Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome

<p>Abstract</p> <p>Background</p> <p>Plasmid-less, engineered <it>Bacillus </it>strains have several advantages over plasmid-carrier variants. Specifically, their stability and potential ecological safety make them of use in industrial applications. As a rule, however, it is necessary to incorporate many copies of a key gene into a chromosome to achieve strain performance that is comparable to that of cells carrying multiple copies of a recombinant plasmid.</p> <p>Results</p> <p>A plasmid-less <it>B. subtilis </it>JE852-based strain secreting glutamyl-specific protease (GSP-the protein product of the <it>mpr </it>gene from <it>B. amyloliquefaciens</it>) was constructed that exhibits decreased levels of other extracellular proteases. Ten copies of an <it>mpr^B.amy</it>cassette in which the GSP gene was placed between the promoter of the <it>B. amyloliquefaciens rplU-rpmA </it>genes and the Rho-independent transcription terminator were ectopically inserted into designated (3 copies) and random (7 copies) points in the recipient chromosome. The resulting strain produced approximately 0.5 g/L of secreted GSP after bacterial cultivation in flasks with starch-containing media, and its performance was comparable to an analogous strain in which the <it>mpr^B.amy</it>cassette was carried on a multi-copy plasmid.</p> <p>Conclusion</p> <p>A novel strategy for ectopically integrating a cassette into multiple random locations in the <it>B. subtilis </it>chromosome was developed. This new method is based on the construction of DNA fragments in which the desired gene, marked by antibiotic resistance, is sandwiched between "front" and "back" portions of random chromosomal DNA restriction fragments. These fragments were subsequently inserted into the targeted sites of the chromosome using double-cross recombination. The construction of a marker-free strain was achieved by gene conversion between the integrated marked gene and a marker-less variant carried by plasmid DNA, which was later removed from the cells.</p

Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure

<p>Abstract</p> <p>Background</p> <p>The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on <it>in vitro </it>and <it>in vivo </it>recombination processes have been developed.</p> <p>Results</p> <p>Here, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant <it>E. coli </it>strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-<it>attB </it>sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-<it>attP</it>-site, and second, the λ system is used for excision of inserted vector part, including the plasmid <it>ori</it>-replication and the marker, flanked by λ-<it>attL/R</it>-sites.</p> <p>Conclusion</p> <p>The developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.</p

Use of the λ Red-recombineering method for genetic engineering of Pantoea ananatis

<p>Abstract</p> <p>Background</p> <p><it>Pantoea ananatis</it>, a member of the <it>Enterobacteriacea </it>family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of <it>Pantoea ananatis </it>have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The λ Red-recombineering technique previously developed in <it>E. coli </it>and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications.</p> <p>Results</p> <p>In this study, the expression of λ Red genes in <it>P. ananatis </it>was found to be highly toxic. A screening was performed to select mutants of <it>P. ananatis </it>that were resistant to the toxic affects of λ Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of λ <it>gam</it>, <it>bet</it>, and <it>exo </it>genes. Using this strain, procedures for fast introduction of multiple rearrangements to the <it>Pantoea ananatis </it>genome based on the λ Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated.</p> <p>Conclusion</p> <p>The λ Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected <it>P. ananatis </it>recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different <it>P. ananatis </it>strains. Combination of these techniques with λ Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.</p

Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization

<p>Abstract</p> <p>Background</p> <p>RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet.</p> <p>Results</p> <p>Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed <it>in vitro </it>linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, <it>repB</it>, the plasmid loci <it>oriT</it>, <it>mobC </it>and <it>mobA </it>were substituted by the DNA fragment containing P_{<it>lac</it>UV5}→<it>lacI</it>. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after <it>lacI </it>elimination. High stability of both constructed plasmids has been demonstrated in <it>Escherichia coli </it>and <it>Pantoea ananatis</it>. Design of RSFmob allows easy substitution of P_{<it>lac</it>UV5}by any desirable promoter for construction of novel derivatives with changed copy number or host range.</p> <p>Conclusion</p> <p>Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in <it>E. coli </it>and <it>P. ananatis </it>have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.</p

The complete genome sequence of Pantoea ananatis AJ13355, an organism with great biotechnological potential

