltransferases RsmB and RsmF and is in fact more similar to known m⁵U (5-methyluridine) RNA methyltransferases. In keeping with the previously proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI.     

Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum

Bacillus subtilis sacB gene with its 463 bp upstream region including its native promoter has been used for marker-free gene deletion in Corynebacterium glutamicum, but the role of this upstream region is not clear. In this study, it was demonstrated that the upstream region of sacB failed to efficiently promote its expression in C. glutamicum, and the native promoter of sacB is weak in C. glutamicum. The expression level of sacB under its native promoter in C. glutamicum is not high enough for cells to confer sucrose sensitivity. Therefore, a new promoter PlacM and a novel vector pDXW-3 were constructed. PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum. The pDXW-3 contains B. subtilissacB with the PlacM fused at the 5′-end, a general Escherichia coli replicon oriE for easy cloning, a kanamycin resistance marker for selection, and a multiple unique restriction sites for XhoI, NotI, EagI, SalI, SacI, BamHI, and NheI, respectively. By using pDXW-3, the aceE gene in the chromosome of C. glutamicum was deleted. This sacB-based system should facilitate gene disruption and allelic exchange by homologous recombination in many bacteria.     

Towards a better understanding of Lactobacillus rhamnosus GG - host interactions

Background

An industrial approach to protein production demands maximization of cloned gene expression, balanced with the recombinant host’s viability. Expression of toxic genes from thermophiles poses particular difficulties due to high GC content, mRNA secondary structures, rare codon usage and impairing the host’s coding plasmid replication. TaqII belongs to a family of bifunctional enzymes, which are a fusion of the restriction endonuclease (REase) and methyltransferase (MTase) activities in a single polypeptide. The family contains thermostable REases with distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI and a few enzymes found in mesophiles. While not being isoschizomers, the enzymes exhibit amino acid (aa) sequence homologies, having molecular sizes of ~120 kDa share common modular architecture, resemble Type-I enzymes, cleave DNA 11/9 nt from the recognition sites, their activity is affected by S-adenosylmethionine (SAM).

Results

We describe the taqIIRM gene design, cloning and expression of the prototype TaqII. The enzyme amount in natural hosts is extremely low. To improve expression of the taqIIRM gene in Escherichia coli (E. coli), we designed and cloned a fully synthetic, low GC content, low mRNA secondary structure taqIIRM, codon-optimized gene under a bacteriophage lambda (λ) P _R promoter. Codon usage based on a modified ‘one amino acid–one codon’ strategy, weighted towards low GC content codons, resulted in approximately 10-fold higher expression of the synthetic gene. 718 codons of total 1105 were changed, comprising 65% of the taqIIRM gene. The reason for we choose a less effective strategy rather than a resulting in high expression yields ‘codon randomization’ strategy, was intentional, sub-optimal TaqII in vivo production, in order to decrease the high ‘toxicity’ of the REase-MTase protein.

Conclusions

Recombinant wt and synthetic taqIIRM gene were cloned and expressed in E. coli. The modified ‘one amino acid–one codon’ method tuned for thermophile-coded genes was applied to obtain overexpression of the ‘toxic’ taqIIRM gene. The method appears suited for industrial production of thermostable ‘toxic’ enzymes in E. coli. This novel variant of the method biased toward increasing a gene’s AT content may provide economic benefits for industrial applications.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

The XmnI restriction-modification system: Cloning,expression, sequence organization and similarity between the R and M genes

The xmnIRM genes from Xanthomonas manihotis 7AS1 have been cloned and expressed in Escherichia coli. The nucleotide (nt) sequences of both genes were determined. The XmnI methyltransferase (MTase)-encoding gene is 1861 by in length and codes for 620 amino acids (aa) (68660 Da). The restriction endonuclease (ENase)-encoding gene is 959 by long and therefore codes for a 319-aa protein (35275 Da). The two genes are aligned tail to tail and they overlap at their respective stop codons. About 4 × 10⁴ units/g wet cell paste of R·XmnI was obtained following IPTG induction in a suitable E. coli host. The xmnIR gene is expressed from the T7 promoter. M·XmnI probably modifies the first A in the sequence, GAA(N)₄TTC. The xmnIR and M genes contain regions of conserved similarity and probably evolved from a common ancestor. M·XmnI is loosely related to M·EcoRI. The XmnI R-M system and the type-I R-M systems probably derived from a common ancestor.     

The NIaIV restriction and modification genes of Neisseria lactamica are flanked by leucine biosynthesis genes

The genes encoding the Neisseria lactamica restriction endonuclease IV (R.NIaIV) and its cognate DNA methyltransferase (M.NlaIV), both of which recognize the sequence GGNNCC, have been cloned in Escherichia coli and overexpressed using the T7 polymerase/promoter system. Analysis of a sequenced 3.58 kb fragment established the gene order, leuD-M.NlaIV-R.NlaIV-leuB. The predicted primary sequence of M.NlaIV (423 amino acids) shows the highest degree of identity to a pair of cytosine-specific methyltransferases, M.BanI (44.9%) and M.HgiCI (44.3%), which recognize the sequence GGYRCC (Y, pyrimidines; R, purines). In contrast, the R.NlaIV protein sequence (243 amino acids) is unique in the existing database, a situation that holds for most endonucleases. Flanking the NlaIV modification and restriction genes are homologues of the leuD and leuB genes of enteric bacteria, which code for enzymes in the leucine biosynthesis pathway. This gene context implies a possible new mode of gene regulation for the RM.NlaIV system, which would involve a mechanism similar to the recently discovered leucine/Lrp regulon in E. coli.Abbreviations R purines - Y pyrimidines - W adenine or thymine - N any base     

Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving l-phenylalanine (l-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of l-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of l-Phe. In contrast, overexpression of aroF ^wt or pheA ^fbr from E. coli significantly increased l-Phe production. Co-overexpression of aroF ^wt and pheA ^fbr improved the titer of l-Phe to 4.46 ± 0.06 g l^?1. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of l-Phe production. Co-overexpression of the mutated pheA ^fbr and the wild-type gene aroH resulted in the production of l-Phe up to 4.64 ± 0.09 g l^?1. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.     

Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.     

Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum

PS2 is one of two major proteins detected in the culture media of various Corynebacterium glutamicum strains. The coding and promoter regions of the cspB gene encoding PS2 were cloned in lambda gt11 using polyclonal antibodies raised against PS2 for screening. Expression of the cspB gene in Escherichia coli led to the production of a major anti-PS2 labelled peptide of 63 000 Da, corresponding presumably to the mature form of PS2. It was detected in the cytoplasm, periplasm and surrounding medium of E. coli. Three other slower migrating bands of 65000, 68 000 and 72 000 Da were detected. The largest one probably corresponds to the precursor form of PS2 in E. coli. Analysis of the nucleotide sequence revealed an open reading frame (ORF) of 1533 nucleotides. The deduced 510-amino-acid polypeptide had a calculated molecular mass of 55 426 Da. According to the predicted amino acid sequence, PS2 is synthesized with a W-terminal segment of 30-amino-acid residues reminiscent of eukaryotic and prokaryotic signal pep-tides, and a hydrophobic domain of 21 residues near the C-terminus. Although no significant homologies were found with other proteins, it appears that some characteristics and the amino acid composition of PS2 share several common features with surface-layer proteins. The cspB gene was then disrupted in C. glutamicum by gene replacement. Freeze-etching electron microscopy performed on the wild-type strain indicated that the cell wall of C. glutamicum is covered with an ordered surface of proteins (surface layer, S-layer) which is in very close contact with other cell-wall components. These structures are absent from the cspB-disrupted strain but are present after reintroduction of the cspB gene on a plasmid into this mutant. Thus we demonstrate that the Slayer protein is the product of the cspB gene.     

Gene Cloning and Characterization of Recombinant RNase HII from a Hyperthermophilic Archaeon

We have cloned the gene encoding RNase HII (RNase HII_Pk) from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an rnh mutant strain of Escherichia coli. This gene was expressed in an rnh mutant strain of E. coli, the recombinant enzyme was purified, and its biochemical properties were compared with those of E. coli RNases HI and HII. RNase HII_Pk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer. Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and E. coli RNase HII proteins, respectively. The enzymatic activity was determined at 30°C and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co²⁺ for RNase HII_Pk, Mn²⁺ for E. coli RNase HII, and Mg²⁺ for E. coli RNase HI. The specific activity of RNase HII_Pk determined in the presence of the most preferred metal ion was 6.8-fold higher than that of E. coli RNase HII and 4.5-fold lower than that of E. coli RNase HI. Like E. coli RNase HI, RNase HII_Pk and E. coli RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3′ bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HII_Pk and E. coli RNases HI and HII are structurally and functionally related to one another.     

Cloning and characterization of the BglII restriction–modification system reveals a possible evolutionary footprint

BglII, a type II restriction–modification (R–M) system from Bacillus globigii, recognizes the sequence 5′-AGATCT-3′. The system has been cloned into E. coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B. globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR). Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R–M system. Oligonucleotide probes specific for the 5′ end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step. The nucleotide sequence of the system has been determined, and several interesting features have been found. The genes are tandemly arranged, with bglIIR preceding bglIIM. The amino acid sequence of M.BglII is compared to those of other known MTases. A third gene encoding a protein with sequence similarity to known C elements of other R–M systems is found upstream of bglIIR. This is the first instance of a C gene being associated with an R–M system where the R and M genes are collinear. In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII. These may shed light on the evolution of the R–M system.     

Chemical synthesis and in vivo hyperexpression of a modular gene coding for Escherichia coli translational initiation factor IF1

Summary An artificial gene encoding the Escherichia coli translational initiation factor IF1 was synthesized based on the primary structure (71 amino acid residues) of the protein. Codons for individual amino acids were selected on the basis of the preferred codon usage found in the structural genes for the initiation factor IF2 of E. coli and Bacillus stearothermophilus, both of which can be expressed at high levels in E. coli cells. We gave the IF1 gene a modular structure by introducing specific restriction enzyme sites into the sequence, resulting in units of three to ten codons. This was conceived to facilitate site-directed mutagenesis of the gene and thus to obtain IF1 with specific amino acid alterations at desired positions. The IF1 gene was assembled by shot-gun ligation of 9 synthetic oligodeoxyri-bonucleotides ranging in size from 31 to 65 nucleotides and cloned into an expression vector to place the gene under the control of an inducible promoter. Upon induction, E. coli cells harbouring the artificial gene were found to produce large amounts (60 mg/100 g cells) of a protein indistinguishable from natural IF1 in both chemecal and biological properties.