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United States Department of Agriculture

Agricultural Research Service

Research Project: NOVEL TECHNOLOGIES FOR PRODUCING RENEWABLE CHEMICALS AND POLYMERS FROM CARBOHYDRATES DERIVED FROM AGRICULTURAL FEEDSTOCKS

Location: Renewable Product Technology Research Unit

Title: Metabolic engineering of Rhizopus oryzae: Effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose

Authors
item Zhang, Baohua -
item Skory, Christopher
item Yang, Shang-Tian -

Submitted to: Metabolic Engineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 6, 2012
Publication Date: September 1, 2012
Citation: Zhang, B., Skory, C.D., Yang, S. 2012. Metabolic engineering of Rhizopus oryzae: Effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose. Metabolic Engineering. 14(5):512-520.

Interpretive Summary: This research describes the metabolic engineering of the fungus Rhizopus for improved production of the industrial chemical fumaric acid. This natural product is used extensively by the food industry and for the manufacturing of synthetic resins. The world demand for fumaric is in excess of 90,000 tons/yr and is expected to increase with the development of new conversion technologies that allow fumaric acid to be used for numerous applications. Rhizopus is able to convert or ferment sugars obtained from agricultural crops to fumaric acid, but improved technologies are necessary to minimize production costs. In this work, fumaric acid yields were increased by over 25% by introducing a novel enzyme that enhances the metabolic conversion pathway in Rhizopus. This work is expected to be a significant contribution towards the development of improved technologies for producing fumaric acid, thereby benefiting the agricultural grower and ultimately the consumer.

Technical Abstract: Fumaric acid, a dicarboxylic acid used as a food acidulant and in manufacturing synthetic resins, can be produced from glucose in fermentation by Rhizopus oryzae. However, the fumaric acid yield is limited by the co-production of ethanol and other byproducts. To increase fumaric acid production, overexpressing endogenous pyruvate carboxylase (PYC) and exogenous phosphoenolpyruvate carboxylase (PEPC) to increase the carbon flux toward oxaloacetate were investigated. Compared to the wild type, the PYC activity in the pyc transformants increased 56%-83%, whereas pepc transformants exhibited significant PEPC activity (3-6 mU/mg) that was absent in the wild type. Fumaric acid production by the pepc transformant increased 26% (0.78 g/g glucose vs. 0.62 g/g for the wild type). However, the pyc transformants grew poorly and had low fumaric acid yields (<0.05 g/g glucose) due to the formation of large cell pellets that limited oxygen supply and resulted in the accumulation of ethanol with a high yield of 0.13-0.36 g/g glucose. This study is the first attempt to use metabolic engineering to modify the fumaric acid biosynthesis pathway to increase fumaric acid production in R. oryzae.

Last Modified: 9/20/2014