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

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Application of leucine dehydrogenase Bcd from Bacillus subtilis for l-valine synthesis in Escherichia coli under microaerobic conditions

Microaerobic cultivation conditions have been shown experimentally and theoretically to improve the performance of a number of bioproduction systems. However, under these conditions, the production of l-valine by Escherichia coli is decreased mainly because of a redox cofactor imbalance and a decreased l-glutamate supply. The synthesis of one mole of l-valine from one mole of glucose generates two moles of NADH via glycolysis but consumes a total of two moles of NADPH, one in the ketol-acid reductoisomerase (KARI) reaction and the other in the regeneration of l-glutamate as an amino group donor for the branched-chain amino acid aminotransferase (BCAT) reaction. The improvement of l-valine synthesis under oxygen deprivation may be due to solving these problems. Increased l-valine synthesis under oxygen deprivation conditions was previously shown in Corynebacterium glutamicum (Hasegawa et al., 2012). In this study, we have proposed the use of NADH-dependent leucine dehydrogenase (LeuDH; EC 1.4.1.9) Bcd from B. subtilis instead of the native NADPH-dependent pathway including aminotransferase encoded by ilvE to improve l-valine production in E. coli under microaerobic conditions. We have created l-valine-producing strains on the base of the aminotransferase B-deficient strain V1 (B-7 ΔilvBN ΔilvIH ΔilvGME::PL-ilvBNN17KDA) by introducing one chromosomal copy of the bcd gene or the ilvE gene. Evaluation of the l-valine production by the obtained strains under microaerobic and aerobic conditions revealed that leucine dehydrogenase Bcd had a higher potential for l-valine production under microaerobic conditions. The Bcd-possessing strain exhibited 2.2-fold higher l-valine accumulation (up to 9.1 g/L) and 2.0-fold higher yield (up to 35.3%) under microaerobic conditions than the IlvE-possessing strain. The obtained results could be interpreted as follows: an altering of redox cofactor balance in the l-valine biosynthesis pathway increased the production and yield by E. coli cells under microaerobic conditions. Thus, the effective synthesis of l-valine by means of “valine fermentation” was shown in E. coli. This methodology has the advantages of being an economical and environmentally friendly process

Engineering Escherichia coli for autoinducible production of L-valine: An example of an artificial positive feedback loop in amino acid biosynthesis.

Artificial metabolically regulated inducible expression systems are often used for the production of essential compounds. In most cases, the application of such systems enables regulating the expression of an entire group of genes in response to any internal signal such as an aerobic/anaerobic switch, a transition to stationary phase, or the exhausting of essential compounds. In this work, we demonstrate an example of another type of artificial autoinducible module, denoted a positive feedback module. This positive feedback module generates an inducer molecule that in turn enhances its own synthesis, promoting an activation signal. Due to the use of acetolactate, an intermediate of the L-valine biosynthetic pathway, as a specific inducer molecule, we realized a positive feedback loop in the biosynthetic pathway of branched chain amino acids. Such positive feedback was demonstrated to improve the production of a target compound

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