Pantoea ananatis AJ13355 is a newly identified member of the Enterobacteriaceae family with promising biotechnological applications. This bacterium is able to grow at an acidic pH and is resistant to saturating concentrations of L-glutamic acid, making this organism a suitable host for the production of L-glutamate. In the current study, the complete genomic sequence of P. ananatis AJ13355 was determined. The genome was found to consist of a single circular chromosome consisting of 4,555,536 bp [DDBJ: AP012032] and a circular plasmid, pEA320, of 321,744 bp [DDBJ: AP012033]. After automated annotation, 4,071 protein-coding sequences were identified in the P. ananatis AJ13355 genome. For 4,025 of these genes, functions were assigned based on homologies to known proteins. A high level of nucleotide sequence identity (99%) was revealed between the genome of P. ananatis AJ13355 and the previously published genome of P. ananatis LMG 20103. Short colinear regions, which are identical to DNA sequences in the Escherichia coli MG1655 chromosome, were found to be widely dispersed along the P. ananatis AJ13355 genome. Conjugal gene transfer from E. coli to P. ananatis, mediated by homologous recombination between short identical sequences, was also experimentally demonstrated. The determination of the genome sequence has paved the way for the directed metabolic engineering of P. ananatis to produce biotechnologically relevant compounds

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells

Specific features of l-histidine production by Escherichia coli concerned with feedback control of AICAR formation and inorganic phosphate/metal transport

Abstract Background In the l-histidine (His) biosynthetic pathway of Escherichia coli, the first key enzyme, ATP-phosphoribosyltransferase (ATP-PRT, HisG), is subject to different types of inhibition. Eliminating the feedback inhibition of HisG by the His end product is an important step that enables the oversynthesis of His in breeding strains. However, the previously reported feedback inhibition-resistant mutant enzyme from E. coli, HisGE271K, is inhibited by purine nucleotides, particularly ADP and AMP, via competitive inhibition with its ATP substrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is formed not only during His biosynthesis but also during de novo purine biosynthesis, acts as a natural analog of AMP and substitutes for it in some enzymatic reactions. We hypothesized that AICAR could control its own formation, particularly through the His biosynthetic pathway, by negatively influencing HisG enzymatic activity, which would make preventing ATP-PRT transferase inhibition by AICAR crucial for His overproduction. Results For the first time, both the native E. coli HisG and the previously described feedback-resistant mutant HisGE271K enzymes were shown to be sensitive to inhibition by AICAR, a structural analog of AMP. To circumvent the negative effect that AICAR has on His synthesis, we constructed the new His-producing strain EA83 and demonstrated its improved histidine production. This increased production was particularly associated with the improved conversion of AICAR to ATP due to purH and purA gene overexpression; additionally, the PitA-dependent phosphate/metal (Me2+-Pi) transport system was modified by a pitA gene deletion. This His-producing strain unexpectedly exhibited decreased alkaline phosphatase activity at low Pi concentrations. AICAR was consequently hypothesized inhibit the two-component PhoBR system, which controls Pho regulon gene expression. Conclusions Inhibition of a key enzyme in the His biosynthetic pathway, HisG, by AICAR, which is formed in this pathway, generates a serious bottleneck during His production. The constructed His-producing strain demonstrated the enhanced expression of genes that encode enzymes involved in the metabolism of AICAR to ATP, which is a substrate of HisG, and thus led to improved His accumulation

H⁺-Translocating Membrane-Bound Pyrophosphatase from <i>Rhodospirillum rubrum</i> Fuels <i>Escherichia coli</i> Cells via an Alternative Pathway for Energy Generation

Inorganic pyrophosphatases (PPases) catalyze an essential reaction, namely, the hydrolysis of PPi, which is formed in large quantities as a side product of numerous cellular reactions. In the majority of living species, PPi hydrolysis is carried out by soluble cytoplasmic PPase (S-PPases) with the released energy dissipated in the form of heat. In Rhodospirillum rubrum, part of this energy can be conserved by proton-pumping pyrophosphatase (H+-PPaseRru) in the form of a proton electrochemical gradient for further ATP synthesis. Here, the codon-harmonized gene hppaRru encoding H+-PPaseRru was expressed in the Escherichia coli chromosome. We demonstrate, for the first time, that H+-PPaseRru complements the essential native S-PPase in E. coli cells. 13C-MFA confirmed that replacing native PPase to H+-PPaseRru leads to the re-distribution of carbon fluxes; a statistically significant 36% decrease in tricarboxylic acid (TCA) cycle fluxes was found compared with wild-type E. coli MG1655. Such a flux re-distribution can indicate the presence of an additional method for energy generation (e.g., ATP), which can be useful for the microbiological production of a number of compounds, the biosynthesis of which requires the consumption of ATP