Co2

IdhA

2D-lactate

2CO2 2Formate

4H 2Acetyl-CoA ^

2ATPÜ 2Acetate

2Ethanol

Figure 5.5 Metabolic engineering of E. coli to produce succinate. See Figure 5.1 for explanation of different types of lines/arrows. Genes involved in engineered steps are indicated by italics. Primary fermentation products are indicated in bold. Strategies include the blocking (genes IdhA, pflB, and ptsG) and amplification (genes ppc and mdh) of homologous enzymes as well as the introduction of heterologous enzymes (R. etli pyc gene). The asterisk (*) indicates that this step does not exist in E. coli. Abbreviations, ldhA, gene encoding D-lactate dehydrogenase; mdh, gene encoding malate dehydrogenase; PEP, phosphoenolpyruvate; pflB, gene encoding pyruvate-formate lyase; ppc, gene encoding PEP carboxylase; ptsG, gene encoding the enzyme IICBGlc of the PTS system; pyc, gene encoding R. etli pyruvate carboxylase.

overexpressing PEP carboxylase results in an increase in succinate percentage yield (molar basis) from 12 to 45% (23). PEP is also a required cosubstrate for glucose transport via the phosphotransferase system (PTS) in wild-type E. coli. Thus, another approach is to direct pyruvate to the succinate branch. This is achieved by transforming a wild-type E. coli strain with plasmid pTrc99A-pyc, which expresses Rhizobium etli pyruvate carboxylase. This strategy results in an increase in both succinate percentage yield (17%) and productivity (0.17 g/L/h) (24). In order to prevent the accumulation of other undesired products, mutations in genes involved in producing lactate (ldhA, encoding lactate dehy-drogenase) and formate (pflB, encoding pyruvate-formate lyase) were introduced, obtaining the strain E. coli NZN111, that grew poorly on glucose under anaerobic conditions. When the gene encoding malic enzyme from Ascaris suum was transformed into NZN111, succinate percentage yield and productivity were further increased to 39% and 0.29 g/L/h, respectively (25,26). Expression of mdh gene encoding malate dehydrogenase also resulted in improved production of succinate by E. coli NZN111 (27).

Donnelly et al. (28) reported an unknown spontaneous chromosomal mutation in NZN111, which permitted anaerobic growth on glucose, and this strain was designated as AFP111. When AFP111 was grown anaerobically under 5% H2 and 95% CO2, a succinate percentage yield of 70% and a succinate-acetate molar ratio of 1.97 were obtained. Further improvements of succinate production with this strain included dual phase fermentation (aerobic growth for biomass generation follow by anaerobic growth for succinate production) that resulted in a succinate percentage yield of 99% and a productivity of 0.87 g/L/h (29). AFP111 mutation was mapped to the ptsG gene, encoding an enzyme of the PTS (30).

Gokarn et al. (31) found that the expression of R. etli pyruvate carboxylase (PYC) in E. coli during anaerobic glucose metabolism caused a 2.7-fold increase in succinate concentration, making it the major product by mass. The increase came mainly at the expense of lactate formation. However, in a mutant lacking lactate dehydrogenase activity, expression of PYC resulted in only a 1.7-fold increase in succinate concentration. An accumulation of pyruvate and NADH, metabolites that affect the interconversion of the active and inactive forms of the enzyme pyruvate formate-lyase, may have caused the decreased enhancement of succinate. The same group (32) had previously shown that the presence of the R. etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Flux calculations indicated that during anaerobic metabolism the pyc(+) strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate than the ppc(+) strain. The results demonstrate that when phosphoenolpyruvate carboxyl-ase (PPC) or PYC are expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.

