Synergistic effect and application of xylanases as accessory enzymes to enhance the hydrolysis of pretreated bagasse.

Recently, the new trend in the second-generation ethanol industry is to use mild pretreatments, in order to reduce costs and to keep higher content of hemicellulose in the biomass. Nevertheless, a high enzyme dosage is still required in the conversion of (hemi)cellulose. The interaction between cellulases and xylanases seems to be an effective alternative to reduce enzyme loading in the saccharification process. At first, to evaluate the synergism of xylanases on bagasse degradation, we have produced two xylanases from glycoside hydrolase family 10 (GH10) and three xylanases from glycoside hydrolase family 11 (GH11), from two thermophilic organisms, Thermobifida fusca and Clostridium thermocellum, and one mesophilic organism, Streptomyces lividans. Peracetic acid (PAA) pretreated bagasse was used as substrate. The combination of XynZ-C (GH10, from C. thermocellum), and XlnB (GH11, from S. lividans) presented the highest degree of synergy after 6h (3.62). However, the combination of XynZ-C and Xyn11A (GH11, from T. fusca) resulted in the highest total yield of reducing sugars. To evaluate the synergism between xylanases and cellulases, commercial cellulase preparation from Trichoderma reesei was combined with the selected xylanases, XynZ-C and Xyn11A. About 2-fold increase was observed in the concentration of reducing sugars, when both xylanases, XynZ-C and Xyn11A, were added together with T. reesei cellulases in the reaction mixture.

Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse.

The effect of pretreatment with peracetic acid (PAA) or an ionic liquid (1-ethyl-3-methylimidazolium acetate, [Emim][OAc]) on the synergism between endoglucanase and endoxylanase in the hydrolysis of bagasse was investigated. An endoglucanase, Cel6A, with a carbohydrate-binding module (CBM) and two endoxylanases, XynZ-C without a CBM and Xyn11A with an intrinsic xylan/cellulose binding module (XBM), were selected. The hemicellulose content, especially arabinan, and the cellulose crystallinity of bagasse were found to affect the cellulase-xylanase synergism. More specifically, higher synergism (above 3.4) was observed for glucan conversion, at low levels of arabinan (0.9%), during the hydrolysis of PAA pretreated bagasse. In contrast, [Emim][OAc] pretreated bagasse, showed lower cellulose crystallinity and achieved higher synergism (over 1.9) for xylan conversion. Ultimately, the combination of Cel6A and Xyn11A resulted in higher synergism for glucan conversion than the combination of Cel6A with XynZ-C, indicating the importance of the molecular architecture of enzymes for metabolic synergism.

Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing.

The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼141 mg/L) than MG1655ΔendAΔrecA (∼48 mg/L) and DH5α (∼40 mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200 mg/L.

Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters.

Insertion specificity of mobile genetic elements is a rather complex aspect of DNA transposition, which, despite much progress towards its elucidation, still remains incompletely understood. We report here the results of a meta-analysis of IS2 target sites from genomic, phage, and plasmid DNA and find that newly acquired IS2 elements are consistently inserted around abrupt DNA compositional shifts, particularly in the form of switch sites of GC skew. The results presented in this study not only corroborate our previous observations that both the insertion sequence (IS) minicircle junction and target region adopt intrinsically bent conformations in IS2, but most interestingly, extend this requirement to other families of IS elements. Using this information, we were able to pinpoint regions with high propensity for transposition and to predict and detect, de novo, a novel IS2 insertion event in the 3' region of the gfp gene of a reporter plasmid. We also found that during amplification of this plasmid, process parameters such as scale, culture growth phase, and medium composition exacerbate IS2 transposition, leading to contamination levels with potentially detrimental clinical effects. Overall, our findings provide new insights into the role of target DNA structure in the mechanism of transposition of IS elements and extend our understanding of how culture conditions are a relevant factor in the induction of genetic instability.

Engineering of Escherichia coli strains for plasmid biopharmaceutical production: scale-up challenges.

Plasmid-based vaccines and therapeutics have been making their way into the clinic in the last years. The existence of cost-effective manufacturing processes capable of delivering high amounts of high-quality plasmid DNA (pDNA) is essential to generate enough material for trials and support future commercialization. However, the development of pDNA manufacturing processes is often hampered by difficulties in predicting process scale performance of Escherichia coli cultivation on the basis of results obtained at lab scale. This paper reports on the differences observed in pDNA production when using shake flask and bench-scale bioreactor cultivation of E. coli strains MG1655ΔendAΔrecA and DH5α in complex media with 20 g/L of glucose. MG1655ΔendAΔrecA produced 5-fold more pDNA (9.8 mg/g DCW) in bioreactor than in shake flask (1.9 mg/g DCW) and DH5α produced 4-fold more pDNA (8 mg/g DCW) in bioreactor than in shake flask (2 mg/g DCW). Accumulation of acetate was also significant in shake flasks but not in bioreactors, a fact that was attributed to a lack of control of pH.

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production.

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.

Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing.

Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains.