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Systems metabolic engineering of Corynebacterium glutamicum for the production of the carbon-5 platform chemicals 5-aminovalerate and glutarate
Microbial Cell Factories volume 15, Article number: 154 (2016)
The steadily growing world population and our ever luxurious life style, along with the simultaneously decreasing fossil resources has confronted modern society with the issue and need of finding renewable routes to accommodate for our demands. Shifting the production pipeline from raw oil to biomass requires efficient processes for numerous platform chemicals being produced with high yield, high titer and high productivity.
In the present work, we established a de novo bio-based production process for the two carbon-5 platform chemicals 5-aminovalerate and glutarate on basis of the lysine-hyperproducing strain Corynebacterium glutamicum LYS-12. Upon heterologous implementation of the Pseudomonas putida genes davA, encoding 5-aminovaleramidase and davB, encoding lysine monooxygenase, 5-aminovalerate production was established. Related to the presence of endogenous genes coding for 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase, 5-aminovalerate was partially converted to glutarate. Moreover, residual l-lysine was secreted as by-product. The issue of by-product formation was then addressed by deletion of the lysE gene, encoding the l-lysine exporter. Additionally, a putative gabT gene was deleted to enhance 5-aminovalerate production. To fully exploit the performance of the optimized strain, fed-batch fermentation was carried out producing 28 g L−1 5-aminovalerate with a maximal space–time yield of 0.9 g L−1 h−1.
The present study describes the construction of a recombinant microbial cell factory for the production of carbon-5 platform chemicals. Beyond a basic proof-of-concept, we were able to specifically increase the production flux of 5-aminovalerate thereby generating a strain with excellent production performance. Additional improvement can be expected by removal of remaining by-product formation and bottlenecks, associated to the terminal pathway, to generate a strain being applicable as centerpiece for a bio-based production of 5-aminovalerate.
Today's petrochemical industry is challenged by the ever-increasing demand for commodity chemicals, polymer materials and related compounds, which is additionally aggravated by the ever-decreasing accessibility of fossil resources as base material [1]. Hence, substantial effort has been made for providing alternative "green" routes to produce platform chemicals through microbial fermentation processes. Compounds of interest comprise organic acids [2] such as succinate [3–5], lactate [6], and itaconic acid [7], diamines including putrescine [8, 9] and cadaverine [10–14] and diols [15–18], all being applicable as building blocks for bio-based plastics. In this regards, also 5-aminovalerate and glutarate are attractive carbon-5 building blocks for the production of nylon from renewable feedstocks [19, 20]. By now, Escherichia coli has been in the focus of interest as platform for either de novo biosynthesis [19] of 5-aminovalerate and glutarate or for biotransformation [20–22] thereof from l-lysine. As 5-aminovalerate and glutarate are degradation products of the proteinogenic amino acid l-lysine, we choose the non-pathogenic Gram-positive soil bacterium Corynebacterium glutamicum, a well-established industrial l-lysine producer [1], as metabolic chassis for the production of these carbon-5 platform chemicals. Beyond the excellent availability of genetic tools [23], and knowledge of its physiology and large-scale fermentation [24, 25], engineered C. glutamicum has only recently been established as centerpiece for the production of l-lysine-derived cadaverine within a pipeline towards the manufacturing of the fully bio-based polyamide PA5.10 [13]. In this work, the metabolic pathway from l-lysine towards 5-aminovalerate was reconstituted by stable genome-based implementation of the Pseudomonas putida KT2440 genes davB, encoding lysine monooxygenase, and davA, encoding 5-aminovaleramide amidase (Fig. 1)—a strategy previously established for E. coli [19, 21]. Glutarate production relied on an endogenous pathway (Fig. 1). Though experimentally shown [26], full and unambiguous annotation of the genes coding for the required enzymes 5-aminovalerate transaminase (GabT) and glutarate semialdehyde dehydrogenase (GabD) is missing so far (Fig. 1). We further engineered the basic producer by elimination of by-product formation and focusing the production flux towards 5-aminovalerate. Production was subsequently benchmarked in fed-batch fermentation.
Recombinant expression of the davBA genes establishes 5-aminovalerate and glutarate production in C. glutamicum
Upon implementation of the P. putida genes davBA in the genome of C. glutamicum LYS-12, this l-lysine-hyper producing strain was re-programmed for 5-aminovalerate and glutarate production. Genome-based integration of the construct (Fig. 1) was first verified by PCR analysis. Cloning-associated mutations were subsequently excluded by sequencing. Functional expression of lysine monooxygenase (DavB) and 5-aminovaleramidase (DavA) was validated by in vitro activity measurement. The underlying assay allowed parallel investigation of l-lysine consumption, related to DavB activity, and 5-aminovalerate production, related to DavA activity. In crude cell extracts of the recombinant AVA-1 strain both lysine consumption (4.1 ± 0.4 mmol L−1 h−1) and 5-aminovalerate production (1.8 ± 0.6 mmol L−1 h−1) was observed. As no conversion took place in the parent strain C. glutamicum LYS-12, this could be specifically attributed to the heterologous davBA expression. Additional proof for the functional operation of the pathway came from cultivation experiments. During growth of C. glutamicum AVA-1 in glucose minimal medium, both 5-aminovalerate and glutarate were produced and secreted into the medium (Fig. 2a). The production was growth-associated. After depletion of glucose, 5-aminovalerate and glutarate accumulation stopped at a titer of 5.4 and 6.5 mM, identifying glutarate as major product in the AVA-1 strain. The pathway precursor l-lysine was also found in the culture supernatant, though it was only secreted to a lower extent of 2.9 mM (Fig. 2a). All products were produced constantly and we could not observe any production shift throughout the cultivation (Fig. 2b), which allowed precise and representative determination of the yields (Table 1). Glutarate was produced most efficiently (123 mmol mol−1). In comparison, the 5-aminovalerate yield was 24 % lower and l-lysine secretion only contributed to about 19 % of the total product formation. The observed production pattern indicated that the l-lysine exporter protein LysE obviously competed with the activity of lysine monooxygenase, catalyzing the initial step of the 5-aminovalerate/glutarate pathway. Similarly, 5-aminovalerate export competed with the endogenous transamination reaction (Fig. 1). The preference of the cell to rather form glutarate than 5-aminovalerate indicated that transamination was more efficient than export. However, once secreted, 5-aminovalerate was not channeled back into the glutarate pathway. Indeed, when C. glutamicum LYS-12 was grown in the presence of 5-aminovalerate, the concentration of 5-aminovalerate did not decrease over the cultivation time and no glutarate formation was observed.
Secretion of the by-production l-lysine is eliminated by the deletion of lysE
To account for the undesired secretion of l-lysine in the basic strain AVA-1, the lysE gene, encoding for the lysine exporter, was deleted from the genome. For this purpose, a shortened DNA fragment, lacking 575 bp of the coding region of lysE, was used to replace the native gene. Correct modification was identified by PCR analysis. Deletion of lysE positively affected the production of the desired C5-compounds in the second-generation strain C. glutamicum AVA-2. Titer (Fig. 2c) and yield (Table 1) were increased for both glutarate and 5-aminovalerate. l-Lysine excretion was fully eliminated. The excess carbon gained from exporter deletion was mostly used in favor of glutarate production. We also observed an increase in the biomass yield, as compared to the parent C. glutamicum AVA-1 strain (Table 1). Obviously, biomass formation appeared as newly arising competitor for the additionally available carbon. The specific growth rate was hardly affected by lysE deletion, whereas the specific glucose uptake rate was 30 % lower (Table 1). Overall, this resulted in a decreased specific production rate for both glutarate and 5-aminovalerate, despite the increased yield (Table 1).
Identification and elimination of 5-aminovalerate transaminase selectively increases the 5-aminovalerate production flux
When aiming at the production of 5-aminovalerate, withdrawal of the product via the glutarate pathway has to be circumvented. The underlying biochemical reactions were, however not fully identified in C. glutamicum so far. For P. putida it is known, that two enzymes namely 5-aminovalerate transaminase, encoded by gabT (davT), and glutarate semialdehyde dehydrogenase, encoded by gabD (davD) are responsible for glutarate formation from 5-aminovalerate [27]. For the glutarate semialdehyde dehydrogenase encoding gene, two potential candidates are annotated in the genome of C. glutamicum (Fig. 1). A gene annotation for the 5-aminovalerate transaminase is, however completely missing. To identify potential genes in the genome of C. glutamicum, a nucleotide BLASTX search was carried out using the gabD and gabT sequence of P. putida as input. From the obtained set of candidates, hits with best E-value were evaluated in more detail, considering existing annotation and genomic localization (Fig. 1). The most promising candidates were the genes NCgl0462 (gabT) and NCgl0463 (gabD) as they show high similarity scores to the P. putida sequence and exhibit the same genomic organization as the P. putida genes as two adjacent open reading frames. For improving 5-aminovalerate production, we selected the gabT gene, annotated as 4-aminobutyrate aminotransferase (Fig. 1), as deletion target to immediately cut the glutarate pathway at the level of 5-aminovalerate and to avoid accumulation of the pathway intermediate glutarate semialdehyde. The coding sequence of the gene was fully deleted as validated by PCR analysis, which revealed a 1347 bp-shortened DNA fragment in C. glutamicum AVA-3 as compared to the parent strain. The strategy for generating the third-generation strain C. glutamicum AVA-3 proved highly beneficial. The product 5-aminovalerate accumulated up to 14 mM (Fig. 2e), which was more than twice as high as compared to the ancestor strains. The mutant exhibited highest yield (275 mmol mol−1) and, in particular, highest specific production rate, despite impaired growth and glucose uptake (Table 1). The latter might partly be related to intracellular accumulation of l-lysine, 5-aminovaleramid and 5-aminovalerate as response to the network perturbation at the level of LysE and GabT. Though small, a secretion flux for glutarate remained indicating residual in vivo transamination of 5-aminovalerate. The strong decrease in the glutarate yield (Table 1), however, clearly showed that the major contributing enzyme had been eliminated by NCgl0462 deletion.
Excellent performance of the 5-aminovalerate-producing C. glutamicum strain AVA-3 under fed-batch conditions
To assess the performance of the designed production strain under conditions more relevant for an industrial process, we benchmarked 5-aminovalerate production by this strain in a fed-batch process on a glucose-molasses medium. In the initial batch phase of the fermentation, we observed exponential growth along with 5-aminovalerate production (Fig. 4a). After a process time of 15 h, the initially supplied sugar (120 g L−1) was depleted and the feed phase was started. Feeding pulses were applied to maintain sugar concentration above 20 g L−1. The level of 5-aminovalerate continuously increased from 12 g L−1 at the end of the batch phase to a final titer of 28 g L−1 after 50 h. Glutarate was concomitantly secreted and accumulated up to 7 g L−1 (Fig. 3a). Proper determination of the optical density as indicator for biomass concentration was hampered in the feed phase due to cell agglomeration and wall-associated growth. The final biomass concentration of 70 g L−1 was hence determined gravimetrically as cell dry mass from the total reactor content at the end of the process. Trehalose was the only other by-product that accumulated in noteworthy amounts (7 g L−1). When evaluating the efficiency for 5-aminovalerate production throughout the process phases (Fig. 3b), slightly different yields were obtained for the batch (0.11 g g−1) and the feed phase (0.13 g g−1). The glutarate yield increased by threefold in the feed phase. The space–time yield for 5-aminovalerate was maximal in the batch phase with a rate up to 0.9 g L−1 h−1. Over the full process time, production occurred at more than half-maximum rate (0.5 g L−1 h−1).
In recent years, l-lysine-derived compounds, e.g. cadaverine [13, 28, 29], glutarate [19, 22, 26], 5-aminovalerate [20, 21] and pipecolic acid [30] have emerged as attractive platform building blocks, to establish bio-based production routes for chemicals and materials [31, 32]. This depicts an excellent possibility to employ existing l-lysine overproducers as starting point for re-engineering. Corynebacterium glutamicum is the major industrial workhorse for the production of l-lysine [1, 33] and—unlike P. putida [27] and E. coli [19]—it naturally misses catabolic reactions for degradation of l-lysine. l-lysine overproducing C. glutamicum strains are hence ideal metabolic chassis for establishing bio-based fermentation processes for these interesting compounds, as there are no side-reactions competing with the target product formation. In this work, we established a de novo production process for the carbon-5 platform chemicals 5-aminovalerate and glutarate on basis of C. glutamicum LYS-12 [34]. These are promising building blocks for bio-based polyamides and polyesters [19], when polymerized with appropriate counterparts such as caprolactame [20] and diamines [13], and beyond serve as platform chemicals [22].
Expression of davBA fills the gap for de novo production of 5-aminovalerate and glutarate in C. glutamicum
The strategy of using heterologous expression of the P. putida genes davBA for re-programming of C. glutamicum LYS-12 for 5-aminovalerate and glutarate production proved highly valuable as more than 80 % of the total production flux was drained towards the newly implemented pathway (Fig. 4). Due to the lack of l-lysine catabolic reactions, additional strain modifications, required for E. coli-based processes [19, 22], were obsolete. About one-third of the rechanneled carbon was secreted as 5-aminovalerate, whereas the major fraction was further metabolized to glutarate via endogenous reactions (Fig. 4). In E. coli, production of 5-aminovalerate and glutarate specifically relied on heterologous expression of the davAB cluster (5-aminovalerate) or a combined expression of the davBA-davDT (gabDT) clusters (glutarate) as, in contrast to C. glutamicum, endogenous genes for davDT are obviously missing [19, 20, 22]. Despite pure production of either of these compounds, E. coli reached maximal yields of 71 mmol mol−1 for 5-aminovalerate and 68 mmol mol−1 for glutarate in a de novo process [19], which is even far below the performance of the basic AVA-1 strain (Table 1). The different production pattern for both species might be related to differences concerning re-uptake of 5-aminovalerate. E. coli efficiently converts externally added 5-aminovalerate into glutarate, when the davBA and davDT (gabDT) gene clusters from P. putida are concomitantly expressed [19]. The obviously active import system for 5-aminovalerate in E. coli can thus explain that there is no mixed production of 5-aminovalerate and glutarate [19, 22]. The constant production characteristics of our novel strains (Fig. 2), however, show that once 5-aminovalerate is secreted, there is no re-import and further conversion to glutarate. In addition, we could not observe any glutarate formation in C. glutamicum LYS-12 when 5-aminovalerate was supplied to the medium. This is attributed to the lack of an active 5-aminovalerate uptake system under the tested conditions. Efficient product secretion without re-uptake—as found in this study for C. glutamicum—is highly desirable and might even become crucial to establish commercially feasible processes [35–37]. The genome-based engineering approach ensured strain stability as previously proven for manifold genetic manipulations of C. glutamicum [38–42]. Overall, production of the target metabolites and the remaining l-lysine production in the AVA-1 strain nicely summed up to the l-lysine production of the parent strain [34]. We took this finding as good indicator that no other by-products arose in noteworthy amounts, as previously shown for cadaverine-producing C. glutamicum [10, 11].
5-aminovalerate production is limited by the in vivo activity of the heterologous pathway
The secretion of l-lysine in the AVA-1 strain revealed limitations in the in vivo activity of the downstream 5-aminovalerate pathway. The newly implemented pathway operated less efficiently than the cadaverine pathway, previously established in C. glutamicum, which fully converted l-lysine into cadaverine by the heterologously implemented lysine decarboxylase [10]. Intracellular l-lysine accumulation in the AVA-1 strain was, however, sufficient to induce expression of the l-lysine exporter [43, 44]. Consequently, the exporter LysE competed with lysine monooxygenase for intracellular l-lysine as substrate, a phenomenon similarly found previously for l-lysine-derived cadaverine production, whereby the cadaverine export protein competed with cytosolic acetyl-transferase reaction that formed N-acetyl-cadaverine [10, 11]. We hence had a closer look at the enzyme activity of lysine monooxygenase and 5-aminovaleramidase. In vitro assays with crude cell extract revealed that the lysine consumption rate, reflecting lysine monooxygenase activity, was two times higher than the production rate of 5-aminovalerate, reflecting 5-aminovaleramidase activity. Expression of both enzymes from the designed operon was obviously not perfectly balanced. For enzymatic production of 5-aminovalerate, however, it was recently shown, that a 1:1 ratio of both enzymes is required for optimal production [21]. On basis of the in vitro activity, the bottleneck of the 5-aminovalerate pathway could on a first glance be assigned to 5-aminovaleramidase. The initial step catalyzed by lysine monooxygenase is, however, massively feedback inhibited by 5-aminovaleramide, 5-aminovalerate, and glutarate at levels of 5 mM [27], likely resulting in a substantially reduced in vivo activity. Moreover, from enzyme activity measurements, carried out in closed tubes, we have additional indication that lysine monooxygenase can become limiting, when oxygen supply is insufficient. In in vitro studies with limited oxygen supply, identical rates for lysine consumption and 5-aminovalerate production were determined (0.56 ± 0.01 mmol L−1 h−1). We hence cannot clearly attribute the bottleneck specifically to one of the enzymes and/or an imbalanced expression thereof. Elimination of LysE activity was therefore most straightforward to avoid loss of the pathway precursor l-lysine. Lacking any secretion system for l-lysine, the additionally available carbon in the AVA-2 strain was partly used in favor of 5-aminovalerate and glutarate formation, and partly supports biomass production, as similarly found recently for ectoine producing C. glutamicum [45]. Despite the higher yields, the specific production rates were not increased as the in vivo activity of the heterologous pathway remained a limiting factor. The rates were actually lower than in the parent strain (Table 1), as the overall fitness (µ, qS) of the AVA-2 strain was slightly impaired. Further improvement of the strain performance can thus be expected from higher davBA gene expression. Moreover, removal of feedback inhibition of lysine monooxygenase is a highly desirable, though challenging task for future strain optimization.
The glutarate pathway is the major competitor for 5-aminovalerate production
The obtained production pattern clearly indicated that the endogenous transaminase reaction of the glutarate pathway is dominating over the 5-aminovalerate export. Deletion of the putative transaminase GabT in the AVA-3 strain successfully redirected the production flux to 5-aminovalerate, though glutarate production was not fully eliminated. Obviously, C. glutamicum possesses more than one gene and enzyme, responsible for 5-aminovalerate transamination. It is interesting to note that P. putida, which can naturally degrade l-lysine via the 5-aminovalerate/glutarate pathway, only possesses a single gabT gene variant, the lack of which prevents growth on l-lysine as sole carbon source [46]. Additional deletion of further gene candidates for gabT and gabD (Fig. 1) appears most straightforward to further diminish glutarate formation in the C. glutamicum AVA-3 strain. However, only few is known about their actual function, leaving a certain risk of undesired side effects upon deletion. As alternative strategy, the enhancement of the 5-aminovalerate export could drive a more efficient product secretion. Indeed, transport processes are a major issue of industrial processes and thus often addressed in the production hosts for improving production of e.g. cadaverine [12, 47], amorphadiene [48], l-lysine [49] and l-arginine [50]. In light of the strong feedback inhibition of lysine monooxygenase, the secretion efficiency appears even more relevant. Similar to E. coli [19], the transport mechanisms for 5-aminovalerate and associated proteins are not yet known for C. glutamicum. The secretory capacity of C. glutamicum for non-natural compounds is, however, admirable as demonstrated in recent years for the production of ectoine [45], cadaverine [12], putrescine [8], and γ-amino butyric acid [51]. As shown for cadaverine [12], export is thereby not necessarily linked to a unique and specific transporter. There are likely transporter families with promiscuous secretory activity for structurally related metabolites, which might be of help for identification.
High-cell density fermentation reveals excellent production performance
To take benefit from high cell density and the generally improved production performance [34, 45], the best 5-aminovalerate producer was investigated during fed-batch operation. C. glutamicum AVA-3 produced 28 g L−1 of 5-aminovalerate within only 50 h, which is the highest titer and productivity described so far for de novo production of this compound [19, 22, 26]. The maximal space–time yield of about 0.9 g L−1 h−1 was even within the range of bio-conversion processes directly producing 5-aminovalerate from l-lysine [19–22]. De novo production as described here obviously benefits over such bio-transformation process, as production can be conducted as one-step process directly from cheap raw material, regularly applied for industrial fermentation [24, 25, 52]. In contrast, biotransformation requires successive production steps for the supply of the biocatalyst, e.g. whole cells or purified enzymes, and a subsequent conversion step for product formation. Moreover, it strictly relies on the pathway precursor l-lysine, additionally being produced by fermentation, requiring subsequent purification. In this regard, de novo production is clearly more flexible, related to the broad natural substrate spectrum of C. glutamicum and additional engineering of assimilation routes for non-natural carbon sources [53, 54]. Depending on price and availability, alternative renewable feedstocks [29, 40, 55] or carbon-containing waste streams from industry [54, 56] might become more attractive, and could hence be used for production.
In this work, we established C. glutamicum as production platform for the polyamide building blocks 5-aminovalerate and glutarate through genetically stable genome manipulation. Following the strategy of heterologous engineering of the 5-aminovalerate pathway from P. putida [19, 22, 26], combined with additional modifications that targeted by-product formation, we generated a strain with the highest de novo production performance concerning titer, yield and productivity. With the remaining issues on the in vivo activity of the davBA pathway, 5-aminovalerate export and glutarate production, we can immediately identify future engineering targets towards strain improvement. Further optimization can be expected applying systems metabolic engineering to unravel and circumvent additional and less obvious bottlenecks through iterative rounds of strain analysis, design and genetic engineering. Similar titer, yield and productivity as already achieved for l-lysine [34] and cadaverine [13, 57] production thus appear feasible to leverage performance of 5-aminovalerate to industrial efficiency. Moreover, the AVA-2 strain already builds an ideal basis for generating glutarate overproducing C. glutamicum strains.
Microorganisms and plasmids
In the present work, the ATCC 13032-derived l-lysine-overproducing C. glutamicum strain LYS-12 [34] was used as host to establish 5-aminovalerate and glutarate production. Reconstruction of the 5-aminovalerate pathway relied on the davBA genes, encoding lysine monooxygenase and 5-aminovaleramidase from P. putida KT2440. Amplification of transformation vectors on basis of the integrative plasmid pClik int sacB [10, 58] was carried out in E. coli strains DH5α and NM522 (Invitrogen, Carlsbad, CA, USA) [34]. All strains and plasmids employed in this study are listed in Table 2.
Molecular design and genetic engineering
For molecular strain, plasmid and primer design, the Clone Manager Professional 9 (Sci-Ed Software, Denver, USA) was used. For sequence similarity search towards identification of gabT and gabD candidates in the genome of C. glutamicum, the corresponding gene sequences from P. putida (PP_0214, gabT, davT and PP_0213, gabD, davD) were BLASTed (BLASTX, http://www.genome.jp/tools/blast/) against the C. glutamicum genome, deposited at KEGG (http://www.genome.jp/kegg/). The genetic construct for genome-based integration of the davBA gene cluster from P. putida KT2440 comprised (i) 500 bp-sized flanking regions as homologous recombination sites for the bioD locus (NCgl2516), (ii) a 200 bp-sized DNA fragment of the promotor of the structural tuf gene (NCgl0480), and (iii) the biosynthetic genes davB (PP_0383) and davA (PP_0382), which were separated by a 20-bp sized ribosomal binding site as inter-genic region (Fig. 1). The putative gabT gene (NCgl0462) was fully deleted from the genome. All DNA fragments were amplified by PCR (2× Phusion Flash PCR Master Mix, Thermo Scientific, Waltham, MA, USA; peQSTAR, PEQLAB Biotechnology GmbH, Erlangen, Germany) from genomic DNA of C. glutamicum ATCC13032 and P. putida KT2440, respectively, with sequence specific primers (Table 3). DNA fragment and vector assembly was carried out by the method of Gibson [59]. The reaction mixture contained 157.5 mM Tris–HCl (pH 7.5), 15.75 mM MgCl2, 15.75 mM DTT, 42 mg µL−1 PEG-800, 0.6 mg µL−1 NAD, 25 mU µL−1 Phusion™ High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Rochester, New York, USA), 7.5 mU µL−1 T5 exonuclease (Epicentre, Madison, USA), 4 U µL−1 Taq Ligase (Thermo Fisher Scientific, Rochester, New York, USA), each 0.3 mM dNTPs. Prior to this, the vector was linearized via restriction with BamHI (FastDigest®, Thermo Fisher Scientific, St. Leon-Roth, Germany). Vector amplification in the E. coli strains DH5α and NM522, purification of plasmid DNA, and its transformation into E. coli and C. glutamicum strains were performed as described previously [39]. PCR and sequence analysis (GATC Biotech AG, Konstanz, Germany) were used for plasmid and strain validation. Deletion of the lysE-gene encoding the l-lysine exporter of C. glutamicum was conducted as described previously [12].
Batch cultivation in shake flasks
Corynebacterium glutamicum was grown in baffled shake flasks with 10 % filling volume at 30 °C and 230 rpm on an orbital shaker (Multitron, Infors AG, Bottmingen, Switzerland; 5 cm diameter). The cultivation procedure involved one pre-culture in complex medium (37 g L−1 BHI) followed by another pre-cultivation in minimal medium to adapt cells to the medium conditions of the main cultivation [60]. The main cultivation was carried out as triplicate in a chemically defined mineral salt medium with glucose as the carbon source [61]. The medium contained: (A) 500 mL salt solution (1 g NaCl, 55 mg MgCl2·7H2O and 200 mg CaCl), (B) 100 mL substrate solution (100 g L−1 glucose), (C) 100 mL buffer solution (2 M potassium phosphate, pH 7.8), (D) 100 mL solution B (150 g L−1 (NH4)2SO4, pH 7.0), (E) 20 mL vitamin solution (25 mg L−1 biotin, 50 mg L−1 thiamine·HCl and 50 mg L−1 pantothenic acid), (F) 10 mL FeSO4-solution (2 g L−1 FeSO4, pH 1.0), (G) 10 mL 100× trace elements [62] and (H) 1 mL DHB-solution (30 mg mL−1 of 3,4-dihydroxybenzoic acid in 0.3 M NaOH) adjusted to 1 L with milliQ. Solutions were separately sterilized by autoclaving (A–D) or by filtration (E–H). The different medium compounds were combined at room temperature freshly before use.
Fed-batch cultivation in stirred tank bioreactors
The production performance of the optimized 5-aminovalerate-producer C. glutamicum AVA-3 was evaluated in a fed-batch process. This was carried out in a complex molasses-based medium, containing per liter: 72.4 g beet molasses, 35 mL corn steep liquor, 40 g (NH4)2SO4, 99 g glucose·H2O, 250 µL H3PO4 (85 %), 11 mg FeSO4·7H2O, 10 mg citrate, 9 mg biotin, 17.5 mg thiamine·HCl, 60 mg pantothenic acid Ca-salt, 18 mg nicotinamid, 6 mg riboflavin, 6 mg FAD, 250 mg KH2PO4, 100 mg MgSO4. The initial batch was started in a volume of 300 mL in a 1000 mL bioreactor (SR0700ODLS, DASGIP AG, Jülich, Germany) and inoculated with cells as previously described [13]. Dosing of the feed (650 g L−1 glucose·H2O, 162.5 g L−1 beet molasses, 40 g L−1 (NH4)2SO4, 6 mg L−1 riboflavin, 6 mg L−1 FAD) was initiated when the sugar concentration dropped below 20 g L−1 and adjusted to maintain glucose concentration constant above 20 g L−1. The cultivation temperature was kept constant at 30 °C via the CWD4 bioblock (DASGIP AG, Jülich, Germany). The pH and the pO2 level were monitored online with a pH electrode (Mettler Toledo, Giessen, Germany) and a pO2 electrode (Hamilton, Höchst, Germany). The pH was kept constant at 6.9 ± 0.1 by automated addition of 25 % NH4OH (MP8 pump system, Eppendorf, Hamburg, Germany). The dissolved oxygen level was maintained at saturation above 30 % by variation of the stirrer speed and the aeration rate. Data acquisition and process operations were controlled by the DAGIP control software (DASGIP AG, Jülich, Germany).
Substrate and product quantification
Amino acids and 5-aminovalerate were quantified by HPLC, using α-aminobutyric acid as internal standard [63] with a modified gradient (0 min: 70 % eluent A; 14 min: 56 % eluent A; 14.5 min: 0 % eluent A; 16.5 min: 0 % eluent A; 17 min: 70 % eluent A; 19 min: 70 % eluent A). Samples from fed-batch fermentation were additionally diluted with MilliQ water prior to dilution with the internal standard to account for the detector limit (<1.5 mM). Quantification of glucose and organic acids was carried out by isocratic HPLC (Agilent 1260 Infinity Series, Waldbronn, Germany) comprising a degasser (G4225A), an autosampler (G1329B) including thermostat (G1330B), an isocratic pump (G1310B), a column oven (G1316C), and a refraction index (RI) detector (G1362A). Separation was conducted on an Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, München, Germany) at 55 °C. As mobile phase 3.5 mM H2SO4 was used at a flow rate of 0.8 mL min−1. The injection volume was 20 µL. For sugar analysis of supernatant samples from fed-batch fermentation, a Metacarb 87C column (300 × 7.8 mm; Agilent, Darmstadt, Germany) with Metacarb 87C guard column (50 × 7.8 mm; Agilent, Darmstadt, Germany) and additional Micro-Guard De-Ashing Refill Cartridges (Bio-Rad, München, Germany) was used at 85 °C with demineralized water as mobile phase at a flow rate of 0.4 mL min−1. If appropriate, samples were diluted prior to analysis. Refraction index was used for detection. Concentration of cell dry mass (CDM) was calculated from the optical density using a correlation factor of CDM [g/L] = 0.32 × OD660. This was determined as described previously [61] for the here used UV-1600PC spectrophotometer (VWR, Hannover, Germany).
Determination of enzyme activity
Crude cell extracts were prepared from exponentially growing cells by mechanical cell disruption. Cultivation and harvest procedure was carried out as previously described [29]. Aliquots of 1 mL cell suspension were then transferred into FastPrep®-24 vials (MP Biomedicals, Illkirch-Graffenstaden, France) containing silica beads (Ø 0.1 mm) and cell disruption was carried out in 2 × 30 s cycles at 5000 rpm (Precellys®-24, PeqLab, Hannover, Germany) including 1 min cooling pauses on ice. Removal of cell debris and protein quantification was performed as previously described [61]. The protocol for subsequent quantification of the conversion rate of l-lysine into 5-aminovalerate, comprising the lumped activity of lysine monooxygenase and 5-aminovaleramide amidase, was based on previous protocols [19, 21]. The final reaction mixture of 5 mL contained 100 mM phosphate buffer (pH 7.5), 15 mM l-lysine, and 50 µL mL−1 of crude cell extract (0.5 mg mL−1 of protein). Incubation was carried out at 30 °C and 230 rpm on an orbital shaker (Multitron, Infors AG, Bottmingen, Switzerland) in 50 mL baffled shake flasks. Samples were regularly taken and thermally inactivated (5 min, 100 °C, Thermomixer F1.5, Eppendorf, Hamburg, Germany). l-lysine consumption and 5-aminovalerate formation were quantified by HPLC as given above. Negative controls were conducted without the addition of crude cell extract and l-lysine, respectively. Moreover, the crude cell extract of the parent strain C. glutamicum LYS-12 was analyzed to account for potential non-DavBA associated enzymatic reactions.
- Ava:
- bioD :
encoding gene of dithiobiotin synthetase
- davA :
encoding gene of aminovaleramide amidase
- davB :
encoding gene of lysine monooxygenase
- gapT :
encoding gene of 5-aminovalerate transaminase
- gabD :
encoding gene of glutarate semialdehyde dehydrogenase
- Glt:
- Glu:
- Lys:
- LysE, lysE :
lysine exporter and corresponding gene
multiple cloning site
origin of replication
polymerase chain reaction
- sacB :
encoding gene of levansucrase from Bacillus subtilis
- tuf :
encoding gene of elongation factor tu
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Authors' contributions
CR constructed the strains and conducted cultivation experiments in shake flasks. GG and MK carried out fed-batch fermentation and analytics. JB performed strain and experimental design, and the enzyme activity studies. JB and CW conceived and structured the work, assessed the data and wrote the manuscript. All authors read and approved the final manuscript.
CW and MK acknowledge the financial support from the BMBF-Grant "Bio2Nylon" (No. 03V0757).
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The dataset(s) supporting the conclusions of this article are all included within the article.
Ethics approval and consent to participate
Not applicable. The manuscript does not contain data collected from humans or animals.
CW and MK acknowledge the financial support from the BMBF-Grant "Bio2Nylon" (No. 03V0757). The funding body did not contribute to the design of the study, data collection, analysis, and interpretation, or writing of the manuscript.
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Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Cite this article
Rohles, C.M., Gießelmann, G., Kohlstedt, M. et al. Systems metabolic engineering of Corynebacterium glutamicum for the production of the carbon-5 platform chemicals 5-aminovalerate and glutarate. Microb Cell Fact 15, 154 (2016). https://doi.org/10.1186/s12934-016-0553-0
DOI: https://doi.org/10.1186/s12934-016-0553-0


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Preston vs Tottenham LIVE: FA Cup result and final score after Son Heung-min double and Arnaut Danjuma debut goal
Tottenham hope to build some momentum for the back half of their season as they travel to Preston in the FA Cup fourth round this evening. After a poor run of games that saw them lose to Arsenal and Manchester City in the Premier League, Spurs got back to winning ways last time out with a 1-0 victory over Fulham thanks to Harry Kane's strike just before half-time.
Antonio Conte's men are still struggling to find any sort of attacking rhythm but today's cup match – against a Championship opponent – could come at just the right time to click them into gear once again. However, the reverse is also true. Should Tottenham lose to Preston today they could will face increased pressure to perform in the league as they attempt to close the gap on the top four.
For their part, Preston will be no pushovers. They're 11th in the Championship and although they ended 2022 in a bad run of form since the turn of the year Preston have won three of their four matches including a 3-1 comeback win against Huddersfield in the previous round of this competition. Can they surprise Tottenham this afternoon?
Follow all the action from Deepdale as Preston host Spurs in the FA Cup:
Preston vs Tottenham
Preston North End host Tottenham in the FA Cup fourth round
Preston have won three of their last four matches, can they shock Spurs today?
87' GOAL! - Danjuma adds a third on his Tottenham debut (PNE 0-3 TOT)
69' GOAL! - Son doubles Spurs' lead (PNE 0-2 TOT)
65' SAVE! - Woodman pushes Perisic's header wide (PNE 0-1 TOT)
50' GOAL! - Son breaks the deadlock (PNE 0-1 TOT)
25' SAVE! - Woodman denies Son with a leaping save (PNE 0-0 TOT)
17' SHOT! - Son's long-ranged effort is smothered by Woodman (PNE 0-0 TOT)
8' CHANCE! - Kulusevski's effort deflected wide by Ledson (PNE 0-0 TOT)
Preston North End FC 0 - 3 Tottenham Hotspur FC
Full-time: Preston 0-3 Tottenham
19:50 , Michael Jones
90+3 mins: The whistle goes and Tottenham advance into the fifth round of the FA Cup. For just over an hour Preston gave the Premier League side a good game but Son Heung-min's deadlock breaker was the deciding factor.
He was brilliant tonight and seems to have found his form in front of goal again.
Preston 0-3 Tottenham
19:47 , Michael Jones
90 mins: Spurs are in cruise control now. They've made light work of things tonight and once that first goal went in for Son Heung-min Preston's willpower started to dwindle.
Three minutes of added time to play.
GOAL! Preston 0-3 Tottenham (Danjuma, 87')⚽
19:45 , Michael Jones
87 mins: It's three! Arnaut Danjuma has a goal on debut for Tottenham!
Spurs work the ball quickly through the middle before Bryan Gil sends the ball out to Dejan Kulusevski on the right wing. He brings the ball into the box then pulls it back to Danjuma who guides his finish into the far bottom corner!
Lovely stuff.
Preston 0-2 Tottenham
19:42 , Michael Jones
84 mins: Son Heung-min takes in the applause of the travelling Tottenham fans. He's been the best player on the pitch tonight.
A couple of shots in the first half were kept out by goalkeeper Freddie Woodman but the forward stuck to his guns and earned his reward - and two goals - in the second half.
Son is replaced with Bryan Gil for Spurs.
Preston 0-2 Tottenham
19:39 , Michael Jones
81 mins: Inside the final 10 minutes now. Can Preston get a goal to set up a tense and exciting finish to this game?
It seems unlikely but this is the FA Cup and strange things happen in this tournament.
Preston 0-2 Tottenham
19:37 , Michael Jones
78 mins: Davinson Sanchez leaps into a tackle on Ched Evans and knocks the ball out for another Preston corner. Can the home side muster something from this?
No! The cross is swept in high and drops over by the back post but it's a comfortable height for Fraser Forster to pluck out of the air.
Preston 0-2 Tottenham
19:32 , Michael Jones
75 mins: Tottenham now have a bunch of players on the pitch that want to impress Antonio Conte. They'll no doubt try to extend this lead and put the game properly out of Preston's reach.
Danjuma almost has the chance to mark his debut with a goal but he's not given the return pass after making a run into the box.
Preston 0-2 Tottenham
19:30 , Michael Jones
72 mins: Antonio Conte now makes his changes. There are three in total as Oliver Skipp and Emerson Royal come on to replace Rodrigo Bentancur and Matt Doherty.
Arnaut Danjuma also makes his debut for Spurs replacing Ryan Sessegnon.
Preston bring on midfielder Ben Woodburn for Ryan Ledson.
GOAL! Preston 0-2 Tottenham (Son, 69')⚽
19:27 , Michael Jones
69 mins: That should be that now! It's brilliant from Son once again. A cutting pass comes into the box and picks out Son Heung-min. He stops the ball spins around Jordan Storey then lifts a left-footed effort into the near top corner.
Lovely finish. Spurs are two goals to the good.
Preston 0-1 Tottenham
19:23 , Michael Jones
65 mins: It's been a decent response from Preston who are seeing more of the ball in Tottenham's half. Spurs remain threatening though as Lenglet whips a cross into the box and finds Perisic.
Perisic guides his header towards goal and Woodman reacts late but manages to push it wide of the target.
Preston 0-1 Tottenham
19:19 , Michael Jones
62 mins: Tom Cannon dribbles the ball into the Tottenham box and cuts back on himself before releasing a pass. Tanganga puts a hand on Cannon's shoulder and the forward dives to the floor looking for a penalty.
The referee has a clear view of the incident and books Cannon for simulation.
Preston 0-1 Tottenham
19:17 , Michael Jones
59 mins: Oh here we go. Alan Browne looks to whip the ball into the box from the right wing and he wins Preston a corner. Robbie Brady takes the set piece and whips it into the penalty area.
It's a poor corner and doesn't beat the first man. Chance wasted for Preston.
Preston 0-1 Tottenham
19:14 , Michael Jones
56 mins: Tottenham have livened up a bit since going in front. They'll know that a second goal takes the tie away from Preston and they are hunting it down.
Sessegnon sweeps down the left wing and wins a corner but Preston deal with it well enough. The home fans are getting louder, they're encouraging their team to make a comeback here.
Preston 0-1 Tottenham
19:10 , Michael Jones
53 mins: Things are going to get difficult for Preston now. At some point they're going to have to loosen up their defensive shape and take the game to Spurs.
The home side need to score to stay in this contest. Will there be a response?
GOAL! Preston 0-1 Tottenham (Son, 50')⚽
19:09 , Michael Jones
50 mins: Glorious strike from Son Heung-min! He drops away from Ryan Ledson and receives the ball from Yves Bissouma.
Son cuts inside, takes the ball to the edge of the 'D' then curls a powerful strike into the far bottom corner! Freddie Woodman tries to get to the effort but can't reach it with his lunging effort.
Tottenham lead!
Preston 0-0 Tottenham
19:07 , Michael Jones
48 mins: Shot! Tottenham pick up where they left off in the first half. They're controlling the ball and passing it confidently up to the Preston final third then run out of ideas.
A hurry of passes sees the ball moved across to Clement Lenglet and he smokes one from range but drills his effort over the bar!
Second half: Preston 0-0 Tottenham
19:02 , Michael Jones
No changes from either manager as the second half gets underway at Deepdale. Will we see a goal in this 45 minutes?
Man Utd vs Reading line-ups
19:00 , Michael Jones
Man Utd XI: De Gea, Wan-Bissaka, Lindelof, Maguire, Malacia, Casemiro, Eriksen, Fernandes, Antony, Weghorst, Rashford
📥 Incoming: tonight's team news! 💪#MUFC || #FACup
— Manchester United (@ManUtd) January 28, 2023
Reading XI: Lumley, Yiadom, Holmes, McIntyre, Hoilett, Ince, Loum, Hendrick, Baba, João, Carroll
This is how the Royals line up for this evening's @EmiratesFACup against @ManUtd.
Team news presented by @toshibatveurope 📺#MUNREA #EmiratesFACup pic.twitter.com/4MMhOQ0dz4— Reading FC (@ReadingFC) January 28, 2023
HT Preston 0-0 Tottenham
19:00 , Michael Jones
How long will Antonio Conte leave it before introducing Harry Kane. The England captain scored the winner against Portsmouth last time out in the competition.
Will Spurs require his services once again?
Will Jadon Sancho play?
18:55 , Michael Jones
As part of Friday's pre-match press conference Erik ten Hag was aksed whether Jadon Sancho would feature tonight and he replied:
"He is training with the team and we will see. I said he's on the way back, he's making steps."
Sancho wasn't seen travelling to Old Trafford with the rest of the players so he may not be included in the matchday squad.
Man Utd vs Reading
18:51 , Michael Jones
Coming up at the conclusion of Preston vs Tottenham we have action from Old Trafford where Manchester United host Reading.
Erik ten Hag's men hope to advance to the fifth round and keep their cup run going. Can Reading give them a game?
The team news for that one will be dropping soon.
HT Preston 0-0 Tottenham
18:49 , Michael Jones
Preston have been organise and resolute in defence tonight. Can they string together a few attacks in the second half and possibly nick a win here?
Half-time: Preston 0-0 Tottenham
18:47 , Michael Jones
45+1 mins: Just the one minute of added time tells you how baldy this game has been lacking in excitement. The teams head into the break on level terms.
Spurs have dominated possession but the Premier League side haven't made the breakthrough.
Preston 0-0 Tottenham
18:45 , Michael Jones
43 mins: Browne and Brady are doing a fine job covering the right side of Preston's and stop Sessegnon fro crossing the ball when he's given it by Clement Lenglet.
Spurs then slip Kulusevski into the left side of the penalty area but his effort is blocked by a sliding Liam Lindsay.
Preston 0-0 Tottenham
18:42 , Michael Jones
40 mins: Bentancur brings the ball through midfield and pass it over to Tanganga. He sends it wide for Doherty on the right wing.
The wingback has space to run into and does so before swinging a high cross into the middle. The ball loops into the penalty area but that allows Freddie Woodman to come off his line and pluck the ball out of the air.
Preston 0-0 Tottenham
18:38 , Michael Jones
37 mins: Shot! Perisic takes the next corner again but slides this one over to Dejan Kulusevski. He brings the ball inside, puts it onto his left foot then laces a shot high and wide!
Preston 0-0 Tottenham
18:37 , Michael Jones
34 mins: Tottenham win a free kick on the right wing but Son hits the one-man wall who deflects the ball out for a corner.
Ivan Perisic takes this set piece but Robbie Brady heads it behind from the near post.
Preston 0-0 Tottenham
18:34 , Michael Jones
31 mins: Ryan Sessegnon gets forward in his role as a wingback but is miles offside when Ivan Perisic sends a through ball behind the lines for him.
Tottenham are a bit toothless at the minute. Preston will be delighted with how the game is going.
Preston 0-0 Tottenham
18:31 , Michael Jones
28 mins: A clearance from Preston sees the ball booted long up the pitch. Tom Cannon sprints forward to put pressure on Davinson Sanchez and the Spurs defender is forced into an error.
He fails to control the ball properly but gets lucky as it bobbles back to Fraser Forster who deals with it.
Preston 0-0 Tottenham
18:26 , Michael Jones
25 mins: Save! Freddie Woodman is called into action again! Son Heung-min finds a pocket of space over on the inside left which allows him to strike one from range.
He takes the shot and looks for the far corner but Woodman leaps across and palms the ball wide.
Preston 0-0 Tottenham
18:25 , Michael Jones
23 mins: Very good defending from Preston as Tottenham work the ball around the front edge of the penalty area. Bentancur, Bissouma and Kulusevski all try to find ways into the box but all three get shut out as Preston moves as one towards the ball.
Spurs maintain possession though.
Preston 0-0 Tottenham
18:22 , Michael Jones
20 mins: Alan Browne dribbles the ball through midfield for Preston as is lucky to earn a free kick after Davinson Sanchez takes the ball off him.
Preston whip the set piece into the penalty area. A scramble happens as both sets of players look to win the ball but Spurs come away with it and Preston immediately drop back to defend.
Preston 0-0 Tottenham
18:17 , Michael Jones
17 mins: Close! Matt Doherty brings the ball inside from the right wing and sends the ball into the feet of Son. Son has no immediate options for a pass so drills a low left-footed effort towards goal.
Freddie Woodman shuffles past the path of the ball and has to leap back on himself to keep it out but the Preston goalkeeper gets two hands to the shot and manages to smother it.
Preston 0-0 Tottenham
18:15 , Michael Jones
14 mins: Son Heung-min receives the ball on the inside left channel and spins away from two defenders before driving towards the box.
He just about makes it to the front edge but McCann sticks in a leg and wins the ball for Preston.
Tottenham are dominating possession as expected but Preston aren't under any pressure in their final third... yet.
Preston 0-0 Tottenham
18:12 , Michael Jones
11 mins: Ali McCann takes a knock to the face and requires a change of shirt after his nose starts to bleed. Preston hoped to challenge the Spurs back line with a counter-attack but the long pass forward flies straight through to Fraser Forster who tidies up.
Preston 0-0 Tottenham
18:08 , Michael Jones
8 mins: Chance! Ivan Perisic takes the free kick and strikes his shot into the wall.
The ball drops kindly into the path of Dejan Kulusevski who looks up and drills an effort at goal but Rayn Ledson is aware of the danger and flings himself at the ball managing to prod it wide of the near post!
Preston 0-0 Tottenham
18:06 , Michael Jones
6 mins: The first corner of the game goes to Spurs with Son Heung-min whipping the ball into the middle of the penalty area after it gets played short.
The ball is cleared as far as Japhet Tanganga who's hit with a late tackle from Ben Whiteman and wins Spurs a free kick in a dangerous area.
Preston 0-0 Tottenham
18:03 , Michael Jones
3 mins: Son Heung-min drops short to collect the ball as Spurs look for a way through the Preston defence.
Dejan Kulusevski moves inside and looks to spin away from Liam Lindsay as the ball comes sliding up the pitch but the defender steps across the Tottenham forward, intercepts the ball and clears his lines.
Kick off: Preston 0-0 Tottenham
18:01 , Michael Jones
The home side get the ball rolling at Deepdale. They punt the ball over to the left wing where the ball hits Japhet Tanganga and goes out of play for a Preston throw in.
Preston vs Tottenham
17:58 , Michael Jones
Here come the teams.
There's a packed house inside the stadium and the Preston fans are singing and chanting already. Tottenham won't have an easy night here.
Kick off is up next.
17:55 , Michael Jones
The warm-ups are done and dusted. The players are gathering in the tunnel and kick off is quickly approaching. Will Spurs dominate against Preston tonight or can the Lillywhites shock the Premier League side?
Harry Kane is up there with the best, says Preston's Spurs loanee Troy Parrott
17:52 , Michael Jones
Troy Parrott has not been asked by his Preston team-mates how to stop Tottenham forward Harry Kane, but admits his answer "would probably be you can't".
Spurs academy graduate Parrott will be reduced to watching on Saturday night after his parent club were drawn away to his current team in the FA Cup fourth round.
It could be a record-breaking occasion for Kane, who needs one more goal to move past Jimmy Greaves and become Tottenham's outright leading scorer, but the striker has not been the sole talk of the Deepdale defenders this week.
Harry Kane is up there with the best, says Preston's Spurs loanee Troy Parrott
Lowe wants Preston to express themselves
17:47 , Michael Jones
Ryan Lowe spoke about tonight's FA Cup clash and says he's excited to take on Spurs and hopes his team will express themselves against the Premier League side.
"I'm really excited, and I think the lads are excited as well," said the Preston manager, "There's a buzz around the place with Tottenham Hotspur coming to town, a Premier League club who are doing fantastically well.
"It's a real opportunity for our lads to test themselves against a fantastic team.
"The message [to the players] is to go out and show what you can do. We're live on the BBC, which everyone knows, so go and show good players how good you can be as well.
"I think the respect will be there mutually. They'll respect us, we'll massively respect them for where they are and what they've done, but I think it's a case of going out and expressing yourselves and showcasing what you can do."
'We'll have a go and see what happens'
17:42 , Michael Jones
Ryan Ledson is aware of how difficult tonight's clash with Tottenham will be be that won't stop Preston playing with intent and confidence tonight.
"They've got, I would say, the best striker in the world in Harry Kane. As I've mentioned, they've got Son too, but they're solid at the back as well." he said before the match,
"Obviously they've got Conte who's an unbelievable manager, who's been everywhere.
"They'll be solid and they'll be good going forward, so we all know it's going to be a tough game, but hopefully we can give them a good game and give a good account of ourselves.
"It's sort of like the pressure's off. No one expects us to go and beat a top four Premier League side, but why not? Let's just go and have a go at them and see what happens."
Will Danjuma make his debut?
17:37 , Michael Jones
Spurs' new signing Arnaut Danjuma has travelled with the squad to Lancashire and he could make his debut at Deepdale.
Danjuma completed his move on Wednesday, sealing a loan until the end of the season from Spanish side Villarreal.
He will be the first player to make his debut in the FA Cup since Dejan Kulusevski and Rodrigo Bentancur came off the bench last season in their fourth round win over Brighton.
Danjuma starts on the bench tonight.
Arnaut Danjuma on joining Spurs
17:32 , Michael Jones
"I'm delighted to be here," said Arnuat Danjuma after signing for Tottenham. "Obviously, this opportunity is very important to me. I'm just really happy to be here.
"To be honest with you, as soon as Tottenham Hotspur came through, it was a no brainer. It is a massive club with a brilliant coach, brilliant staff and the facilities are obviously unbelievable. So, for me, I'm very, very excited to be part of the team.
"I hope to be positive, hope to leave a good impact, hope to be able to prove my qualities as a player, hope to give something new and something enthusiastic for the fans.
"And obviously aim for the highest possible in every competition we are competing in."
The history between Preston and Tottenham
17:27 , Michael Jones
Spurs first played Preston North End competitively in the FA Cup on 27th January, all the way back in 1900.
Then in the Southern League, Tottneham lost that first round clash 1-0 to in front of 7,000 fans at Deepdale.
But, just a year later, they were back in Lancashire at the same stage of the competition and produced a stunning 4-2 win in a replay on their way to writing history by becoming the first and only non-league team to lift the famous trophy.
Harry Kane's bid to become Tottenham's record scorer faces delay
17:22 , Michael Jones
Illness could see Harry Kane miss out on the chance to become Tottenham Hotspur's all-time leading scorer in Saturday's FA Cup tie at Preston.
The Spurs forward went level with Jimmy Greaves on Monday when he hit the winner at Fulham to score his 266th goal for the club.
Boss Antonio Conte revealed afterwards Kane had struggled with a fever and he missed training on Wednesday and Thursday this week before he returned on Friday ahead of the weekend clash at Sky Bet Championship outfit Preston.
Harry Kane's bid to become Tottenham's record scorer faces delay
'Anything can happen'
17:17 , Michael Jones
Spurs will arrive at Deepdale to face off against Preston tonight for the first time since their meeting at the same venue in 2009 and Ryan Ledson believes his Lancashire side can cause an FA Cup shock.
Th midfielder said: "Obviously we're coming up against some of the best players in the world. You only have to look at the front three with Harry Kane, Son, and maybe Richarlison.
"They've all just been at the World Cup so obviously they're unbelievable players, and it's good to test yourself. At Deepdale, hopefully it's going to be a full house and the fans can get behind us and we can give it a real good go.
"Anything can happen; it's football. In the FA Cup especially, you've seen some bigger shocks than Preston beating Tottenham. We'll go there, it's a free hit for us.
"We'll go and enjoy ourselves, we'll enjoy the day and give it a right good go."
Preston vs Tottenham team changes
17:12 , Michael Jones
Preston make two changes to the team that defeated Birmingham City in the Championship last time out. Ben Whiteman and Ched Evans return in place of Liam Delap and Ben Woodburn.
Antonio Conte meanwhile makes six changes to the Spurs side that defeated Portsmouth in the third round. Ben Davies, Emerson Royal, Pape Sarr, Oliver Skipp, Bryan Gil and Harry Kane all drop out with Clement Lenglet, Matt Doherty, Yves Bissouma, Rodrigo Bentancur, Ivan Perisic and Dejan Kulusevski brought in.
Kane starts on the bench after missing training with an illness.
Preston vs Tottenham line-ups
17:00 , Michael Jones
Preston XI: Woodman, Whiteman, Storey, Lindsay, Hughes, Browne, McCann, Ledson, Brady, Cannon, Evans
📋 TEAM NEWS: This is how North End line up against @SpursOfficial this evening! 👇#COYW #pnefc pic.twitter.com/mvU8oPkUfm
— Preston North End FC (@pnefc) January 28, 2023
Tottenham XI: Forster, Tanganga, Sanchez, Lenglet, Doherty, Bissouma, Bentancur, Sessegnon, Kulusevski, Son, Perisic
Your @EmiratesFACup starting XI 👊 pic.twitter.com/5GU0PH9wXb
— Tottenham Hotspur (@SpursOfficial) January 28, 2023
Preston vs Tottenham
16:58 , Michael Jones
The lights are on at Deepdale ahead of the 6pm kick off between Preston and Tottenham. The official team news will be dropping shortly.
Will Arnaut Danjuma start for Spurs?
16:55 , Michael Jones
Cristian Stellini was asked whether Tottenham's new signing Arnaut Danjuma would start for Spurs today and he was non-committal with his reply saying:
"It depends on the system we use. In the system we use normally he could be a number 10, or a striker. This is the way we want to work with him. This season we lost Lucas for a long time, and we need a player to add to our rotation and it's good to have him.
"He has experience, because he's played good Champions League last season. He is a player that can play like a striker, like a winger. He has a lot of positions he can cover, so we are happy, and we are looking forward to working with him.
"It's normal in January, you need time to give him all the information to play in our team. We are happy, we want to work with him and we are looking forward to working with him."
Jack Harrison rocket inspires Leeds past Accrington in FA Cup
16:50 , Michael Jones
Leeds avoided another FA Cup upset by winning 3-1 at Accrington to reach the fifth round for just the fourth time in 20 years.
Jack Harrison's stunning strike gave the Premier League side an interval lead and two goals in as many second-half minutes from Junior Firpo and Luis Sinisterra killed the tie.
But it was not all plain sailing for Jesse Marsch's side and Sky Bet League One side Accrington deservedly pulled one back through teenage substitute Leslie Adekoya's first senior goal.
But Leeds, dumped out of the cup by the likes of Hereford, Histon, Rochdale, Sutton, Newport and Crawley in recent years, had too much quality for their hosts and reached the last 16 for the first time in seven years.
Jack Harrison rocket inspires Leeds past Accrington in FA Cup
Kelechi Iheanacho continues fine FA Cup form as Leicester edge past Walsall
16:45 , Michael Jones
Kelechi Iheanacho was Leicester's FA Cup saviour again as the Foxes squeezed past Walsall.
The substitute's deflected strike clinched a 1-0 win to send the visitors into the fifth round and spare Youri Tielemans' blushes. The skipper missed a second-half penalty before Iheanacho ended the Saddlers' stubborn resistance.
Iheanacho, who also scored the winner in the 1-0 third-round victory at Gillingham, now has 17 goals in 23 FA Cup appearances.
Kelechi Iheanacho continues fine FA Cup form as Leicester edge past Walsall
Harry Kane a doubt
16:40 , Michael Jones
Harry Kane is a doubt for tonight's FA Cup match against Preston after missing training due to illness earlier in the week. Antonio Conte's assistant Cristian Stellini took the pre-match press conference and confirmed that Kane has travelled with the squad but may not feature.
"The most important thing we have to analyse is the condition of Harry [Kane]." he said, "He played our last match with illness. This week, he trained only today.
"He's not perfect, but he wants to travel with us, and he wants to be involved in the game. He wants to play in every game.
"We're happy he's back, but the last three days were not so perfect for him, because he pushed himself a lot in the last game."
Early team news for Preston
16:35 , Michael Jones
Preston will be without Tottneham loanee Troy Parrott as the forward is unavailable for selection against his parent club.
Liam Delap is also cup tied having represented Stoke City in their third round victory over Hartlepool United. Ben Whiteman and Brad Potts were back in training, and therefore should be in contention to feature.
How to watch Preston vs Tottenham
16:30 , Michael Jones
Preston vs Tottenham in the fourth round of the FA Cup is due to kick off at 6pm GMT on Saturday 28th January at Deepdale in Preston.
Viewers in the United Kingdom can watch the game live on BBC One, with coverage from 5.40pm. The match can be streamed via the BBC iplayer as well.
Preston vs Tottenham
15:57 , Michael Jones
Hello and welcome to The Independent's coverage of this evening's FA Cup action.
There are two late kick offs tonight with the first one seeing Championship side Preston host the Premier League's Tottenham at Deepdale. Preston have won three of their last four matches since the turn of the year and are in decent form to welcome Spurs tonight. Harry Kane missed training on Thursday and is a doubt for the match which kicks off at 6pm.
Following the conclusion of that game, Manchester United then take on Reading at Old Trafford with this match kicking off at 8pm. Erik ten Hag is targeting the latter stages of all the domestic competitions and will want his team to put on a position display against Reading tonight. Jadon Sancho has returned to first team training and good feature whilst Andy Carroll is fit again for the Championship side.
We'll have all the build-up, team news and updates throughout the evening so stick with us.

subset: commoncrawl


text:
According to the extraordinary budget submitted recently to the Legislative Assembly, the government would cut expenses in 25,800 million.
The biggest impact will hit ministries' budgets: 19,491 million colones less than the original amount.
Other major cuts would hit other government institutions that will lose 2,219 million colones.
Pension expenses would also be reduced in 4,102 million.
During the approval of the 2016 regular budget, the government pledged to reduce between 42 and 50 billion in public spending throughout 2016.
Source: La Nación.


text:
the best course sales software. Wcnonline.Learnworlds. that offer the students with on time courses a handles the concerns related to course handling. It has a demonstration that can make the user to feel simple in working.
Whether you've never ever heard of before, or you have actually invested ages searching the internet for a reliable review, you're in the ideal place.
Listed below we've composed an extensive evaluation of what offers, its interface, and the quality of its customer service. Hopefully, with all this information at your disposal, you'll be in a better position to judge whether is the right course creator solution for you.
Concentrate on the left-hand and leading side margins (this is the simplest way to tackle it). These are where all the necessary tools lie, so utilize these as your base. For instance, you'll find critical functions like "Develop course" and "Sneak peek page" from here.
As you work your method through' dashboard, you'll see that every step is accompanied by visual and textual ideas. This provides easy access to the extra assistance you require to make the most out of.
Under the "Active Students" tab, you'll see which of your trainees have actually visited real-time. This enables you to engage with your trainees there and then, while they're going through your course.
This makes it quite easy to recognize sales trends and patterns. Once again, the analytics section is a little frustrating (at very first) however stick with it, when you get to grips with it, it's a robust reporting tool.
Developing a School
At the top of the control panel, you'll find the "School wizard." Be sure to check this out prior to trying to develop your own course. This takes you through whatever you need to understand step-by-step.
All in all, creating your school is reasonably uncomplicated; it's simply a two-step procedure. First, you name your course, and then you define your language, time zone, and currency.
You can then start producing your digital courses as soon as you've set up your online school. Through the 'Creation Wizard' you'll be triggered to:
Give your course a name
Customize your course's URL
Select the 'type' obviously you wish to produce (free, paid, upcoming, unnoticeable draft, and so on).
If paid), Select a rate (.
Publish an image.
Set a description of the course.
This might seem like a lot, but don't worry– all of this is extremely simple. Learnworlds assumes you do not have any anticipation, so they attempt to make it as basic as possible for you to bring your online course to life.
explains the course modifying process as 'effortless.' And from a quick scan of some of the user evaluates online, they verify that it is. The drag and drop course contractor and content import tool makes creating your e-course extremely simple.
As we've currently alluded to, this is where LearnWorlds' learning management system (LMS) enters into its own. Here you'll discover most of its trainee interactivity and engagement tools, including:.
An interactive video gamer.
Interactive eBooks.
Online testing engines.
Let's take a look at the interactive video player in more detail. If you're an educator wanting to deal with every learning design, this tool is the one to utilize. You can utilize interactive videos to engage students aesthetically, auditorially, verbally, and kinesthetically– how neat is that?!
By consisting of any (or all) of these in your videos:.
Well, your trainees can highlight the text and make annotations as they check out your ebook. Simply like the interactive video, you can embed questionnaires and evaluations within your eBooks.
These interactive functions are somewhat distinct to LearnWorld; not many other online course platforms offer such tools.
It's up to you whether you examine students at the end of a module, lesson, upon course conclusion, or, if at all. You can insert your: logo design, course name, as well as date of problem.
As soon as you enter LearnWorlds, what you get is a fully white label site, your own domain and limitless branding abilities. On the other hand, with you need to buy a domain and a hosting environment, set up WordPress and lastly, subscribe to, to access their plug-in.
Leaving aside the pre-setup expense, your school needs a lot more to get up and running. As quickly as you sign-up with Learndash, you will probably require a designer to help you configure your school and a designer to provide it the look that you prefer.
Now, if we assume that your tech skills are excellent or you can manage hiring a team to assist you develop your website, your school will not be functional unless you set up all the needed plug-ins that are compatible with Learndash.
On the other hand, whether you need to develop a landing page or an entire school,' drag-and-drop contractor enables you to set up and personalize your site quickly, with absolutely no coding skills.
You can pick from 400+ design templates and widgets, include powerful kinds and pop-ups and even produce communities that look like an integrated social network. All these functionalities are readily available right in your school's environment, without any external combination or plug-in.
From monitoring how your students engage with your courses and following their development to tracking profits from your affiliates and sales, analytics are crucial to optimizing your school's performance.
With advanced integrated analytics tools, LearnWorlds empowers you to keep your students engaged and offer more courses with total user progress reports and deep course insights, right on your control panel. For a fast summary, you can access advanced visualizations and charts that will assist you transform your courses and take your school to the next level.
As an example, you can see your most interesting courses, the performance of your learning activities and essential metrics about your trainees' interaction with your school. All these pieces of information exist in a structured way and can be easily exported, so you can share them with your group or use them in your own systems.
Learndash does not have a built-in analytics tool, though you can integrate your school with plug-ins and tools like Google Analytics and Propanel. Learndash's integrations will help you get there if connecting numerous external apps to get a glance of your school's efficiency is not a concern for you.
On the other hand, LearnWorlds features its unique Mobile App Contractor, allowing you to create your own white-labeled app for both iOS and Android, at an extra fee, and take your business mobile.
Setting up your app is quite simple, as you can leverage the click-and-edit visual home builder functionality to bring your brand to life, without a line of code required. Also, you can keep your course curriculum constant without feeling overwhelmed. Your school's content is automatically integrated for your school's web and mobile variations at the same time. Wcnonline.Learnworlds
The Mobile App Home builder comes packed with incredible features to boost your organization and boost your trainees' engagement. You can harness the power of push notifications to keep your users engaged and trigger them to do something about it. However also, you can provide learners with a quick and easy method to purchase your online courses through in-app purchases and supercharge your school's growth.


text:
System Maintenance! On Sunday, January 21, between 10:00am and noon, WRA will perform maintenance, temporarily disrupting access to the WRA website as well as access to online coursework. Please plan accordingly. Thank you for your patience.
4801 Forest Run Road, Suite 201
Madison, WI 53704-7337
In recent months, the WRA has been in communication with legal counsel from the National Association of REALTORS® (NAR) as to the application of the federal e-consent law, also known as the federal Electronic Signatures in Global and National Commerce Act (E-Sign).A good portion of practitioners and consumers live in an electronic world. And because of this, the WRA regularly receives questions about handling the situation of one party providing electronic consent while the other cannot or may not want to provide electronic consent because the agent is the one who will receive documents on his or her behalf in the transaction.
With guidance from NAR, the WRA can now provide some clarification as to a common and frustrating situation in which one consumer wants to conduct the real estate transaction in the electronic world — which could include electronic signatures, electronic records and email delivery — while the other does not have the ability to use electronic means or will not or does not provide electronic consent.
The discussion with NAR provided two clarifications:
To help simplify and present this discussion in as straightforward a manner as possible, I present you with a series of questions and answers:
Question: Can agents' email addresses be used for delivery in transactional documents instead of consumers' email addresses?
Answer: Yes.
Per NAR's guidance, an agent can do this presuming the agent has authorization from the consumer to use the agent's email address on the consumer's behalf. However, keep in mind that electronic consent is required if the agent forwards transaction documents on to the consumer's email address.
Question: Can agents use their email addresses instead of the consumers' email addresses for delivery in the offer to purchase even though one or both of the consumers cannot or will not provide electronic consent?
Rather than shutting down one party's use of technology when the other party does not or cannot use technology and thus will not provide electronic consent, the offer allows each party to authorize delivery of documents in a practical method.
Question: When a party authorizes the agent to include the agent's email address in the offer to purchase or some other transaction document, does that authorization have to be in writing?
Answer: No.
However, like most things in life and real estate, it's typically easier to defend when there is written evidence. While obtaining the consumer's authorization allowing the agent to use the agent's email address for delivery in the transaction is preferred, it is not required by the law. Company policy will provide guidance for the agent on how to proceed relating to the authorization.
For instance, when someone says they have the authority under a power of attorney to act on behalf of someone else, the person claiming to have the authority is the one who must bear the burden of proving they have the proper authority to act in the capacity of power of attorney.
The easiest way to prove authority to act as a power of attorney is by providing written evidence. The same would be true if a consumer later claims never to have authorized the use of the agent's email address on the consumer's behalf in a real estate transaction. One way to obtain written authorization could be to use the following language in the offer or other transaction document:
If the agent's email address appears in the email delivery section of the offer to purchase or other transaction document and the consumer has not provided electronic consent, the consumer authorizes the use of that email address on the consumer's behalf.
Another way would be to use the following language in a listing contract, buyer agency agreement or other agency agreement:
Client authorizes the use of the email address of an agent associated with the Firm in the email delivery section of the offer to purchase or other transaction document and hereby authorizes the use of that email address on the consumer's behalf for delivery of documents and written notices.
Keep in mind, if you forward the transaction documents and/or notices you received from the other agent in these circumstances to your party by email, then arguably you would need to obtain electronic consent from the consumer because you have triggered the E-Sign law. For example, if a listing agent sends a lead-based paint (LBP) disclosure to the buyer's agent, the buyer's agent must have the buyer's electronic consent to forward the LBP disclosure to the buyer's email. If the agent is going to print out and deliver the LBP disclosure to the buyer personally, by fax or by mail as agreed upon in the offer, the agent does not need to obtain electronic consent.
Question: Is there a risk to include my email address as the agent in the delivery section for my party?
If an agent's email address is used, the agent becomes responsible to make sure the documents are timely forwarded and/or presented to the party. An agent's acceptance of this role creates responsibilities on the part of the agent to make sure that notices and other documents received on behalf of a party are promptly and competently processed and forwarded to the party. The agent often must act swiftly because the party often is required to give a quick response to the other party. Arguably there may be liability when an agent does not timely process and deliver critical materials. The same risk exists now when a party asks an agent to use the agent's fax number or address or to act as the party's recipient for delivery.
Question: Can I ignore federal law now?
The laws still exist and therefore you still need to be concerned about electronic consent. This clarification offers some relief, but the federal law cannot be ignored. See the "Overview of the Law" box for more information.
The WRA created a one-page cheat sheet that provides an overview of the federal e-sign law, how to meet the law's requirements and a handy chart about electronic consent. Download the PDF of the one-page cheat sheet at the link below.
Download electronic consent cheat sheet >>
Cori Lamont is Director of Corporate and Regulatory Affairs for the WRA.


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1-1 Coaching
Create the life you want!
- 1 hr1 hour
- 350 US dollars$350
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Service Description
Using various techniques, we will work together to create the life you have been dreaming of.
Cancellation Policy
I'm pretty flexible, but ask that you let me know the day before if possible of needing to reschedule.
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