Development of quantitative metabolomics for <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism.     

One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.     

Characterization of two Synechococcus sp. PCC7002‐related cyanobacterial strains in relation to 16S rDNA,crtR gene,lipids and pigments

Two Synechococcus strains from the Culture Collection of the Institute for Marine Sciences of Andalusia (Cádiz, Spain), namely Syn01 and Syn02, were found to be closely related to the model strain Synechococcus sp. PCC7002 according to 16S rDNA (99% identity). Pigment and lipid profiles and crtR genes of these strains were ascertained and compared. The sequences of the crtR genes of these strains were constituted by 888 bp, and showed 99% identity between Syn01 and Syn02, and 94% identity of Syn01 and Syn02 to Synechococcus sp. PCC7002. There was coincidence in photosynthetic pigments between the three strains apart from the pigment synechoxanthin, which could be only observed in Synechococcus sp. PCC7002. Species of sulfoquinovosyl‐diacyl‐glycerol (SQDG), phosphatidyl‐glycerol (PG), mono‐ and di‐galactosyl‐diacyl‐glycerol (MGDG and DGDG) were detected by high performance liquid chromatography‐mass spectrometry analysis of lipid extracts. The most abundant species within each lipid class were those containing C18:3 together with C16:0 fatty acyl substituents in the glycerol backbone of the same molecule. From these results it is concluded that these cyanobacterial strains belong to group 2 of the lipid classification of cyanobacteria.     

Automated fast filtration and on‐filter quenching improve the intracellular metabolite analysis of microorganisms

To reliably determine intracellular metabolite concentrations in microorganisms, accurate sampling and sample inactivation strategies are crucial. Here, we present a method for automated fast filtration and on‐filter quenching of microbial samples to overcome metabolite leakage induced by cold shock and significantly reduce the sampling and treatment time compared to manual filtration methods. The whole process of sampling, sample filtration, filter wash, and quenching of the filter with liquid nitrogen was finished in less than 6–15 s, depending on the experimental setup. By integration into an automated fast sampling device, we compared our method to the conventional methanol quenching method and showed that intracellular amino acid contents in Escherichia coli were significantly increased (≥75%) with our fast filtration and on‐filter quenching method. Furthermore, we investigated different filter types for the fast filtration and the efficiency of metabolite extraction from cells held on filters. Additionally, we found that the fast filtration behaves considerably different during exponential and nonexponential growth, probably due to variations of cell morphologies. Overall, we demonstrated that the automation of the fast filtration method significantly reduces the time for filtration and quenching and hence enlarge the number of metabolites that can be quantified with this leakage‐free sampling method.     

Engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of light-sensitive riboflavin (vitamin B2)

Due to their capability of photosynthesis and autotrophic growth, cyanobacteria are currently investigated with regard to the sustainable production of a wide variety of chemicals. So far, however, no attempt has been undertaken to engineer cyanobacteria for the biotechnological production of vitamins, which is probably due to the light-sensitivity of many of these compounds. We now describe a photoautotrophic bioprocess to synthesize riboflavin, a vitamin used as a supplement in the feed and food industry. By overexpressing the riboflavin biosynthesis genes ribDGEABHT from Bacillus subtilis in the marine cyanobacterium Synechococcus sp. PCC 7002 riboflavin levels in the supernatant of the corresponding recombinant strain increased 56-fold compared to the wild-type. Introduction of a second promoter region upstream of the heterologous ribAB gene – coding for rate-limiting enzymatic functions in the riboflavin biosynthesis pathway – led to a further increase of riboflavin levels (211-fold compared to the wild-type). Degradation of the light-sensitive product riboflavin was prevented by culturing the genetically engineered Synechococcus sp. PCC 7002 strains in the presence of dichromatic light generated by red light-emitting diodes (λ = 630 and 700 nm). Synechococcus sp. PCC 7002 naturally is resistant to the toxic riboflavin analog roseoflavin. Expression of the flavin transporter pnuX from Corynebacterium glutamicum in Synechococcus sp. PCC 7002 resulted in roseoflavin-sensitive recombinant strains which in turn could be employed to select roseoflavin-resistant, riboflavin-overproducing strains as a chassis for further improvement.     

Nucleotide Sequence of Plasmid pAQ1 of Marine Cyanobacterium Synechococcus sp. PCC7002

We have determined the complete nucleotide sequence of pAQ1,the smallest plasmid of the unicellular marine cyanobacteriumSynechococcus sp. PCC7002. The plasmid consists of 4,809 bpand has at least four open reading frames that potentially encodepolypeptides of 50 or more amino acids. We found that a palindromicelement, the core sequence of which is G(G/A)CGATCGCC, is over-representednot only in plasmid pAQ1 but also in the accumulated cyanobacterialgenomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942,vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBLdatabases. It suggests that this sequence might mediate generearrangement, thus increasing genetic diversity, since recombinationevents are frequent in cyanobacteria.     

Leakage of adenylates during cold methanol/glycerol quenching of Escherichia coli

Effective and rapid inactivation of cellular metabolism is a prerequisite for accurate metabolome analysis. Cold methanol quenching is commonly applied to stop any metabolic activity and, at the same time remaining the cells’ integrity. However, it is reported that especially prokaryotic cells like Escherichia coli and Corynebacterium glutamicum tend to leak intracellular metabolites during cold methanol quenching. In this work leakage of adenylates is quantified for different quenching fluids. Further, a methanol/glycerol based quenching fluid is proposed, which reduces leakage drastically compared to the commonly applied methanol/water solution (16% ATP leakage compared to more than 70%).     

Global metabolomics characterization of bacteria: pre-analytical treatments and profiling

Pre-analytical treatments of bacteria are crucial steps in bacterial metabolomics studies. In order to achieve reliable samples that can best represent the global metabolic profile in vivo both qualitatively and quantitatively, many sample treatment procedures have been developed. The use of different methods makes it difficult to compare the results among different groups. In this work, E. coli samples were tested by using NMR spectroscopy. Both liquid N₂ and cold methanol quenching procedures reduce the cell membrane integrity and cause metabolites leakage. However, liquid N₂ quenching affected the cell viability and the NMR metabolites’ profile less than cold methanol procedure. Samples obtained by metabolite extraction were significantly superior over cell suspensions and cell lysates, with a higher number of detectable metabolites. Methanol/chloroform extraction proved most efficient at extraction of intracellular metabolites from both qualitative and quantitative points of view. Finally, standard operating procedures of bacterial sample treatments for NMR metabolomics study are presented.     

An Organic Acid Based Counter Selection System for Cyanobacteria

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).     

Computational evaluation of Synechococcus sp. PCC 7002 metabolism for chemical production

Cyanobacteria are ideal metabolic engineering platforms for carbon-neutral biotechnology because they directly convert CO₂ to a range of valuable products. In this study, we present a computational assessment of biochemical production in Synechococcus sp. PCC 7002 (Synechococcus 7002), a fast growing cyanobacterium whose genome has been sequenced, and for which genetic modification methods have been developed. We evaluated the maximum theoretical yields (mol product per mol CO₂ or mol photon) of producing various chemicals under photoautotrophic and dark conditions using a genome-scale metabolic model of Synechococcus 7002. We found that the yields were lower under dark conditions, compared to photoautotrophic conditions, due to the limited amount of energy and reductant generated from glycogen. We also examined the effects of photon and CO₂ limitations on chemical production under photoautotrophic conditions. In addition, using various computational methods such as minimization of metabolic adjustment (MOMA), relative metabolic change (RELATCH), and OptORF, we identified gene-knockout mutants that are predicted to improve chemical production under photoautotrophic and/or dark anoxic conditions. These computational results are useful for metabolic engineering of cyanobacteria to synthesize value-added products.     

