Browsing by Author "Thakker, Chandresh"
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Item Increasing bacterial succinate productivity(2014-08-05) San, Ka-Yiu; Bennett, George N.; Balzer, Grant; Zhu, Jiang; Thakker, Chandresh; Sánchez, Ailen; Rice University; United States Patent and Trademark OfficeImproved bacteria for making succinate and other 4 carbon dicarboxylates from the Krebs cycle have modifications to reduce acetate, lactate, EtOH and formate, as well as turn on the glyoxylate shunt, produce more NADH and overexpress In one embodiment, the bacteria are ΔadhEΔldhAΔiclRΔack-pta plus PYC+ and NAD+-dependent FDH+.Item Making C4+ products in bacteria(2017-05-16) Bennett, George; Thakker, Chandresh; Rice University; United States Patent and Trademark OfficeMethods of making C4+ hydrocarbon feedstocks using anaerobic microbes are described.Item Metabolic engineering of carbon and redox flow in the production of small organic acids(Springer, 2014) Thakker, Chandresh; Martínez, Irene; Li, Wei; San, Ka-Yiu; Bennett, George N.The review describes efforts toward metabolic engineering of production of organic acids. One aspect of the strategy involves the generation of an appropriate amount and type of reduced cofactor needed for the designed pathway. The ability to capture reducing power in the proper form, NADH or NADPH for the biosynthetic reactions leading to the organic acid, requires specific attention in designing the host and also depends on the feedstock used and cell energetic requirements for efficient metabolism during production. Recent work on the formation and commercial uses of a number of small mono- and diacids is discussed with redox differences, major biosynthetic precursors and engineering strategies outlined. Specific attention is given to those acids that are used in balancing cell redox or providing reduction equivalents for the cell, such as formate, which can be used in conjunction with metabolic engineering of other products to improve yields. Since a number of widely studied acids derived from oxaloacetate as an important precursor, several of these acids are covered with the general strategies and particular components summarized, including succinate, fumarate and malate. Since malate and fumarate are less reduced than succinate, the availability of reduction equivalents and level of aerobiosis are important parameters in optimizing production of these compounds in various hosts. Several other more oxidized acids are also discussed as in some cases, they may be desired products or their formation is minimized to afford higher yields of more reduced products. The placement and connections among acids in the typical central metabolic network are presented along with the use of a number of specific non-native enzymes to enhance routes to high production, where available alternative pathways and strategies are discussed. While many organic acids are derived from a few precursors within central metabolism, each organic acid has its own special requirements for high production and best compatibility with host physiology.Item Production of succinic acid by engineered E. coli strains using soybean carbohydrates as feedstock under aerobic fermentation conditions(Elsevier, 2013) Thakker, Chandresh; San, Ka-Yiu; Bennett, George N.Escherichia coli strains HL2765 and HL27659k harboring pRU600 and pKK313 were examined for succinate production under aerobic conditions using galactose, sucrose, raffinose, stachyose, and mixtures of these sugars extracted from soybean meal and soy solubles. HL2765(pKK313)(pRU600) and HL27659k(pKK313)(pRU600) consumed 87 mMand 98 mMhexose of soybean meal extract and produced 83 mM and 95 mM succinate, respectively. While using soy solubles extract, HL2765(pKK313)(pRU600) and HL27659k(pKK313)(pRU600) consumed 160 mM and 187 mM hexose and produced 158 mM and 183 mMsuccinate, respectively. Succinate yield of HL2765(pKK313)(pRU600)was low as compared to that of HL27659k(pKK313)(pRU600) while using acid hydrolysate of soybean meal or soy solubles extracts. Maximum succinate production of 312 mMwith a molar yield of 0.82 mol/mol hexose was obtained using soy solubles hydrolysate by HL27659k(pKK313)(pRU600). This study demonstrated the use of soluble carbohydrates of the renewable feedstock, soybean as an inexpensive carbon source to produce succinate by fermentation.Item Use of transposase and ends of IS608 enables precise and scarless genome modification for modulating gene expression and metabolic engineering applications in Escherichia coli(Wiley, 2015) Thakker, Chandresh; Lin, Kevin; Martini-Stoica, Heidi; Bennett, George N.Various methods have been developed for gene disruption in bacteria; however, extra in vitro manipulation steps or the residual presence of a scar in the host chromosome limits the use of such methods. By utilizing the unique properties of ISHp608, we have developed a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The λ Red mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. We demonstrated lacZ gene point mutation repair, two precise disruptions of the lacZ gene and constructed a library of lacZ variants having variable β-galactosidase activity by changing its ribosome binding site sequences using the ISHp608 system. This technique can be used in E. coli genome modification and could be extended for use in other bacteria.