title: Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase.
abstract: Succinic acid is a specialty chemical having numerous applications in industrial, pharmaceutical and food uses. One of the major challenges in the succinate fermentation process is eliminating the formation of byproducts. In this study, we describe eliminating byproduct formate and improving succinate productivity by reengineering a high succinate producing E. coli strain SBS550MG-Cms243(pHL413Km). The NAD(+)-dependent formate dehydrogenase gene (fdh1) of Candida boidinii was coexpressed with Lactococcus lactis pyruvate carboxylase (pycA) under the control of Ptrc and PpycA promoters in plasmid pHL413KF1. The newly introduced fdh1 converts 1 mol of formate into 1 mol of NADH and CO2. The reengineered strain SBS550MG-Cms243(pHL413KF1) retains the reducing power of formate through an increase in NADH availability. In anaerobic shake flask fermentations, the parent strain SBS550MG-Cms243(pHL413Km) consumed 99.86 mM glucose and produced 172.38 mM succinate, 16.16 mM formate and 4.42 mM acetate. The FDH bearing strain, SBS550MG-Cms243(pHL413KF1) consumed 98.43 mM glucose and produced 171.80 mM succinate, 1mM formate and 5.78 mM acetate. Furthermore, external formate supplementation to SBS550MG(pHL413KF1) fermentations resulted in about 6% increase in succinate yields as compared to SBS550MG(pHL413Km). In an anaerobic fed-batch bioreactor process, the average glucose consumption rate, succinate productivity, and byproduct formate concentration of SBS550MG(pHL413Km) was 1.40 g/L/h, 1g/L/h, and 17 mM, respectively. Whereas, the average glucose consumption rate, succinate productivity and byproduct formate concentration of SBS550MG(pHL413KF1) was 2 g/L/h, 2 g/L/h, 0-3 mM respectively. A high cell density culture of SBS550MG(pHL413KF1) showed further improvement in succinate productivity with a higher glucose consumption rate. Reduced levels of byproduct formate in succinate fermentation broth would provide an opportunity for reducing the cost associated with downstream processing, purification, and waste disposal.
authors.affiliation: Department of Bioengineering, Rice University, Houston, TX 77005, USA.
authors.forename: Grant J
authors.initials: GJ
authors.lastname: Balzer
authors.forename: Chandresh
authors.initials: C
authors.lastname: Thakker
authors.forename: George N
authors.initials: GN
authors.lastname: Bennett
authors.forename: Ka-Yiu
authors.initials: KY
authors.lastname: San
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics; D005561:Formates / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods; D009243:NAD / Q000235:genetics / Q000378:metabolism*; D011766:Pyruvate Carboxylase / Q000096:biosynthesis / Q000235:genetics; D019802:Succinic Acid / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C499417:FDH1 protein, Candida; D005561:Formates; D005656:Fungal Proteins; D009243:NAD; C030544:formic acid; D019802:Succinic Acid; D005560:Formate Dehydrogenases; D011766:Pyruvate Carboxylase
keywords: Byproduct; E. coli; Formate; Formate dehydrogenase; NADH; Succinate