Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases.

Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.

Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering.

Escherichia coli strains are widely used as host for the production of recombinant proteins. Compared to E. coli K12, E. coli BL21 (DE3) has several biotechnological advantages, such as a lower acetate yield and a higher biomass yield, which have a beneficial effect on protein production. In a previous study (BMC Microbiol. 2011, 11:70) we have altered the metabolic fluxes of a K12 strain (i.e. E. coli MG1655) by deleting the regulators ArcA and IclR in such a way that the biomass yield is remarkably increased, while the acetate production is decreased to a similar value as for BL21 (DE3). In this study we show that the increased biomass yield beneficially influences recombinant protein production as a higher GFP yield was observed for the double knockout strain compared to its wild type. However, at higher cell densities (>2 g L(-1) CDW), the GFP concentration decreases again, due to the activity of proteases which obstructs the application of the strain in high cell density cultivations. By further deleting the genes lon and ompT, which encode for proteases, this degradation could be reduced. Consequently, higher GFP yields were observed in the quadruple knockout strain as opposed to the double knockout strain and the MG1655 wild type and its yield approximates the GFP yield of E. coli BL21 (DE3), that is, 27±5 mg g(CDW)(-1) vs. 30±5 mg g(CDW)(-1), respectively.

Biocatalytic production of novel glycolipids with cellodextrin phosphorylase.

Glycolipids have gained increasing attention as natural surfactants with a beneficial environmental profile. They are typically produced by fermentation, which only gives access to a limited number of structures. Here we describe the biocatalytic production of novel glycolipids with the cellodextrin phosphorylase from Clostridium stercorarium. This enzyme was found to display a broad donor and acceptor specificity, allowing the synthesis of five different products. Indeed, using either α-glucose 1-phosphate or α-galactose 1-phosphate as glycosyl donor, sophorolipid as well as glucolipid could be efficiently glycosylated. The transfer of a glucosyl moiety afforded a mixture of products that precipitated from the solution, resulting in near quantitative yields. The transfer of a galactosyl moiety, in contrast, generated a single product that remained in solution at thermodynamic equilibrium. These glycolipids not only serve as a new class of biosurfactants, but could also have applications in the pharmaceutical and nanomaterials industries.

Effect of iclR and arcA deletions on physiology and metabolic fluxes in Escherichia coli BL21 (DE3).

Deletion of both iclR and arcA in E. coli profoundly alters the central metabolic fluxes and decreases acetate excretion by 70%. In this study we investigate the metabolic consequences of both deletions in E. coli BL21 (DE3). No significant differences in biomass yields, acetate yields, CO(2) yields and metabolic fluxes could be observed between the wild type strain E. coli BL21 (DE3) and the double-knockout strain E. coli BL21 (DE3) ΔarcAΔiclR. This proves that arcA and iclR are poorly active in the BL21 wild type strain. Noteworthy, both strains co-assimilate glucose and acetate at high glucose concentrations (10-15 g l(-1)), while this was never observed in K12 strains. This implies that catabolite repression is less intense in BL21 strains compared to in E. coli K12.

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering.

Different hosts have been used for recombinant protein production, ranging from simple bacteria, such as Escherichia coli and Bacillus subtilis, to more advanced eukaryotes as Saccharomyces cerevisiae and Pichia pastoris, to very complex insect and animal cells. All have their advantages and drawbacks and not one seems to be the perfect host for all purposes. In this review we compare the characteristics of all hosts used in commercial applications of recombinant protein production, both in the area of biopharmaceuticals and industrial enzymes. Although the bacterium E. coli remains a very often used organism, several drawbacks limit its possibility to be the first-choice host. Furthermore, we show what E. coli strains are typically used in high cell density cultivations and compare their genetic and physiological differences. In addition, we summarize the research efforts that have been done to improve yields of heterologous protein in E. coli, to reduce acetate formation, to secrete the recombinant protein into the periplasm or extracellular milieu, and to perform post-translational modifications. We conclude that great progress has been made in the incorporation of eukaryotic features into E. coli, which might allow the bacterium to regain its first-choice status, on the condition that these research efforts continue to gain momentum.

Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3).

BACKGROUND: Gene expression is regulated through a complex interplay of different transcription factors (TFs) which can enhance or inhibit gene transcription. ArcA is a global regulator that regulates genes involved in different metabolic pathways, while IclR as a local regulator, controls the transcription of the glyoxylate pathway genes of the aceBAK operon. This study investigates the physiological and metabolic consequences of arcA and iclR deletions on E. coli K12 MG1655 under glucose abundant and limiting conditions and compares the results with the metabolic characteristics of E. coli BL21 (DE3). RESULTS: The deletion of arcA and iclR results in an increase in the biomass yield both under glucose abundant and limiting conditions, approaching the maximum theoretical yield of 0.65 c-mole/c-mole glucose under glucose abundant conditions. This can be explained by the lower flux through several CO2 producing pathways in the E. coli K12 ΔarcAΔiclR double knockout strain. Due to iclR gene deletion, the glyoxylate pathway is activated resulting in a redirection of 30% of the isocitrate molecules directly to succinate and malate without CO2 production. Furthermore, a higher flux at the entrance of the TCA was noticed due to arcA gene deletion, resulting in a reduced production of acetate and less carbon loss. Under glucose limiting conditions the flux through the glyoxylate pathway is further increased in the ΔiclR knockout strain, but this effect was not observed in the double knockout strain. Also a striking correlation between the glyoxylate flux data and the isocitrate lyase activity was observed for almost all strains and under both growth conditions, illustrating the transcriptional control of this pathway. Finally, similar central metabolic fluxes were observed in E. coli K12 ΔarcA ΔiclR compared to the industrially relevant E. coli BL21 (DE3), especially with respect to the pentose pathway, the glyoxylate pathway, and the TCA fluxes. In addition, a comparison of the genome sequences of the two strains showed that BL21 possesses two mutations in the promoter region of iclR and rare codons are present in arcA implying a lower tRNA acceptance. Both phenomena presumably result in a reduced ArcA and IclR synthesis in BL21, which contributes to the similar physiology as observed in E. coli K12 ΔarcAΔiclR. CONCLUSIONS: The deletion of arcA results in a decrease of repression on transcription of TCA cycle genes under glucose abundant conditions, without significantly affecting the glyoxylate pathway activity. IclR clearly represses transcription of glyoxylate pathway genes under glucose abundance, a condition in which Crp activation is absent. Under glucose limitation, Crp is responsible for the high glyoxylate flux, but IclR still represses transcription. Finally, in E. coli BL21 (DE3), ArcA and IclR are poorly expressed, explaining the similar fluxes observed compared to the ΔarcAΔiclR strain.

Validation study of 24 deepwell microtiterplates to screen libraries of strains in metabolic engineering.

In this study we validated the use of 24 square deepwell microtiterplates to screen large libraries of metabolically engineered strains by investigating the optimization of succinate production. Wild type E. coli MG1655 and 11 derived mutants were physiologically evaluated by growth in 24 deepwell MTPs and 2L benchtop bioreactors. Growth parameters, product yields and byproduct formation were determined for all mutants. The results show that similar average values and standard deviations for these parameters were obtained. Especially a high correlation was noticed for the acetate byproduct yield and the succinate production rate. For these parameters there was no significant difference for 8 out of 12 strains between MTPs and 2L bioreactors. However a lower maximum growth rate was observed in 2L reactors as opposed to 24 deepwell plates for 9 out of 12 mutants which could be linked to the higher amount of dead cells in the benchtop bioreactors (12% vs. 2% in MTPs). Finally, a cluster-based approach was used to select good producer strains, i.e. strains with a high succinate yield and succinate production rate. Bad, intermediate and good producer strains were clustered in the same groups for MTPs and benchtop bioreactors for 11 out of the 12 investigated strains.

Transient metabolic modeling of Escherichia coli MG1655 and MG1655 DeltaackA-pta, DeltapoxB Deltapppc ppc-p37 for recombinant beta-galactosidase production.

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, among other reasons because its genetics are far better characterized than those of any other microorganism. To improve the understanding of recombinant protein synthesis in E. coli, the production of a model recombinant protein, beta-galactosidase, was studied in response to the constitutive overexpression of the anaplerotic reaction afforded by PEP carboxylase. To this end, an IPTG wash-in experiment was performed starting from a well-defined steady-state condition for both the wild-type E. coli and a mutant with a defective acetate pathway and a constitutively overexpressed ppc. In order to compare the dynamics of the fluxes over time during the wash-in experiment, a method referred to as transient metabolic flux analysis, which is based on steady-state metabolic flux analysis, was used. This allowed us to track the intracellular changes/fluxes in both strains. It was observed that the flux towards fermentation products was 3.6 times lower in the ppc overexpression mutant compared to the wild-type E. coli. In the former on the other hand, the PPC flux is in general higher. In addition, the flux towards beta-galactosidase was higher (12.4 times), resulting in five times more protein activity. These results indicate that by constitutively overexpressing the anaplerotic ppc gene in E. coli, the TCA cycle intermediates are increasingly replenished. The additional supply of these protein precursors has a positive result on recombinant protein production.

Catching prompt metabolite dynamics in Escherichia coli with the BioScope at oxygen rich conditions.

The design and application of a BioScope, a mini plug-flow reactor for carrying out pulse response experiments, specifically designed for Escherichia coli is presented. Main differences with the previous design are an increased volume-specific membrane surface for oxygen transfer and significantly decreased sampling intervals. The characteristics of the new device (pressure drop, residence time distribution, plug-flow behavior and O2 mass transfer) were determined and evaluated. Subsequently, 2.8 mM glucose perturbation experiments on glucose-limited aerobic E. coli chemostat cultures were carried out directly in the chemostat as well as in the BioScope (for two time frames: 8 and 40 s). It was ensured that fully aerobic conditions were maintained during the perturbation experiments. To avoid metabolite leakage during quenching, metabolite quantification (glycolytic and TCA-cycle intermediates and nucleotides) was carried out with a differential method, whereby the amounts measured in the filtrate were subtracted from the amounts measured in total broth. The dynamic metabolite profiles obtained from the BioScope perturbations were very comparable with the profiles obtained from the chemostat perturbation. This agreement demonstrates that the BioScope is a promising device for studying in vivo kinetics in E. coli that shows much faster response (< 10 s) in comparison with eukaryotes.