Construction of broad-host-range plasmids for the expression of heterologous genes in Acetobacter methanolicus B58

The construction of different plasmids reported here on the basis of a broad-host-range RSF1010 replicon allows an efficient expression of heterologous genes in the acidophilic methanol-assimilating bacterium Acetobacter methanolicus B58. The promoter-probe vector pRS201 was used for the identification and isolation of the promoter containing sequences derived from the DNA of the Acetobacter phage Acm1. Further, this plasmid was coupled with the Escherichia coli promoters tac and p_r creating the expression vectors pRS201tac and pRS201p_r, respectively. After the insertion of the chloramphenicol acetyltransferase (cat) gene of the cloned promoters downstream, the chloramphenicol acetyltransferase (CAT) was determined in a cell-free extract of both E. coli and A. methanolicus. Using E. coli promoters as well as promoters of the Acetobacter phage Acm1 arranged in tandem with the promoters of the heterologous genes to be expressed, the pectat lyase gene (ptlB) of Erwinia carotovora and the threonine A gene (thrA) of E. coli were successfully expressed in A. methanolicus. The stability of recombinant plasmids under various conditions in A. methanolicus strains was tested using antibiotic-free media.     

Development of a Plasmid Vector for Easy Selection of Strong Promoters

A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of β-lactamase activity in liquid or solid media. Received: 26 July 1999 / Accepted: 22 November 1999     

Requirements for the high-level expression of murine interleukin-3 cDNA inEscherichia coli

Summary Murine interleukin-3 (Mu IL-3) cDNA was previously expressed inEscherichia coli using atac promoter and a constitutive high copy number plasmid vector. We found that significant increases in expression levels could be realized by using thetac promoter for the expression of Mu IL-3 in a plasmid vector possessing a temperature-inducible runaway-replicon. In contrast, use of anlpp promoter under similar conditions did not result in an increase in the Mu IL-3 expression level. Significant differences were observed when the expression levels of IL-3 were monitored in variousE. coli hosts having different genetic backgrounds. A mutant ofE. coli which lacks the protease La was found to increase the level of IL-3 produced. This report describes the effect of a specific protease-deficientE. coli host strain, as well as the effect of different promoters and plasmid replicons on the expression levels and stability of a heterologous gene product.     

Tuning the Expression Level of a Gene Located on a Bacterial Chromosome

A new method of constructing a set of bacterial cell clones varying in the strength of a promoter upstream of the gene of interest was developed with the use of Escherichia coli MG1655 and lacZ as a reporter. The gist of it lies in constructing a set of DNA fragments with tac-like promoters by means of PCR with the consensus promoter P _tac and primers ensuring randomization of the four central nucleotides in the ?35 region. DNA fragments containing the tac-like promoters and a selective marker (Cm ^R) were used to replace lacI and the regulatory region of the lactose operon in E. coli MG1655. Direct LacZ activity assays with independent integrant clones revealed 14 new promoters (out of 4⁴ = 256 possible variants), whose strength varied by two orders of magnitude: LacZ activity in the corresponding strains gradually varied from 10² Miller units with the weakest promoter to 10⁴ Miller units with consensus P _tac Sequencing of the modified promoters showed that randomization of three positions in the ?35 region is sufficient for generating a representative promoter library, which reduces the number of possible variants from 256 to 64. The method of constructing a set of clones varying in expression of the gene or operon of interest is promising for modern metabolic engineering.     

Temperature-regulated expression of the tac/lacI system for overproduction of a fungal xylanase in Escherichia coli

 Temperature-regulated expression of recombinant proteins in the tac promoter (P_tac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the P_tac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42°C were about 4.5 times higher than those of the cells grown at 23°C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl β-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated P_tac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacI ^q, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the λP_L system, the P_tac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best λP_L-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated P_tac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated P_tac system can be used for overproduction of some non-toxic recombinant proteins. Received: 27 June 1995/Received revision: 13 September 1995/Accepted: 30 September 1995     

Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to <Emphasis Type="Italic">Escherichia coli</Emphasis> Genome

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P _trc and P _araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.     

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P _lac , P _tac , or P _BAD derived from Escherichia coli, or promoter regions of phaC1 (P _phaC ) or phaP1 (P _phaP ) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P _lac , P _tac , P _phaC , and P _phaP mediated constitutive gene expression, among which P _tac was the strongest promoter. lacI-P _tac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P _BAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P _phaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P _phaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.     

Comparison of promoter activities of heterologous and homologous promoters in Bacillus subtilis

Summary We have constructed promoter probe vectors with the Escherichia coli galactokinase monitoring system that can be used in Bacillus subtilis. In vivo studies with these vectors demonstrated that the E. coli trp and tac(trp::lac) promoter regions could be utilized in B. subtilis. These promoter regions and the promoter region for the erythromycin resistance gene originating from Staphylococcus aureus were preferentially utilized during the stationary growth phase of B. subtilis, whereas the B. subtilis P21K and P29K promoters were utilized during the exponential growth phase and decreased rapidly during the stationary phase. The apparent strength of these promoters of E. coli in B. subtilis, in terms of galactokinase units, was comparable with those of the B. subtilis promoters.     

Promoter engineering to optimize recombinant periplasmic Fab′ fragment production in Escherichia coli

Fab’ fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab’ fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab’ fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the P_tac promoter, present in the plasmid, pTTOD‐A33 Fab’, to a P_tic promoter which has been shown by others to direct expression at a 35% reduced rate compared to P_tac. We characterized the resultant production trains in which either P_tic or P_tac promoters direct Fab’ fragment expression. The P_tic promoter strain showed a 25?30% reduction in Fab’ expression relative to the original P_tac strain. Reduced Fab’ leakage and increased viability over the course of a fed‐batch fermentation were also observed for the P_tic promoter strain. We conclude that cell design steps such as the P_tac to P_tic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab’ fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840–847, 2016     

Single-step linker-based combinatorial assembly of promoter and gene cassettes for pathway engineering

A linker-based approach for combinatorial assembly of promoter and gene cassettes into a biochemical pathway is developed. A synthetic library containing 144 combinations, with 3 promoters and 4 gene variants, was constructed for the ackA and pta genes of the acetate utilization pathway in E. coli. The in vitro isothermal assembled library was then introduced into E. coli mutant (acs-, pta-, ackA-) and selected for restoration of acetate utilization. 81% of the colonies screened contained the complete functional pathway. Thirty positive clones were analyzed and accounted for 10% of the 144 promoter?Cgene combinations.     

Molecular cloning and expression ofThiobacillus ferrooxidans chromosomal ribulose bisphosphate carboxylase genes inEscherichia coli

The genes coding ford-ribulose-1,5-bisphosphate carboxylase (RuBPCase) from an iron-oxidizing bacterium,Thiobacillus ferrooxidans, were cloned into anEscherichia coli plasmid, pUC18. The recombinant plasmid, termed pTR11, contained a 4.0-kb PstI fragment including the entire coding regions for both large and small subunits of RuBPCase.Escherichia coli carrying pTR11 did not show any CO₂-fixing activity. However, a derivative plasmid with an appropriate deletion, which was placed under the control of atac promoter, conferred ribulose bisphosphate-dependent CO₂-fixing activity on the host cell. Analysis of gel-filtration chromatography of the RuBPCase synthesized inE. coli revealed that it had a hexadecameric form like the native enzyme ofT. ferrooxidans.     

Comparison of the cell surface barrier and enzymatic modification system inBrevibacterium flavum andB. lactofermentum

To investigate impediments to plasmid transformation inBrevibacterium flavum BF4 andB. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties.B. lactofermentum was more sensitive to growth inhibition by glycine thanB. flavum. Release of cellular proteins during sonication was more rapid forB. lactofermentum than forB. flavum. Plasmid DNA (pCSL17) isolated fromB. flavum transformed recipient McrBC⁺ strains ofEscherichia coli with lower efficiency than McrBC⁻.McrBC digestion of this DNA confirmed thatB. flavum contain methylated cytidines in the target sequence ofMcrBC sequences butB. lactofermentum contained a different methylation pattern. DNA derived from theB. lactofermentum transformed recipient EcoKR⁺ strains ofE. coli with lower efficiency than EcoKR⁻, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.     

l-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum

Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for l-valine production by over-expressing ilvEBN ^r C genes at 31?°C in 72?h fermentation. Different strategies were carried out to reduce the by-products’ accumulation in l-valine fermentation and also to increase the availability of precursor for l-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of l-isoleucine. Effect of different relative dissolved oxygen on l-valine production and by-products’ formation was recorded, indicating that 15?% saturation may be the most appropriate relative dissolved oxygen for l-valine fermentation with almost no l-lactic acid and l-glutamate formed. To minimize l-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of l-alanine accumulated by alaT inactivated strains harboring ilvEBN ^r C genes, l-alanine concentration was reduced to 0.18?g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, and 0.22?g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. Meanwhile, l-valine production and conversion efficiency were enhanced to 31.15?g/L and 0.173?g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, 38.82?g/L and 0.252?g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. This study provides combined strategies to improve l-valine yield by minimization of by-products’ production.     

Metabolic Engineering of Carotenoid Biosynthesis in Escherichia coli by Ordered Gene Assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 μg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p_R promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.