Roles for heme–copper oxidases in extreme high-light and oxidative stress response in the cyanobacterium Synechococcus sp. PCC 7002

The ctaCIDIEI and ctaCIIDIIEII gene clusters that encode heme–copper cytochrome oxidases have been characterized in the marine cyanobacterium Synechococcus sp. PCC 7002 and the inactivation of ctaDI was shown to affect high-light adaptation. In this study, Synechococcus sp. PCC 7002 wild-type, ctaDI, ctaDII, and ctaDI–ctaDII double mutants were grown under extreme high-light and oxidative stress to further assess the roles of cytochrome oxidases in cyanobacteria. Cells of the ctaDI mutant strain barely grew under extreme high-light illumination of 4.5 mE m⁻² s⁻¹, suggesting that CtaDI is required for high-light acclimation in Synechococcus sp. PCC 7002. The ctaDI–ctaDII double mutant cells unexpectedly tolerated extreme high-light intensity, indicating that the disruption of ctaDII gene suppresses the high-light sensitivity phenotype of the ctaDI single mutant. The ctaDII mutant cells also exhibited higher tolerance to the oxidative stress compound, methyl viologen, in the growth media. The ctaDII mutant and the ctaDI–ctaDII double mutant cells had approximately twofold higher levels of superoxide dismutase (SOD) activity, indicating that the disruption of ctaDII gene increased the capacity to decompose active oxygen species. These results suggest that the CtaII cytochrome oxidase may be involved with the oxidative stress response, including the control of SOD expression.     

Leakage-free rapid quenching technique for yeast metabolomics

Accurate determination of intracellular metabolite levels requires reliable, reproducible techniques for sampling and sample treatment. Quenching in 60% (v/v) methanol at −40°C is currently the standard method for sub-second arrest of metabolic activity in microbial metabolomics but there have been contradictory reports in the literature on whether leakage of metabolites from the cells occurs. We have re-evaluated this method in S. cerevisiae using a comprehensive, strictly quantitative approach. By determining the levels of a large range of metabolites in different sample fractions and establishing mass balances we could trace their fate during the quenching procedure and confirm that leakage of metabolites from yeast cells does occur during conventional cold methanol quenching, to such an extent that the levels of most metabolites have been previously underestimated by at least twofold. In addition, we found that the extent of leakage depends on the time of exposure, the temperature and the properties of the methanol solutions. Using the mass balance approach we could study the effect of different quenching conditions and demonstrate that leakage can be entirely prevented by quenching in pure methanol at ≤−40°C, which we propose as a new improved method. Making use of improved data on intracellular metabolite levels we also re-evaluated the need of sub-second quenching of metabolic activity and of removing the extracellular medium. Our findings have serious implications for quantitative metabolomics-based fields such as non-stationary ¹³C flux analysis, in vivo kinetic modeling and thermodynamic network analysis.

Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum

Background

A reliable quenching and metabolite extraction method has been developed for Lactobacillus plantarum. The energy charge value was used as a critical indicator for fixation of metabolism.

Results

Four different aqueous quenching solutions, all containing 60% of methanol, were compared for their efficiency. Only the solutions containing either 70 mM HEPES or 0.85% (w/v) ammonium carbonate (pH 5.5) caused less than 10% cell leakage and the energy charge of the quenched cells was high, indicating rapid inactivation of the metabolism. The efficiency of extraction of intracellular metabolites from cell cultures depends on the extraction methods, and is expected to vary between micro-organisms. For L. plantarum, we have compared five different extraction methodologies based on (i) cold methanol, (ii) perchloric acid, (iii) boiling ethanol, (iv) chloroform/methanol (1:1) and (v) chloroform/water (1:1). Quantification of representative intracellular metabolites showed that the best extraction efficiencies were achieved with cold methanol, boiling ethanol and perchloric acid.

Conclusion

The ammonium carbonate solution was selected as the most suitable quenching buffer for metabolomics studies in L. plantarum because (i) leakage is minimal, (ii) the energy charge indicates good fixation of metabolism, and (iii) all components are easily removed during freeze-drying. A modified procedure based on cold methanol extraction combined good extractability with mild extraction conditions and high enzymatic inactivation. These features make the combination of these quenching and extraction protocols very suitable for metabolomics studies with L. plantarum.     

Respiratory terminal oxidases alleviate photo-oxidative damage in photosystem I during repetitive short-pulse illumination in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Oxygenic phototrophs are vulnerable to damage by reactive oxygen species (ROS) that are produced in photosystem I (PSI) by excess photon energy over the demand of photosynthetic CO₂ assimilation. In plant leaves, repetitive short-pulse (rSP) illumination produces ROS to inactivate PSI. The production of ROS is alleviated by oxidation of the reaction center chlorophyll in PSI, P700, during the illumination with the short-pulse light, which is supported by flavodiiron protein (FLV). In this study, we found that in the cyanobacterium Synechocystis sp. PCC 6803 P700 was oxidized and PSI was not inactivated during rSP illumination even in the absence of FLV. Conversely, the mutant deficient in respiratory terminal oxidases was impaired in P700 oxidation during the illumination with the short-pulse light to suffer from photo-oxidative damage in PSI. Interestingly, the other cyanobacterium Synechococcus sp. PCC 7002 could not oxidize P700 without FLV during rSP illumination. These data indicate that respiratory terminal oxidases are critical to protect PSI from ROS damage during rSP illumination in Synechocystis sp. PCC 6803 but not Synechococcus sp. PCC 7002.     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Metabolic quenching of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>: efficiency of methods and impact of cold shock

Representative and valid cytoplasmic concentrations are essential for ensuring the significance of results in the field of metabolome analysis. One of the most crucial points in this respect is the sampling itself. A rapid and sudden stopping of the metabolism on a timescale that is much faster than the conversion rates of investigated metabolites is worthwhile. This can be achieved by applying of cold methanol quenching combined with reproducible, fast, and automated sampling. Unfortunately, quenching the metabolism by a sharp temperature shift leads to what is known as cold shock or the cell-leakage effect. In the present work, we applied a microstructure heat exchanger to analyze the cold shock effect using Corynebacterium glutamicum as a model microorganism. Using this apparatus together with a silicon pipe, it was possible to assay the leakage effect on a timescale starting at 1 s after cooling cell suspension. The high turnover rates not only require a rapid quenching technique, but also the correct application. Moreover, we succeeded in showing that even when the required appropriate setup of methanol quenching is not used, the metabolism is not stopped within the required timescale. By applying robust techniques like rapid sampling in combination with reproducible sample processing, we ensured fast and reliable metabolic inactivation during all steps.     

Characterization of the heme–histidine cross-link in cyanobacterial hemoglobins from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. PCC 6803 and<Emphasis Type="Italic"> Synechococcus</Emphasis> sp. PCC 7002

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the ¹H, ¹⁵N, and ¹³C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N₂-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal pK _a whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations DQF-COSY double-quantum-filtered correlated spectroscopy - GlbN cyanoglobin - Hb hemoglobin - hx hexacoordinate - MALDI matrix-assisted laser desorption ionization - NOE nuclear Overhauser effect - NOESY two-dimensional nuclear Overhauser effect spectroscopy - rHb recombinant hemoglobin - rHb-A recombinant hemoglobin with covalently attached heme - rHb-R recombinant heme-reconstituted hemoglobin - S6803 Synechocystis sp. PCC 6803 - S7002 Synechococcus sp. PCC 7002 - TOCSY totally correlated two-dimensional spectroscopy - TPPI time-proportional phase incrementation - trHb truncated hemoglobin - WATERGATE water suppression by gradient-tailored excitation - WEFT water elimination Fourier transform     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus     

Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol

Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical diversity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5 °C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells.