Genome engineering of the Corynebacterium glutamicum chromosome by the Extended Dual-In/Out strategy.

A novel genome editing method for repeated introduction of foreign DNA, including insertion of rather large DNA fragments, into predesigned points in the Corynebacterium glutamicum chromosome was developed. The method is based on the implementation of the Dual-In/Out strategy, which was previously provided in Escherichia coli according to recombineering-based methods (Minaeva et al., 2008) and allowed step-by-step construction of marker-less plasmid free recombinant strains. The strategy, suggested in the current study, is based on (i) E. coli Rac prophage RecE564/RecT-dependent recombineering; (ii) corynephage ϕ16 (Int/Xis)- and E. coli phage P1 Cre-mediated site-specific recombination systems; and (iii) the development of a C. glutamicum electrotransformation protocol with donor chromosomal DNA for combining of obtained modifications. It was found, that for each tested C. glutamicums strain, the efficiency of the different modifications for electrotransformation fluctuated significantly (up to two orders of magnitude), likely due to the recombinogenic accessibility of the corresponding locus of the bacterial chromosome. To avoid this difficulty, we proposed the phage Mu-driven transposition as a powerful approach for pre-selection of chromosomal regions convenient for single insertions and their further combination in a one strain. Additionally, it was found that the expression of RecE564/RecT coding genes in the recipient strain facilitated the inheritance of the penetrated DNA. It is proposed that the developed strategy in general and its separate elements should be helpful for broadening the genetic toolbox needed for genome editing of targeted C. glutamicum strains.

Efficient production of long double-stranded RNAs applicable to agricultural pest control by Corynebacterium glutamicum equipped with coliphage T7-expression system.

RNA-based pesticides exert their function by suppressing the expression of an essential gene in the target pest through RNA interference caused by double-stranded RNA (dsRNA). Here, we selected target genes for growth suppression of the solanaceous crop pests ladybird beetle (Henosepilachna vigintioctopunctata) and Colorado potato beetle (Leptinotarsa decemlineata)-the death-associated inhibitor of apoptosis protein 1 gene (diap1), and an orthologous gene of the COPI coatomer protein complex (copI), respectively. We constructed a cost-competitive overproduction system for dsRNA using Corynebacterium glutamicum as a host bacterium. The dsRNA expression unit was equipped with two sets of promoters and terminators derived from coliphage T7, and the convergent expression system was designed to be selectively transcribed by T7 RNA polymerase. This expression system efficiently overproduced both target dsRNAs. On culture in a jar fermentor, the yield of diap1-targeting dsRNA (approximately 360 bp) was > 1 g per liter of culture. Long-chain diap1-targeting dsRNAs (up to around 1 kbp) could be produced without a substantial loss of efficiency. dsRNA accumulated in C. glutamicum significantly suppressed larval growth of H. vigintioctopunctata. The dsRNA expression technology developed here is expected to substantially reduce dsRNA production costs. Our method can be applied for a wide range of industrial uses, including agricultural pest control. KEY POINTS: • Overexpression of dsRNA was achieved in C. glutamicum using a coliphage T7 system. • The best strain produced > 1 g/L of the target dsRNA species, for use as an insecticide. • The developed system efficiently produced long dsRNA species, up to ~ 1 kbp.

Universal Actuator for Efficient Silencing of Escherichia coli Genes Based on Convergent Transcription Resistant to Rho-Dependent Termination.