5.3.2 Pyruvic Acid

Several bacterial strains have been used to produce pyruvic acid via the fermentation of different sugars. These include strains of Escherichia, Pseudomonas, Enterococcus, Acinetobacter, and Corynebacterium (33). The most successful processes (highest yield and concentrations, and shortest fermentation times) are those involving E. coli strains. The main strategy used has been limiting pyruvate consumption by pyruvate dehydrogenase complex (PDH) under aerobic conditions. Considering that lipoic acid is a cofactor of PDH, a screen by Yokota et al. (34) identified a lipoic acid auxotroph, E. coli W1485lip2, which accumulated 25.5 g/L pyruvate from 50 g/L glucose at 32 hours. Pyruvate production was further improved by introducing a mutation in F1-ATPase, obtaining strain TBLA-1 (35). Strain TBLA-1 exhibited a higher capacity for producing pyruvate, more than 30 g/L of pyruvate were produced from 50 g/L of glucose in only 24 hours. The growth of TBLA-1 decreased to 67% of that in the parent strain, due to lower energy production (TBLA-1 is an F1-ATPase-defective mutant). The enhanced pyruvate productivity in strain TBLA-1 was thought to be linked to increased activities of some glycolytic enzymes (36). Although there was an increase in glycolytic flux due to a decrease in ATP production (i.e., an F1-ATPase mutation), the physiological mechanism mediating these changes was not identified. Recently, it was shown that the glycolytic flux in E. coli is controlled by the demand for ATP (37). By increasing the ATP hydrolysis, the glycolytic flux was increased by approximately 70%, indicating that glycolytic flux is mainly (> 75%) controlled by reactions hydrolyzing ATP. In light of these results, it is clear that increased pyruvate production in strain TBLA-1 when compared to its wild type E. coli W1485lip2 is due to lower ATP production in TBLA-1, which in turn is driving glycolysis and resulting in higher glycolytic fluxes.

5.3.3 Lactic Acid

Many microorganisms produce D-lactic acid (d-LAC), and some LABs, such as Lactobacillus bulgaricus, produce highly pure d-LAC (38). l-LAC has also been produced using other LABs, such as Lactobacillus helveticus, Lactobacillus amylophilus, and Lactobacillus delbruekii (39). Since LABs have complex nutritional requirements and low growth rates, and exhibit incomplete or negligible pentose utilization (40), other bacterial strains have been engineered to produce optically pure d- or l-LAC including E. coli (41,42), Rhizopus orizae (43), and B. subtilis (44). Chang et al. (41) engineered E. coli to produce optically pure d- or l-LAC. A pta mutant of E. coli RR1, which was deficient in the phosphotransacetylase of the Pta-AckA pathway, was found to metabolize glucose to d-LAC and to produce a small amount of succinate byproduct under anaerobic conditions. An additional mutation in ppc (encoding PPC) made the mutant produce D-LAC like a homofermentative LAB. In order to produce l-LAC, a nonindigenous fermentation product, an L-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain, which lacked phosphotransacetylase and d-LAC dehydrogenase. This recombinant strain was able to metabolize glucose to l-LAC as the major fermentation product, and produced up to 45 g/L of l-LAC. Zhou et al. (42) constructed derivatives of E. coli W3110 capable of producing D-LAC in a mineral salts medium. They eliminated competing pathways by chromosomal inactivation of genes encoding fumarate reductase (frdABCD), alcohol/aldehyde dehydrogenase (adhE), and pyruvate formate lyase (pflB). d-LAC production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule and a chemical purity of d-LAC of ~98% with respect to soluble organic compounds. The cell yield and LAC productivity were increased by a further mutation in the acetate kinase gene (ackA). The aforementioned ME strategies used to engineer homolactic pathways in E. coli are summarized in Figure 5.6.

5.3.4 Acetic Acid

Acetic acid obtained through fermentation is mainly used in the food market (vinegar, meat preservative). Microorganisms currently used in its production are Saccharomyces cerevisiae, Acetobacter aceti, and Clostridia species. E. coli W3110 was recently engineered to produce acetic acid from glucose (45). The resulting strain (TC36) converted 60 g/L of glucose into 34 g/L of acetate in 18 h. Strain TC36 was constructed by sequentially assembling deletions that inactivated oxidative phosphorylation (AatpFH), disrupted the cyclic function of the tricarboxylic acid pathway (AsucA), and eliminated native fermentation pathways (AfocA-pflB, AfrdBC, AldhA, and AadhE). These mutations minimized the loss of substrate carbon and the oxygen requirement for redox balance. Although TC36 produces only four ATPs per glucose, this strain grows well in a mineral salts medium and

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