Fumaric acid is a natural organic acid widely found in nature. With a chemical structure of two acid carbonyl groups and a trans-double bond, fumaric acid has extensive applications in the polymer industry, such as in the manufacture of polyesters, resins, plasticizers and miscellaneous applications including lubricating oils, inks, lacquers, styrenebutadiene rubber, etc. It is also used as acidulant in foods and beverages because of its nontoxic feature. Currently, fumaric acid is mainly produced via petrochemical processes with benzene or n-butane as the feedstock. However, with the increasing crude oil prices and concerns about the pollution problems caused by chemical synthesis, a sustainable, bio-based manufacturing process for fumaric acid production has attracted more interests in recent years.
Rhizopus oryzae is a filamentous fungus that has been extensively studied for fumaric acid production. It produces fumaric acid from various carbon sources under aerobic conditions. Malic acid and ethanol are produced as byproducts, and the latter is accumulated mainly when oxygen is limited. Like most organic acid fermentations, the production of fumaric acid is limited by low productivity, yield and final concentration influenced by many factors including the microbial strain used and its morphology, medium composition, and neutralizing agents. Many efforts have been done to improve fumaric acid production through optimization of the fermentation process. The goal of
this project was to use metabolic engineering to modify the fumaric acid biosynthesis pathway to increase fumaric acid production in R. oryzae. This is the first attempt to apply genetic engineering strategies to change the metabolic flux towards fumaric acid biosynthesis.
The biosynthesis of fumaric acid in R. oryzae takes place in the cytosol and three enzymes: pyruvate carboxylase (PYC), malate dehydrogenase and fumarase are involved in the reaction. PYC is situated at the branch point of pyruvate metabolism, and its role in affecting fumaric acid and various metabolites accumulation was thus studied in this work. An expression plasmid containing native R. oryzae pyc gene, encoding pyruvate carboxylase, was transformed into the uracil auxotroph R. oryzae M16. Two transformants were obtained: pyc3 and pyc5, and were verified by Southern hybridization. Compared to the wild type, the PYC activity in the pyc tranformants increased 56%-83%. However, the pyc transformants grew poorly and had a low fumaric acid yield of less than 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.
An exogenous phosphoenolpyruvate carboxylase (PEPC) was introduced in R. oryzae with the aim to increase CO2 fixation and the carbon flux toward Oxaloacetate. The pepc gene encoding PEPC was expressed in R. oryzae under the endogenous pgk1 promoter and pdcA terminator. The obtained pepc transformants exhibited significant PEPC activity of 3-6 mU/mg that was absent in the wild type. Compared to the fermentation kinetics of the wild type, the fumaric acid production by the pepc transformant increased 26% (0.78 g/g glucose vs. 0.62 g/g for the wild type).
Fumarase catalyzing the final step of fumaric acid biosynthesis in R. oryzae was overexpressed to investigate its effects on cell growth and fumaric acid production. Three fumR fragments with different lengths of 5’ and 3’ untranslated regions (UTR) were used to express the fumR gene in R. oryzae. All transformants showed significantly increased fumarase activity during both the seed culture and fermentation stages. However, fumarase overpression yielded more malic acid, instead of fumaric acid in the fermentation. It was attributed to the catalytic prevalence on the direction of fumaric acid to malic acid by the overexpressed fumarase. The results suggested that the overepxressed fumarase by itself was not responsible for the overproduction of fumaric acid in R. oryzae.
Fumaric acid is an intermediate in the succinic acid biosynthesis pathway in E. coli fermentation under anaerobic conditions. The fumarate reductase, encoded by frd, was disrupted in E. coli KJ060, a high succinic acid producer, to study its effect on the metabolic flux distribution. The frd gene was removed from E. coli chromosome through homologous recombination. Under anaerobic conditions, the frd disrupted mutant of E. coli KJ060M produced little succinic acid. However, it did not produce much fumaric acid either. Under aerobic conditions, the mutant produced malic acid as the main product, with a high yield of 0.72 g/g glucose, higher than that (0.65 g/g) produced by the parental strain. A high malic acid concentration of 48.4 g/L was produced in fed-batch fermentation in a 5-liter fermenter with a productivity of 2.38 g/L•h. The mutant thus has the potential for use in industrial production of malic acid, which is widely used as acidulants in foods and beverages.