This C4 dicarboxylic acid is one of the key intermediates of glucose catabolism in aerobic organisms (including Homo sapiens) but can also be formed anaerobically in fermentative microbes (figure 8.4). In either case, CO2 is required to be “fixed” into organic chemicals; in classical microbial texts, this is described as “anaplerosis,” acting to replenish the pool of dicarboxylic and tricarboxylic acids when individual com­pounds (including the major industrial products citric, itaconic, and glutamic acids) are abstracted from the intracellular cycle of reactions and accumulated in the extracellular medium. Under anaerobic conditions, and given the correct balance of fermentation

GLUCOSE

products, a net “dark” fixation of CO2 can occur, and it is this biological option that has been most exploited in the development of modern biosynthetic routes:

• The rumen bacterium Actinobacillus succinogenes was discovered at Michigan State University and commercialized by MBI International, Lansing, Michi — gan.1617 Succinate yields as high as 110 g/l have been achieved from glucose.

• At the Argonne National Laboratory, Argonne, Illinois, a mutant of E. coli unable to ferment glucose because of inactivation of the genes encod­ing lactate dehydrogenase and pyruvate formate lyase spontaneously gave rise to a chromosomal mutation that reestablished glucose fermentative capacity but with an unusual spectrum of products: 1 mol succinate and 0.5 mol each of acetate and ethanol per mole of glucose consumed.1819 The second mutation was later mapped to a glucose uptake protein that, when inactivated or impaired, led to slow glucose transport into the cells and avoided any repression of genes involved in this novel fermentation.20 The result is a curious fermentation in which redox equivalents are balanced by a partition of carbon between the routes to succinate and that to acetate and ethanol (in equal measures), pyruvate being “oxidatively” decarboxylated rather than being split by pyruvate formate lyase activity or reduced to lac­tic acid, both routes lost from wild-type E. coli biochemistry in the paren­tal strain (figure 8.5). The maximum conversion of glucose to succinate by this route is 1 mol/mol, a carbon conversion of 67%; succinate titers have

with PTS

glucose

uptake

Oxaloacetic acid

Succinic acid oxidation value = 0 0.5 mole

oxidation value = +1
1 mole

FIGURE 8.5 Redox balance in the fermentation of glucose to succinic acid by Escherichia coli.

reached 75 g/l. Because E. coli is only facultatively anaerobic, biomass in the fermentation can be generated rapidly and to a high level under aerobic conditions, O2 entry then being restricted to transform the process to one of anaerobic metabolism.21 The same organism can successfully utilize both glucose and xylose in acid hydrolysates of corn straw and generate succi­nate as a fermentation product.22

• In wild-type E. coli, glucose fermentations produce complex mixtures of acid and nonacidic products, in which succinate may be only a minor component (chapter 2, section 2.2). Nevertheless, the succinate titer can be greatly increased by process optimization, and Indian researchers achieved more than 24 g/l within 30 hours with laboratory media and

17 g/l in 30 hours in a fermentor with an economical medium based on corn steep liquor and cane sugar molasses.23 24 The same group at the University of Delhi have enhanced succinate productivity with Bacteroides fragilis, another inhabitant of the human gut and intestine but an obli­gately anaerobic species.25 26

• In complete contrast, an aerobic system for succinate production was designed with a highly genetically modified E. coli, using the same glu­cose transport inactivation described above but also inactivating possible competing pathways and expressing a heterologous (Sorghum vulgare) gene encoding PEP carboxylase, another route for anaplerosis.2728 A succinate yield of 1 mol/mol glucose consumed was demonstrated, with a high pro­ductivity (58 g/l in 59 hours) under fed-batch aerobic reactor conditions. The biochemistry involved in this production route entails directing carbon flow via anaplerotic reactions to run “backward” through the tricarboxylic acid cycle, that is, in the sequence:

PEP ^ oxaloacetate ^ malate ^ fumarate ^ succinate

• At the same time, succinate is produced in the “forward” direction by block­ing the normal workings of the cyclic pathway (with a necessary loss of carbon as CO2) and the activation of a pathway (the “glyoxylate shunt”) nor­mally only functioning when E. coli grows on acetate as a carbon source:

citrate ^ isocitrate ^ succinate + glyoxylate

• The enzymes catalyzing the final two steps in the pathway from PEP, malate dehydratase and fumarate reductase, can be overexpressed in bacterial spe­cies and are the subjects of two recent patent applications from Japan and

Korea.29,30

• To return to anaerobic rumen bacteria, Anaerobiospirillum succiniciprodu — cens had a short but intense history as a candidate succinate producer.3133 Fermenting glucose in a medium containing corn steep liquor as a cost effective source of nitrogen and inorganic nutrients, succinate titers reached

18 g/l from 20.2 g/l of glucose, equivalent to a conversion efficiency of 1.35 mol/mol.34

• The same research group at the Korean Advanced Institute of Science and Technology, Daejeon, Republic of Korea, then isolated a novel rumen bacterial species, Mannheimia succiniciproducens, and has determined its complete genomic sequence as well as constructing a detailed metabolic network for the organism.35-37 Mutants of this microbe can produce suc­cinate with much reduced amounts of other acids and can anaerobically ferment xylose and wood hydrolysate to succinate.38 39

There are grounds to predict that overexpressing genes for anaplerotic pathway enzymes would enhance succinate production (and, in other genotypes or fermenta­tion conditions, the accumulation of other acids of the tricarboxylic cycle); experi­mental evidence amply confirms this prediction.40-45 With the capabilities to perform metabolic computer-aided pathway analysis with known gene arrays, comparison of succinate producers and nonproducers and between different species would be expected to greatly accelerate progress toward constructing the “ideal” microbial cell factory. Comparison of E. coli and M. succiniciproducens suggested five target genes for inactivation but combinatorial inactivation did not result in succinate over­production in E. coli; two of the identified genes — ptsG (the glucose transport system) and pykF (encoding pyruvate kinase, the enzyme interconverting PEP and pyruvic acid), together with the second pyruvate kinase gene (pykA) — increased succinate accumulation by more than sevenfold, although succinate was still greatly outweighed by the other fermentation products (formate, acetate, etc.).46 Eliminating the glycolytic pathway below PEP will clearly aid succinate production, but the pro­ducing cells will by then be highly dependent on organic nutrients (including many amino acids) for growth and maintenance.

The combination of several related technologies is particularly appealing for commercial production of succinic acid:

• Optimization of the Argonne National Laboratory strains for succinate pro­duction by the Oak Ridge National Laboratory, Oak Ridge, Tennessee

• Innovations for succinate recovery from the fermentation broth at Argonne National Laboratory

• An improved succinic acid purification process47

• The development of catalytic methods for converting succinic acid to 1,4- butanediol and other key derivatives at Pacific Northwest National Labora­tory, Richmond, Washington48

These advances have moved biologically derived succinic acid close to commercial­ization as a component of the first genuine biorefinery.