Fumaric acid from glucose

Early experiments concerned the enzymatic hydrogenation of fumaric acid to succinic acid, catalyzed by either yeast 24,55 or enzyme extracts from yeast cells56. Much later, strains of Escherichia coli, Aerobacter aerogenes and Aerobacter cloacae were shown to have the same ability 57,58. An interesting mixed culture fermentation has been developed, where the fungus Rhizopus chinensis produces fumaric acid from glucose and a selected E. coli converts the previously formed fumaric acid into succinic acid58. 

Strain improvements for increased fumaric acid production with laser irradiation on R. oryzae were carried out to induce mutations. Following exposure to the irradiation, the mutant strain FM19 exhibited a 56.3% increased titer to produce 49.4g/L of fumaric acid from glucose. The mutant strain followed carbon and amino acid metabolism and provided new insights into the metabolic characterization of a high-yielding fumaric acid strain (Yu et al., 2012). 

Although the production of fumaric acid from either glucose, sucrose, starch, or molasses by fermentation using Rhizopus was in commercial operation during the 1940s, it was discontinued due to low productivity and the cheap source of petroleum-derived feedstock. 

Battat et al. [87] used A. flavus ATCC 13697 as the biocatalyst for the production of malic acid from glucose in a 16-1 stirred-tank fermentor. The optimal fermentation conditions are as follows agitation rate, 350 rpm Fe +, 12 mg/1 nitrogen (as ammonium sulfate), 271 mg/1 phosphate, 1.5 mM. Total amount of CaCOj added was 90 g/1. Fermentation was carried out at 32 °C for up to 200 h. Under the aforementioned conditions, 113 g/1 of L-malic acid were produced from 120 g/1 glucose utilized with an overall productivity of 0.59 g/l/h. Based on the molar yield, it was 128% for mahc acid and 155% for total acid (malic, fumaric and succinic acid). The increase in acid accumulation during the course of incubation coincides with the increase in the activities of NAD -malate dehydrogenase, fumarase and citrate synthase. 

Commercially, fumaric acid may be prepared from glucose by the action of fungi such as Rhizopus nigricans, as a by-product in the manufacture of maleic and phthalic anhydrides, and by the isomerization of maleic acid using heat or a catalyst. 

Fumaric acid can also be produced from xylose. The rate of xylose fermentation is much slower than with glucose with a specific productivity of only about 0.075 g fumaric acid/h/g biomass. Kautola and Linko [73] used immobilized R. arrhizus with polyurethane foam to ferment xylose. A specific productivity of 0.087 g/l/h was obtained when the initial xylose concentration was 100 g/1 and the resident time was 10.25 days. 

Fumaric acid production using immobilized Rhizopus cells has also been studied. Petruccioli et al. [74] immobilized R. arrhizus NRRL 1526 on polyurethane sponge to carry out repeated batch fumaric acid production from glucose syrup with KOH/KCO3 as the neutralization agent and CO2 source. Although the yield (12.3 g/1) is low, it provides the possibility of using immobilized Rhizopus for the continuous production of fumaric acid. 

L-Malic acid can also be produced from glucose using a combination of a fumaric acid producer Rhizopus arrhizus) and an organism with a high fumarase activity in the same fermentor [89,90]. 

Succinic acid can also be generated from fumarate [100] or citrate [101] in the presence of a readily metabolizable carbon source to serve as the hydrogen donor. When citrate is the hydrogen acceptor, it is split into oxaloacetate and acetate by citrate lyase. Oxaloacetate is in turn converted into succinate [102]. The rate of conversion and yield of succinate from fumarate can be enhanced by the amplification of genes that synthesize fumarate reductase [103, 104]. Table 1 shows the fermentation results reported by Wang et al. [104]. In addition, succinic acid can be generated from glucose with mixed culture fermentation, in which fumarate produced by a Rhizopus culture is converted into succinate by a bacterial culture [105]. 

Over 500 different a-amino acids have now been synthesized or isolated. About 20 of them form the main components of proteins (see also Chapter 30). a-Amino acids are commerically obtained by fermentation of glucose (arg, asp, gin, glu, his, ile, lys, pro, val, thr) or glycine (ser), or enzymatic attack on aspartic acid (ala) or fumaric acid (asp), by hydrolysis, for example, of casein or sugar beet waste (arg, cys, his, hyp, leu, tyr), by transformation of ornithine (arg) or glutamic acid (gin), or, alternatively, by complete synthesis from aldehydes using the Strecker synthesis (ala, gly, leu, met, phe, thr, trp, val), from acrylonitrile (gly, lys), or from caprolactam (lys). The racemates are obtained by total synthesis, but L-amino acids are produced by all the other processes. The racemates are separated and the D-isomers produced are again racemized. 

Direct proof that this reductive TCA pathway participates in fumaric acid production came from C NMR experiments using [1- C] and [U- C] glucose as a carbon source for R. oryzae (Kenealy et al., 1986). The unexpectedly high fumaric acid molar yields can thus be explained in terms of pyruvate carboxylation as the initial reaction of a reductive TCA pathway (Figure 15.1). 

Zhang B, Yang ST. (2012). Metabolic engineering of Rhizopus oryzae Effects of overexpress-ing fumR gene on cell growth and fumaric acid biosynthesis from glucose. Proc Biochem, 47, 2159-2165. 

According to Tsuchida et al. [2], for a culture medium containing 13% glucose, 1% ammonium sulfate, and 1.2% fumaric acid (plus other trace nutrients etc.) the yield of L-phenylalanine was 21.7 mg/ml after 72 hr of cultivation at a temperature of 31.5°C. This represents a yield of approximately 16.7% from glucose by weight. Other amino acids are also produced in small quantities (<5 kg/m ), with Lysine making up approximately 50%. 

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