Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets

Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions.     

Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli

The identification of genetic targets that are effective in bringing about a desired phenotype change is still an open problem. While random gene knockouts have yielded improved strains in certain cases, it is also important to seek the guidance of cell-wide stoichiometric constraints in identifying promising gene knockout targets. To investigate these issues, we undertook a genome-wide stoichiometric flux balance analysis as an aid in discovering putative genes impacting network properties and cellular phenotype. Specifically, we calculated metabolic fluxes such as to optimize growth and then scanned the genome for single and multiple gene knockouts that yield improved product yield while maintaining acceptable overall growth rate. For the particular case of lycopene biosynthesis in Escherichia coli, we identified such targets that we subsequently tested experimentally by constructing the corresponding single, double and triple gene knockouts. While such strains are suggested (by the stoichiometric calculations) to increase precursor availability, this beneficial effect may be further impacted by kinetic and regulatory effects not captured by the stoichiometric model. For the case of lycopene biosynthesis, the so identified knockout targets yielded a triple knockout construct that exhibited a nearly 40% increase over an engineered, high producing parental strain.     

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

Tn916 delta E: a Tn916 transposon derivative expressing erythromycin resistance

The tetracycline resistance gene encoded within the transposon Tn916 was replaced with the gene encoding erythromycin resistance from the plasmid pVA838. The derivative transposon of Tn916 was designated Tn916 delta E and was introduced into the Streptococcus faecalis chromosome by protoplast transformation. The conjugation/transposition functions of Tn916 delta E were similar to those observed for Tn916 in S. faecalis and Tn916 delta E was capable of self-conjugation at frequencies similar to those of other S. faecalis and Group B Streptococcus. This transposon will be useful for mutagenesis studies in gram-positive organisms, especially in those species where erythromycin resistance is a more desirable selectable marker.     

Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli

Identification of multiple gene targets that exhibit different modes of action toward a desired phenotype is a crucial step in strain improvement. Target identification methods based on traceable genetic perturbations and stoichiometric modeling have been employed before for the mining of putative overexpression and knock-out targets. Most search methods are sequential and, as such, quite limited in the space they can explore. In this study, we investigate a multi-dimensional search approach whereby unknown interactions of gene targets identified by different search methods are assessed by employing orthogonal search strategies. To this end, we combined knock-out and overexpression gene targets, identified through systematic and combinatorial approaches, respectively, in order to improve lycopene production in Escherichia coli. Specifically, we first identified multiple overexpression targets by screening genomic libraries of E. coli in a sequential-iterative manner. Targets so identified confirmed previously amplified genes in the non-mevalonate pathway (dxs and idi) and some regulatory genes (rpoS and appY). Additionally, this method revealed novel gene targets (yjiD, ycgW, yhbL, purDH, and yggT). A two-dimensional search was subsequently undertaken, whereby the selected overexpression targets were combined with the knock-out targets predicted by stoichiometric modeling. All combinations of single (rpoS, appY, yjiD, ycgW, and yhbL), double (yjiD-ycgW) and triple (yjiD-ycgW-yhbL) overexpressions with four gene deletion backgrounds, including single (delta gdhA, or delta aceE), double (delta gdhA delta aceE), and triple (delta gdhA delta aceE delta fdhF) knockouts, were constructed and evaluated for lycopene production. Investigation of the metabolic landscape spanned by these 40 strains identified the best-engineered strain (T5(P)-dxs, T5(P)-idi, rrnB(P)-yjiD-ycgW, delta gdh delta aceE delta fdhF, pACLYC), which accumulated 16,000 ppm (16 mg/g cell) of lycopene within 24 h in a batch shake flask with 5 g/L of glucose in M9 minimal medium.     

Characterization of lycopene-overproducing E. coli strains in high cell density fermentations

Previous work identified two recombinant strains of Escherichia coli capable of significant lycopene overproduction. These strains were constructed by superimposing the deletion of three genes, selected through combinatorial and systematic searches of the metabolic landscape, onto a previously engineered strain over-expressing critical genes in the lycopene biosynthesis pathway. In this paper, we characterize the performance of these two strains in comparison to the parental, pre-engineered strain. Specifically, high cell density fermentations were performed after identifying optimized putative operating parameters. High oxygen levels and increased pH values were found to be critical for increasing both specific and volumetric product titers. Carbon balances suggest linkages between glutamate, NADPH, formate, and alanine levels with lycopene overproduction. Furthermore, lycopene production reached nearly 220 mg/l from approximately 27 g dry cell weight/l in these reactors, which is the highest value reported to date for E. coli.     

Isolation of mutants ofClostridium acetobutylicum ATCC 824 deficient in protease activity

In this study we report the isolation of mutants ofClostridium acetobutylicum ATCC 824 that are deficient in protease activity. The mutants were formed by use of Tn916 transposon mutagenesis, and this study further indicates this technique as a viable method for studying the genetics ofC. acetobutylicum. The reduced protease phenotype can be distinguished on milk agar plates and was confirmed by enzyme activity measurements with the fluorescent protease substrate FITC-casein. The protease-deficient strains are capable of normal spore formation and solvent production.     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.     

Conjugative transposition of Tn916: preferred targets and evidence for conjugative transfer of a single strand and for a double-stranded circular intermediate

Transposition of conjugative transposons proceeds by excision and formation of a covalently closed circular Intermediate that includes at its joint the six flanking bases from its previous host (coupling sequences). To elucidate the role of the coupling sequences in this process and to determine the sequence of targets used by Tn916, we studied its insertion into a plasmid following conjugation. The results differ from those previously observed when Tn916 was introduced by transformation. They suggest that only one specific strand of the transposon molecule is transferred during the conjugation event and that complementary strand synthesis produces a double-stranded transposon circle with no mismatches which serves as the reaction intermediate. Tn916 inserts preferentially at specific sites and the same targets are used when Tn916 comes from donors with different coupling sequences. An analysis of the sequences of preferred targets is presented.     

Inter- and intrageneric transfer of Tn916 between Streptococcus faecalis and Clostridium tetani

We report that the streptococcal resistance transposon, Tn916, is conjugally transferred to Clostridium tetani (Utrecht) in intergenic matings. Streptococcus faecalis CG180, harboring a 41-kb plasmid (pAM180) containing Tn916 (15 kb), transferred the transposon-associated tetracycline resistance (Tcr) to C. tetani in filter matings at a frequency of about 10(-4)/donor. An erythromycin resistance marker carried by pAM180 was not transferred, indicating lack of plasmid conjugation or stable inheritance of plasmid sequences. DNA extracted from C. tetani transconjugants was probed with radiolabeled Tn916 using Southern blot analysis and these results indicated that the transposon integrated at multiple host genomic sites. Tn916-carrying C. tetani strains were able to transfer Tcr to suitable recipient strains of C. tetani as well as to S. faecalis recipients. These results indicate that this transposon is able to be disseminated and expressed in obligately anaerobic gram-positive bacteria. Moreover, this system opens avenues for the implementation of transposon mutagenesis in this important pathogenic species.     

Selection of independent Ds transposon insertions in somatic tissue of potato by protoplast regeneration

Potato is an autotetraploid crop plant that is not very amenable to the deployment of transposon tagging for gene cloning and gene identification. After diploidisation it is possible to get potato genotypes that grow well, but they are self-incompatible. This prevents the production of selfed progeny that are normally used in gene tagging approaches to select for parental lines with the target gene to be tagged in a homozygous stage. We describe here an alternative selection method for directed transposon tagging for a gene of interest in a heterozygous background. Diploid potato plants with a Ds transposon linked to the desired gene of interest (the Phytophthora infestans R1 resistance locus) in a heterozygous stage were used for the development of this directed transposon tagging strategy. After crossing to a diploid Ac transposon-containing genotype, 22 ’interesting’ seedlings (R1Ds/r–; Ac/–) were selected that showed active Ds transposition as displayed by DNA blot hybridisation, empty donor site PCR and sequencing. Protoplast isolation and the use of the hygromycin gene as a cell-specific selection marker of Ds excision enabled the direct selection of Ds excision sectors in these highly chimaeric seedlings. This somatic selection of Ds transpositions and the regeneration through protoplasts resulted in the development of a large population of almost 2000 hygromycin-resistant plants. Southern blot analysis confirmed the insertion of Ds at independent positions in the genome. Every selected plant displayed independent Ds excisions and re-insertions due to the expression of the Ac transposase throughout development. This population, which is developed from seedlings with the desired R1 gene in a heterozygous stage, is directly useful for searching for transposon-tagged R1 mutants. In general, this approach for selecting for somatic transpositions is particularly suitable for the molecular isolation of genes in a heterozygous crop like potato. Received: 29 November 1999 / Accepted: 30 December 1999     

Highly efficient Staphylococcus carnosus mutant selection system based on suicidal bacteriocin activation

Strains from various staphylococcal species produce bacteriocin peptides, which are thought to play important roles in bacterial competition and offer interesting biotechnological avenues. Many bacteriocins are secreted as inactive prepeptides with subsequent activation by specific proteolytic cleavage. By deletion of the protease gene gdmP in Staphylococcus gallinarum Tü3928, which produces the highly active lanthionine-containing bacteriocin gallidermin (lantibiotic), a strain was created producing inactive pregallidermin. On this basis, a new suicidal mutant selection system in the food-grade bacterium Staphylococcus carnosus was developed. Whereas pregallidermin was inactive against S. carnosus, it exerted potent bactericidal activity toward GdmP-secreting S. carnosus strains. To take advantage of this effect, gdmP was cloned in plasmid vectors used for random transposon mutagenesis or targeted allelic replacement of chromosomal genes. Both mutagenesis strategies rely on rare recombination events, and it has remained difficult and laborious to identify mutants among a vast majority of bacterial clones that still contain the delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2 enabled very fast, easy, and reliable identification of transposon and gene replacement mutants, respectively. Mutant selection in the presence of pregallidermin caused suicidal inactivation of all clones that had retained the plasmids and allowed growth of only plasmid-cured mutants. Efficiency of mutant identification was several magnitudes higher than standard screening for the absence of plasmid-encoded antibiotic resistance markers and reached 100% specificity. Thus, the new pregallidermin-based mutant selection system represents a substantial improvement of staphylococcal mutagenesis methodology.     

