Irina Vladimirovna Biryukova

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

BackgroundThe 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 in vitro and in vivo recombination processes have been developed.ResultsHere, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant E. coli strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-attB 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-attP-site, and second, the λ system is used for excision of inserted vector part, including the plasmid ori-replication and the marker, flanked by λ-attL/R-sites.ConclusionThe 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.

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 Ptac and primers ensuring randomization of the four central nucleotides in the −35 region. DNA fragments containing the tac-like promoters and a selective marker (CmR) 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 Ptac 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.

A new method for the construction of translationally coupled operons in a bacterial chromosome

A new method for the construction of translationally coupled operons in a bacterial chromosome was developed on the basis of the recombineering approach. The method includes the in vitro construction of an artificial operon with an efficiently translated proximal cistron, its insertion into the Escherichia coli chromosome, the modification of the operon via Red-driven insertion of a special “Junction” with an excisable selective marker into the intercistronic region of the initial operon, and the excision of the marker. The Junction structure was designed and tested. The Junction consists of three components. The first component is the E. coli rplC-rplD intercistronic region and serves for placing the TAA codon of the proximal gene in the SD sequence (TAAGGAG) of rplD. The second component is the CmR gene flanked by λattL/R sites in such a fashion that the residual λattB site after λInt/Xis-driven excision of the marker does not contain termination codons in frame with ATG of rplD. The third component is the E. coli trpE-trpD intercistronic region which is added so that TGA of trpE acts a termination codon of the new open reading frame (ORF), while the overlapping (TGATG) ATG of trpD is in the position of the initiation codon of the distal gene of the original operon. The general design of the Junction provides the conversion of the original two-cistron operon into a three-cistron operon with translationally coupled genes, where the coupling of the artificial ORF (rplD’-λattB-’trpE) with the proximal gene is due to the rplC-rplD intercistronic region and its coupling with the distal gene is due to trpE-trpD. The strategy was experimentally implemented to construct an artificial operon Ptac-aroG4-serA5, where the expression the distal serA5 gene was optimized owing to translational coupling in a three-cistron operon.

Pho regulon promoter-mediated transcription of the key pathway gene aroGFbr improves the performance of an l-phenylalanine-producing Escherichia coli strain

DAHP synthase (EC 4.1.2.15) is one of the key enzymes involved in aromatic amino acid biosynthesis in Escherichia coli. An approximately twofold decrease in DAHP synthase activity level was detected in the late growth phase of the l-phenylalanine (Phe)-producing E. coli strain, in which this enzyme encoded by aroG4 is resistant to feedback inhibition. An additional copy of aroG4 that is controlled by promoters of E. coli phoA or pstS genes was integrated into the chromosome of the Phe producer. The choice of promoter was based on the detected activation of the Pho regulon that occurs in response to the depletion of soluble inorganic orthophosphate (Pi) in the medium, provided that the optical density of the Phe-producing culture did not exceed 70% of its maximum value. Pho-mediated aroG4 transcription increased both the accumulation of Phe and the level of DAHP synthase activity in the late stage of batch cultivation on glucose in Pi-limited conditions. Disruption of rpoS led to the improved performance of a PphoA-aroG4 strain. The pstS promoter that is recognized by the σ70/σS-associated core RNA polymerase resulted in the stable maintenance of DAHP synthase activity during long-drawn fed-batch cultivation of the RpoS+ strain carrying the PpstS-aroG4.

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

BackgroundRSF1010 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.ResultsNovel 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 in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5→lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range.ConclusionNovel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis 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.

Metabolic engineering of Escherichia coli for L-tryptophan production

The review summarizes the main approaches applied during the creation of L-tryptophan producing strains based on Escherichia coli for the industrial production of this amino acid. In addition, some prospects for the further improvement of tryptophan producers to increase their productivity and improve their technological characteristics based on systems metabolic engineering approaches are outlined in the review. These approaches can be used to obtain the producers of other aromatic amino acids and tryptophan precursors or derivatives.

Conditional Silencing of the Escherichia coli pykF Gene Results from Artificial Convergent Transcription Protected from Rho-Dependent Termination

PykF is one of two pyruvate kinases in Escherichia coli K-12. λPL was convergently integrated into the chromosome of the MG1655 strain, downstream of pykF, face-to-face with its native promoter. In the presence of λcIts857, efficient pykF ts-silencing was achieved when the 5′-terminus of the PL-originated antisense RNA (asRNA), consisting of the rrnG-AT sequence, converted elongation complexes of RNA polymerase to a form resistant to Rho-dependent transcription termination. pykF silencing was detected by the following features: (a) impaired growth of the strain when pykA was also disrupted and when using ribose as a non-phosphotransferase system-transporting carbon source; (b) a pattern of reduced synthesis of the full-sized pykF mRNA, mediated by reverse transcription PCR, and (c) a significant decrease in PykF activity. The advantages of anti-terminated convergent transcription were clearly manifested in the strains where the rho_a-terminator was inserted specifically to interrupt asRNA synthesis. Most likely, the target gene was silenced by transcriptional interference due to collisions between converging RNA polymerases, although, strictly, the role of cis-asRNA effects could not be excluded. While details of the mechanisms have yet to be determined, anti-terminated convergent transcription is a promising new technique for silencing other target genes.