TY - JOUR TI - Parsing in vivo and in vitro contributions to microcrystalline cellulose hydrolysis by multidomain glycoside hydrolases in the Caldicellulosiruptor bescii secretome AU - Conway, Jonathan M. AU - Crosby, James R. AU - McKinley, Bennett S. AU - Seals, Nathaniel L. AU - Adams, Michael W. W. AU - Kelly, Robert M. T2 - BIOTECHNOLOGY AND BIOENGINEERING AB - Six multidomain glycoside hydrolases (GHs), CelA (Athe_1867), CelB (Athe_1859), CelC (Athe_1857), CelD (Athe_1866), CelE (Athe_1865), and CelF (Athe_1860) are encoded in the Caldicellulosiruptor bescii glucan degradation locus (GDL). Each GH was affinity-tagged, overexpressed, and purified from recombinant C. bescii for side-by-side characterization in vitro and to examine the contribution of each of these enzymes to microcrystalline cellulose hydrolysis in vivo. All six recombinant GDL GHs were glycosylated, and deletion of glycosyltransferase Athe_1864 eliminated this posttranslational modification. A simplex centroid mixture experimental design revealed that in vitro optimal mixtures of the GDL GHs were predominantly CelA, CelC, and CelE, had low to moderate proportions of CelB and CelD, and minimal CelF. The best binary mixture contained CelA + CelB in a 3:2 molar ratio, whereas the best ternary mixture was composed of CelA + CelC + CelE in equimolar amounts. Neither the native C. bescii secretome nor cocktails of GDL GHs in vitro exceeded 25% of cellulose hydrolysis observed for wild-type C. bescii in vivo. C. bescii deletion strains lacking specific GDL GHs could be restored to wild-type degradation levels with the exogenous addition of either 5 µg/ml of recombinant GDL GH cocktails based on the natural secretome or mixtures optimized in vitro. Also, the addition of CelA up to 100 µg/ml provided no significant additional benefit. These results suggest that the C. bescii secretome is naturally balanced to achieve optimal synergy for cellulose degradation. They also reinforce the importance of microbial contributions to microcrystalline cellulose hydrolysis and suggest that mass action effects from glucan fermentation shift equilibria to drive degradation. DA - 2018/10// PY - 2018/10// DO - 10.1002/bit.26773 VL - 115 IS - 10 SP - 2426-2440 SN - 1097-0290 KW - Caldicellulosiruptor berci KW - cellulase KW - extreme thermophile KW - glycoside hydrolase KW - lignocellulose KW - secretome ER - TY - JOUR TI - Bacteria and archaea as the sources of traits for enhanced plant phenotypes AU - Smith-Moore, Caroline M. AU - Grunden, Amy M. T2 - BIOTECHNOLOGY ADVANCES AB - Rising global demand for food and population increases are driving the need for improved crop productivity over the next 30 years. Plants have inherent metabolic limitations on productivity such as inefficiencies in carbon fixation and sensitivity to environmental conditions. Bacteria and archaea inhabit some of the most inhospitable environments on the planet and possess unique metabolic pathways and genes to cope with these conditions. Microbial genes involved in carbon fixation, abiotic stress tolerance, and nutrient acquisition have been utilized in plants to enhance plant phenotypes by increasing yield, photosynthesis, and abiotic stress tolerance. Transgenic plants expressing bacterial and archaeal genes will be discussed along with emerging strategies and tools to increase plant growth and yield. DA - 2018/11/15/ PY - 2018/11/15/ DO - 10.1016/j.biotechadv.2018.07.007 VL - 36 IS - 7 SP - 1900-1916 SN - 1873-1899 KW - Abiotic Stress KW - Archaea KW - Bacteria KW - Carbon Fixation KW - Genetic Engineering KW - Nitrogen Acquisition KW - Phosphate Acquisition KW - Yield ER - TY - JOUR TI - Purification of human erythropoietin by affinity chromatography using cyclic peptide ligands AU - Kish, William S. AU - Roach, Matthew K. AU - Sachi, Hiroyuki AU - Naik, Amith D. AU - Menegatti, Stefano AU - Carbonell, Ruben G. T2 - JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES AB - Prior work described the identification and characterization of erythropoietin-binding cyclic peptides SLFFLH, VVFFVH, FSLLHH and FSLLSH (all of the form cyclo[(Nα-Ac)Dap(A)-X1-X6-AE], wherein X1-X6 is the listed sequences). In this work, the peptide ligands were synthesized on Toyopearl chromatographic resins and utilized for purifying recombinant human erythropoietin (rHuEPO) from complex sources. Elution buffer pH and composition were optimized to maximize the recovery of standard rHuEPO from the peptide resins. The peptide-based adsorbents were employed for separating rHuEPO from a mixture of albumin, myoglobin, and IgG to examine their selectivity. When using FSLLHH, the inclusion of low amounts of surfactants in the wash and elution buffers facilitated the recovery of rHuEPO with high yield and purity. Specifically, FSLLSH and VVFFVH afforded the most efficient separation of rHuEPO, with yield and purity of 85% and 95–97%, respectively. The affinity resins were also utilized to purify rHuEPO from spiked CHO cell culture fluid. In particular, FSLLSH provided the most successful separation from CHO, with yield and purity above 90%, and 1.0 log10 reduction of host cell proteins. The influence of conductivity and pH in the CHO-rHuEPO load was investigated. Finally, FSLLSH-based resins were used to purify rHuEPO spiked into a Pichia pastoris cell culture fluid, resulting in product yield and purity of 96% and 84%, respectively, and 1.3 log10 reduction of host DNA. These results compare well with values obtained using wheat germ agglutinin agarose and clearly indicate the potential of the cyclic peptide resins as a viable tool for rHuEPO purification. DA - 2018/5/15/ PY - 2018/5/15/ DO - 10.1016/j.jchromb.2018.03.039 VL - 1085 SP - 1-12 SN - 1873-376X KW - Cyclic peptide ligand KW - Erythropoietin purification KW - Affinity chromatography KW - Chinese hamster ovary KW - Pichia pastoris ER -