Dynamic control is a distinguished strategy in modern metabolic engineering, in which inducible convergent transcription is an attractive approach for conditional gene silencing. Instead of a simple strong "reverse" (r-) promoter, a three-component actuator has been developed for constitutive genes silencing. These actuators, consisting of r-promoters with different strengths, the ribosomal transcription antitermination-inducing sequence rrnG-AT, and the RNase III processing site, were inserted into the 3'-UTR of three E. coli metabolic genes. Second and third actuator components were important to improve the effectiveness and robustness of the approach. The maximal silencing folds achieved for gltA, pgi, and ppc were approximately 7, 11, and >100, respectively. Data were analyzed using a simple model that considered RNA polymerase (RNAP) head-on collisions as the unique reason for gene silencing and continued transcription after collision with only one of two molecules. It was previously established that forward (f-) RNAP with a trailing ribosome was approximately 13-times more likely to continue transcription after head-on collision than untrailed r-RNAP which is sensitive to Rho-dependent transcription termination (RhoTT). According to the current results, this bias in complex stabilities decreased to no more than (3.0-5.7)-fold if r-RNAP became resistant to RhoTT. Therefore, the developed constitutive actuator could be considered as an improved tool for controlled gene expression mainly due to the transfer of r-transcription into a state that is resistant to potential termination and used as the basis for the design of tightly regulated actuators for the achievement of conditional silencing.

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

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.

Mu-driven transposition of recombinant mini-Mu unit DNA in the Corynebacterium glutamicum chromosome.

A dual-component Mu-transposition system was modified for the integration/amplification of genes in Corynebacterium. The system consists of two types of plasmids: (i) a non-replicative integrative plasmid that contains the transposing mini-Mu(LR) unit bracketed by the L/R Mu ends or the mini-Mu(LER) unit, which additionally contains the enhancer element, E, and (ii) an integration helper plasmid that expresses the transposition factor genes for MuA and MuB. Efficient transposition in the C. glutamicum chromosome (≈ 2 × 10-4 per cell) occurred mainly through the replicative pathway via cointegrate formation followed by possible resolution. Optimizing the E location in the mini-Mu unit significantly increased the efficiency of Mu-driven intramolecular transposition-amplification in C. glutamicum as well as in gram-negative bacteria. The new C. glutamicum genome modification strategy that was developed allows the consequent independent integration/amplification/fixation of target genes at high copy numbers. After integration/amplification of the first mini-Mu(LER) unit in the C. glutamicum chromosome, the E-element, which is bracketed by lox-like sites, is excised by Cre-mediated fashion, thereby fixing the truncated mini-Mu(LR) unit in its position for the subsequent integration/amplification of new mini-Mu(LER) units. This strategy was demonstrated using the genes for the citrine and green fluorescent proteins, yECitrine and yEGFP, respectively.

Gyroscopic effect detection in the colliding-pulse hybridly mode-locked erbium-doped all-fiber ring soliton laser.

We report on the gyroscopic effect detection in the bidirectional ultra-short pulse hybridly mode-locked erbium-doped all-fiber ring soliton laser. Owing to the Kerr nonlinearity contribution through self-phase modulation and self-steepening effects to the carrier-to-envelope phase slip of both clockwise and counterclockwise solitons, wideband controllable tuning of a gyroscope bias point has been demonstrated by means of appropriate adjustment of either intracavity polarization or pump power. Angular velocity detected ranges from 0.12 deg/s to 90 deg/s while rotation sensitivity reaches 7 kHz/(deg/s) for 0.79 m2 single-coil ring gyroscope in agreement with a calculated scale factor value. The bias point drift responsible for the gyroscope resolution capabilities has been studied on long (during 35-h-long continuous operation experiment) and short (∼1 min) time scales.

Complete nucleotide sequence and annotation of the temperate corynephage ϕ16 genome.

The complete genome of ϕ16, a temperate corynephage from Corynebacterium glutamicum ATCC 21792, was sequenced and annotated (GenBank: KY250482). The electron microscopy study of ϕ16 virion confirmed that it belongs to the family Siphoviridae. The ϕ16 genome consists of a linear double-stranded DNA molecule of 58,200 bp (G+C = 52.2%) with protruding cohesive 3'-ends of 14 nt. Four major structural proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting technique. Using bioinformatics analysis, 101 putative ORFs and 5 tRNA genes were predicted. Only 27 putative gene products could be assigned to known biological functions. The ϕ16 genome was divided into functional modules. Seven putative promoters and eight putative unidirectional intrinsic terminators were predicted. One site of putative «-1¼ programmed ribosomal frameshifting was proposed in the phage tail assembly genome region. C. glutamicum genetic tools could be broadened by exploiting the known integrase gene (gp33) and the newly identified excisionase gene (gp47), participating in site-specific recombination between ϕ16-attP/attB.

Generation regimes of bidirectional hybridly mode-locked ultrashort pulse erbium-doped all-fiber ring laser with a distributed polarizer.

We report on the stable picosecond and femtosecond pulse generation from the bidirectional erbium-doped all-fiber ring laser hybridly mode-locked with a coaction of a single-walled carbon nanotube-based saturable absorber and nonlinear polarization evolution that was introduced through the insertion of the short-segment polarizing fiber. Depending on the total intracavity dispersion value, the laser emits conservative solitons, transform-limited Gaussian pulses, or highly chirped stretched pulses with almost 20 nm wide parabolic spectrum in both clockwise (CW) and counterclockwise (CCW) directions of the ring. Owing to the polarizing action in the cavity, we have demonstrated for the first time, to the best of our knowledge, an efficient tuning of soliton pulse characteristics for both CW and CCW channels via an appropriate polarization control. We believe that the bidirectional laser presented may be highly promising for gyroscopic and other dual-channel applications.

High-energy, sub-100 fs, all-fiber stretched-pulse mode-locked Er-doped ring laser with a highly-nonlinear resonator.

We report on ultra-short stretched pulse generation in an all-fiber erbium-doped ring laser with a highly-nonlinear germanosilicate fiber inside the resonator with a slightly positive net-cavity group velocity dispersion (GVD). Stable 84 fs pulses were obtained with a 12 MHz repetition rate at a central wavelength of 1560 nm with a 48.1 nm spectral pulse width (full width at half maximum, FWHM) and 30 mW average output power; this corresponds to the 29.7 kW maximum peak power and 2.5 nJ pulse energy obtained immediately from the oscillator.

Oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases.

Brevibacterium lactofermentum and Corynebacterium glutamicum are important biotechnology species of the genus Corynebacterium. The single-strand DNA annealing protein (SSAP)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains B. lactofermentum AJ1511 and C. glutamicum ATCC13032. When the rpsL gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×10(3) and (6.7±1.3)×10(3) streptomycin-resistant colonies per 10(8) viable cells for the corresponding strains. We tested the influence of several parameters that are known to enhance the efficiency of oligonucleotide-mediated recombination in other bacterial species. Among them, increasing the concentration of oligonucleotides and targeting the lagging strand of the chromosome have proven to have positive effects on both of the tested species. No difference in the efficiency of recombination was observed between the oligonucleotides phosphorothiorated at the 5' ends and the unmodified oligonucleotides or between the oligonucleotides with four mutated nucleotides and those with one mutated nucleotide. The described approach demonstrates that during the adaptation of the recombineering technique, testing SSAP-independent oligonucleotide-mediated recombination could be a good starting point. Such testing could decrease the probability of an incorrect interpretation of the effect of exogenous protein factors (such as SSAP and/or corresponding exonucleases) due to non-optimal experimental conditions. In addition, SSAP-independent recombination itself could be useful in combination with suitable selection/enrichment methods.

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 (P(i)) 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 P(i)-limited conditions. Disruption of rpoS led to the improved performance of a P(phoA)-aroG4 strain. The pstS promoter that is recognized by the σ(70)/σ(S)-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 P(pstS)-aroG4.

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. lambdaP(L) was convergently integrated into the chromosome of the MG1655 strain, downstream of pykF, face-to-face with its native promoter. In the presence of lambdacIts857, efficient pykF ts-silencing was achieved when the 5'-terminus of the P(L)-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.