Transfer of Tn1545 and Tn916 to Clostridium acetobutylicum

Tn1545, a conjugative transposon originally discovered in Streptococcus pneumoniae, has been transferred from Enterococcus faecalis and Bacillus subtilis to Clostridium acetobutylicum NCIB 8052. Transfer between different strains of C. acetobutylicum has also been observed. Insertion of Tn1545 into the C. acetobutylicum chromosome occurred at multiple sites, as shown by Southern hybridization. Although ermAM (erythromycin-resistance) was the most satisfactory marker for primary selection of transconjugants, all three Tn1545-encoded antibiotic resistance genes (aphA-3, ermAM, and tetM) were apparently expressed in C. acetobutylicum. Our results indicate that Tn1545 is potentially useful for undertaking mutagenesis and mutational cloning in this industrially important organism. Transfer of another conjugative transposon, Tn916, from E. faecalis to C. acetobutylicum NCIB 8052 was also apparently detected. Circumstantial evidence suggests that there may be a hot spot for Tn916 insertion in the C. acetobutylicum NCIB 8052 chromosome.     

Within-host competitive interactions as a mechanism for the maintenance of parasite diversity

Variation among parasite strains can affect the progression of disease or the effectiveness of treatment. What maintains parasite diversity? Here I argue that competition among parasites within the host is a major cause of variation among parasites. The competitive environment within the host can vary depending on the parasite genotypes present. For example, parasite strategies that target specific competitors, such as bacteriocins, are dependent on the presence and susceptibility of those competitors for success. Accordingly, which parasite traits are favoured by within-host selection can vary from host to host. Given the fluctuating fitness landscape across hosts, genotype by genotype (G×G) interactions among parasites should be prevalent. Moreover, selection should vary in a frequency-dependent manner, as attacking genotypes select for resistance and genotypes producing public goods select for cheaters. I review competitive coexistence theory with regard to parasites and highlight a few key examples where within-host competition promotes diversity. Finally, I discuss how within-host competition affects host health and our ability to successfully treat infectious diseases.     

Molecular Characterization of Acanthamoeba spp. Occurring in Water Bodies and Patients in Poland and Redefinition of Polish T16 Genotype

Acanthamoeba genus is divided into 20 genotypes (T1–T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near‐complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water.     

Combinatorial network of transcriptional regulation and microRNA regulation in human cancer

Background

In order to reduce time and efforts to develop microbial strains with better capability of producing desired bioproducts, genome-scale metabolic simulations have proven useful in identifying gene knockout and amplification targets. Constraints-based flux analysis has successfully been employed for such simulation, but is limited in its ability to properly describe the complex nature of biological systems. Gene knockout simulations are relatively straightforward to implement, simply by constraining the flux values of the target reaction to zero, but the identification of reliable gene amplification targets is rather difficult. Here, we report a new algorithm which incorporates physiological data into a model to improve the model??s prediction capabilities and to capitalize on the relationships between genes and metabolic fluxes.

Results

We developed an algorithm, flux variability scanning based on enforced objective flux (FVSEOF) with grouping reaction (GR) constraints, in an effort to identify gene amplification targets by considering reactions that co-carry flux values based on physiological omics data via ??GR constraints??. This method scans changes in the variabilities of metabolic fluxes in response to an artificially enforced objective flux of product formation. The gene amplification targets predicted using this method were validated by comparing the predicted effects with the previous experimental results obtained for the production of shikimic acid and putrescine in Escherichia coli. Moreover, new gene amplification targets for further enhancing putrescine production were validated through experiments involving the overexpression of each identified targeted gene under condition-controlled batch cultivation.

Conclusions

FVSEOF with GR constraints allows identification of gene amplification targets for metabolic engineering of microbial strains in order to enhance the production of desired bioproducts. The algorithm was validated through the experiments on the enhanced production of putrescine in E. coli, in addition to the comparison with the previously reported experimental data. The FVSEOF strategy with GR constraints will be generally useful for developing industrially important microbial strains having enhanced capabilities of producing chemicals of interest.     

A system of transposon mutagenesis for bacteriophage T4

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation(Ds) transposon lines with insertions on all 5 Arabidopsischromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsisgenome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsislines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

Central carbon metabolism influences cellulase production in Bacillus licheniformis

Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for ¹³C metabolic flux analysis. A significant change was observed in metabolic fluxes after ¹³C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering.

Significance and Impact of the Study

Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering.     

Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

Background  

Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern.