Bioprocess considerations for expanded-bed chromatography of crude canola extract: sample preparation and adsorbent reuse

Compared to the conventional microbial and mammalian systems, transgenic plants produce proteins in a different matrix. This provides opportunities and challenges for downstream processing. In the context of the plant host Brassica napus (canola), this work addresses the bioprocessing challenges of solid fractionation, resin fouling by native plant components (e.g., oil, phenolics, etc.), hydrodynamic stability, and resin reuse for expanded bed adsorption for product capture. Plant tissue processing and subsequent protein extraction typically result in an extract with a high content of solids containing a wide particle-size distribution. Without removal of larger particles, the column inlet distributor plugged. The larger particles (> 50 microm) were easily removed through centrifugal settling comparable to that attainable with a scroll decanter. The remaining solids did not affect the column performance. Less than 4% of the lipids and phenolics in the fed extract bound to STREAMLINE trade mark DEAE resin, and this small proportion could be satisfactorily removed using recommended clean-in-place (CIP) procedures. Hydrodynamic expansion and adsorption kinetics of the STREAMLINE trade mark DEAE resin were maintained throughout 10 cycles of reuse, as was the structural integrity of the resin beads. No significant accumulation of N-rich (e.g., proteins) and C/O-rich components (e.g., oil and phenolics) occurred over the same period.     

Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.     

Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of L-leucine and L-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in alpha-proteobacteria but not in other prokaryotes analyzed.     

Constitutive expression of the beta-ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants

Polyhydroxybutyrate (PHB) is a member of a class of thermoelastic polymers called polyhydroxyalkanoates that serve many bacteria as intracellular storage molecules for carbon and energy. Transgenic plants provide a potential means of producing this polymer cost-effectively. To date, however, few reports of the successful production of this polymer have been published, with the exception of work with transgenic Arabidopsis. Using a variety of chimeric constructs, we have determined that the constitutive, chloroplast-localized expression of one of the genes involved in PHB production-the beta-ketothiolase (phbA) gene-is detrimental to the efficient production of transgenic PHB. The alternate use of either inducible or somatically activated promoters allowed the construction of transgenic PHB-producing potato (Solanum tuberosum) and tobacco (Nicotiana tabacum) plants, although the amount of PHB formed was still rather low. Taking advantage of an inducible promoter, the maximal amount of PHB produced in transgenic potato was 0.09 mg g(-1) dry weight. In transgenic tobacco using a somatically activated promoter, up to 3.2 mg g(-1) dry weight was accumulated. In Arabidopsis, the formation of high levels of PHB had previously been shown to be accompanied by severe negative effects on growth and development of the plant. Phasins are proteins known from PHB-producing bacteria speculated to serve as protectants against the highly hydrophobic surface of the PHB granules in the bacterial intracellular milieu. Co-expression of the phasin gene in parallel with the PHB synthesis genes, however, did not lead to reduced symptom development.     

Antisense repression of cytosolic phosphoglucomutase in potato (Solanum tuberosum) results in severe growth retardation, reduction in tuber number and altered carbon metabolism

The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.     

Potato hexokinase 2 complements transgenic Arabidopsis plants deficient in hexokinase 1 but does not play a key role in tuber carbohydrate metabolism

Potato plants (Solanum tuberosum L. cv. Désirée) transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 2 exhibited altered enzyme activities and expression of hexokinase 2 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed an 11-fold variation in leaf (from 48% of the wild-type activity in antisense transformants to 446% activity in sense transformants) and an 8-fold variation in developing tubers (from 35% of the wild-type activity in antisense transformants to 212% activity in sense transformants). Despite the wide range of hexokinase activities, no substantial change was found in the fresh weight yield, starch, sugar and metabolite levels of transgenic tubers. However, both potato hexokinases 1 and 2 were able to complement the hyposensitivity of antisense hexokinase 1 Arabidopsis transgenic plants to glucose. In an in vitro bioassay of seed germination in a medium with high glucose levels, double transformants showed the same sensitivity to glucose as that of the wild-type ecotype, displaying a stunted phenotype in hypocotyls, cotyledons and roots.     

Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake

Sulfo-N-succinimidyl esters of LCFAs are a powerful tool to investigate the functional significance of plasmalemmal proteins in the LCFA uptake process. This notion is based on the following observations. First, sulfo-N-succinimidyl oleate (SSO) was found to inhibit the bulk of LCFA uptake into various cell types, i.e. rat adipocytes, type II pneumocytes and cardiac myocytes. Second, using cardiac giant membrane vesicles, in which LCFA uptake can be investigated in the absence of mitochondrial -oxidation, SSO retained the ability to largely inhibit LCFA uptake, indicating that inhibition of LCFA transsarcolemmal transport is its primary action. Third, SSO has no inhibitory effect on glucose and octanoate uptake into giant membrane vesicles derived from heart and skeletal muscle, indicating that its action is specific for LCFA uptake. Finally, SSO specifically binds to the 88 kDa plasmalemmal fatty acid transporter FAT, a rat homologue of human CD36, resulting in an arrest of the transport function of this protein.In addition to its inhibitory action at the plasma membrane level, evidence is presented for the lack of a direct inhibitory effect on subsequent LCFA metabolism. First, the relative contribution of oxidation and esterification to LCFA uptake is not altered in the presence of SSO. Second, isoproterenol-mediated channeling of LCFAs into oxidative pathways is not affected by sulfo-N-succinimidyl palmitate (SSP). As an example of its application we used SSP to study the role of FAT/CD36 in contraction- and insulin-stimulated LCFA uptake by cardiac myocytes , showing that this transporter is a primary site of regulation of cellular LCFA utilization.     

A novel beta-diketone-cleaving enzyme from Acinetobacter johnsonii: acetylacetone 2,3-oxygenase

A novel Fe+Zn containing oxygenase from Acinetobacter johnsonii catalyses 2,3-cleavage of acetylacetone to acetate and methylglyoxal has been purified. The stoichiometry of reactants and products conforms to a classical dioxygenase. The pure protein is a homotetramer of 64kD with variable amounts of Fe(2+) and Zn(2+). Activity of the enzyme is more closely related to the Fe(2+) content than to the amount of protein. A purification of acetylacetone 2,3-oxygenase, some of its physical properties, and the preference for some analogous substrates are described.     

Characterisation of preYvaY export reveals differences in the substrate specificities of Bacillus subtilis and Escherichia coli leader peptidases

Translocation, processing and secretion of YvaY, a Bacillus subtilis protein of unknown function, were characterised both in B. subtilis and in Escherichia coli. In its natural host B. subtilis, YvaY was transiently synthesised at the end of the exponential growth phase. It was efficiently secreted into the culture supernatant in spite of a calculated membrane spanning domain in the mature part of the protein. In E. coli, despite the high conservation of Sec-dependent transport components, processing of preYvaY was strongly impaired. To uncover which elements of E. coli and B. subtilis translocation systems are responsible for the observed substrate specificity, components of the B. subtilis Sec-system were co-expressed besides yvaY in E. coli. Expression of B. subtilis secA or secYEG genes did not affect processing, but expression of B. subtilis signal peptidase genes significantly enhanced processing of preYvaY in E. coli. While the major signal peptidases SipS or SipT had a strong stimulatory effect on preYvaY processing, the minor signal peptidases SipU, SipV or SipW had a far less stimulatory effect in E. coli. These results reveal that targeting and translocation of preYvaY is mediated by the E. coli Sec proteins but processing of preYvaY is not performed by E. coli signal peptidase LepB. Thus, differences in substrate specificities of E. coli LepB and the B. subtilis Sip proteins provide the bottleneck for export of YvaY in E. coli. Significant slower processing of preYvaY in absence of SecB indicated that SecB mediates targeting of the B. subtilis precursor.