Strain improvements

Although at this time, there is no commercial-scale succinic acid bioprocess, interest remains high, as shown by the activity in the patent and scientific literature. Three different organisms which all show promise have been the focus of the strain improvement research and process development. 

Anaerobiospirillum succiniciproducens is another high succinate producer that has been the focus of several patents and process development. One patent describes a method for 

coli does not contain pyruvate carboxylase, a heterologous gene from Rhizobium etli was introduced into E. coli. A 50 % increase in succinate levels and about a 20 % decrease in lactate concentration were observed. Glucose uptake was not affected, and thus, this approach may have advantages over increased PEP-carboxylase, which may have undesirable 

Organic Acid and Soivent Production Acetic, Lactic, Giuconic, Succinic, and Poiyhydroxyaikanoic Acids 

Another overexpression strategy was tried with the NAD -dependent malic enzyme of E. coli Thermodynamically, the reduction of pyruvate to malate is favored, but in nature this reaction does not occur. A double mutant of E. coli, NZNlll, which is blocked in both pyruvate formate lyase pjT) and lactate dehydrogenase (Idh), was used as the host. It is unable to grow anaerobically because its pyruvate metabolism is blocked by the fermentation end products acetate, formate, ethanol, and lactic acid. The mutant NZNl 11 with multiple copies of malic enzyme accumulated succinic acid as a major end product only when the cells were switched to anaerobic metabolism gradually by metabolic depletion of oxygen in a sealed tube (Clark et al. 1988). Mutant strains blocked in either pfl or Idh did not alter their distribution of fermentation products when overexpressing malic enzyme. 

The genetic engineering of the microorganism for amino acid overproduction proceeds through various ways. 

This involves the engineering of endpoint or branch point enzymes in the amino acid biosynthetic pathway leading to the accumulation of amino acids of interest. Feedback resistance of enzymes also led to amino acid overproduction. Amplification of rate-limiting enzymes has led to the increase in phenylalanine production. Engineering the branch point enzymes has led to the conversion of tryptophan to tyrosine or phenylalanine. 

The pathways in the central carbon metabolism involve TCA cycle and glyoxylate shunt, glycolysis, phosphotransferase system (PTS), gluconeogenesis, pentose phosphate pathway (PPP).The carbon flux partitions at different nodes in the central metabolism and major flux partitioning for the product of interest may occur at principal branch points. Engineering of the enzymes in these branch points of the biosynthetic pathways will direct the carbon flux toward the product of interest leading to maximal product yield. 

Improvement in ATP synthesis has led to the overproduction of amino acids. The disruption of an inefficient branch of cytochrome complex has led to the increase in lysine production in Corynebacterium without increased consumption of sugars.  

5 Engineering Anaplerotic Pathways or Precursor Supply Chain 

To obtain industrially useful properties, an original isolate of a microorganism is greatly modified in the laboratory. Most of the organisms are altered 

In the best-documented example of the formation of lysine, the product is formed from aspartate which reacts via aspartylphosphate and aspartate semialdehyde to lysine. The wild type of Brevibacterium lactofermentum does not produce any lysine. With the following steps the yield could be increased to 50 g IT1  

S-(2-Aminoethyl)-L-cysteine (AEC), H2N-CH2-CH2-S-CH2-CH(NH2)-COOH, a lysine analog, acts as a false feedback inhibitor on aspartokinase, which produces aspartylphosphate from aspartate. The inhibitor simulates, for aspartokinase, the absence of lysine and threonine, and as a consequence the AEC insensitive mutant is no longer inhibited by lysine and threonine. The result was a yield increase from 0 to 16 g L 1. 

Lysine is formed from aspartate and pyruvate pyruvate, however, is also consumed for the synthesis of alanine. The discovery of an alanine auxotroph, a mutant which needs external alanine addition for growth because it cannot catalyze a precursor step to alanine, was responsible for a yield increase from 16 to 33 g L 1. 

a-Chlorocaprolactam and y-methyl-i-lysine inhibit enzymes which are on the metabolic path to lysine. After the respective mutants were found, the yield could be increased to 43 g L 1. 

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Refs. 21, 22, 155. Abbreviations AHV, a-amino-(3-hydroxyvaleric acid Horn, L-homoserine AEG, (3 -(2-aminoethyl)-L-cysteine ppc, phosphoenolpymvate carboxylase the strain improvement largely depends on the transduction technology sensitive resistant —, auxotroph or deficient , leaky auxotroph +, prototrophic revertant. Table 7. Amino Acid Production from Hydrocarbons ... 

Superior penicillin producing cultures ate capable of producing in excess of 30 mg/mL of penicillin G (154). Cephalosporin producing strains, however, generally grow poorly and cephalosporin C production is not as efficient as is that of penicillin. Factors such as strain maintenance, strain improvement, fermentation development, inoculum preparation, and fermentation equipment requkements ate discussed in the hterature (3,154). 

Natural isolates usually produce commercially important products in very low concentrations and the potential productivity of the organism is controlled by the genome. In practice, the process of strain improvement involves the continual genetic modification of the culture, followed by reappraisals of its cultural requirements. Genetic modification may be achieved by ... 

After a strain improvement and development programme similar to, but more complicated than that of penicillin, the D-a-aminoadipyl side chain containing cephalosporin C was obtained by large scale fermentation. However, cephalosporin C could not be isolated as easily as penicillin G or V. Due to its amphoteric nature it is soluble at any pH in the fermentation broth. Several costly isolation procedures involving ion-exchange chromatography have been developed, as a result of which cephalosporin C is much more expensive than penicillin G. 

There comes a time when sequential improvements in penicillin productivity obtained by standard strain improvement techniques (physical and chemical mutagenesis in conjunction with a variety of selection techniques that apply pressure for high-yielding variants) become subject to rate-limiting returns. At first, it is easy to double the titre with each campaign later in the genealogy even a 5% improvement would be regarded as excellent. 

Once the targets for rational strain improvement have been determined, the genes can be modified using recombinant DNA technologies, with the endpoint of overexpression or inactivation of these genes in suitable host organisms, such as E. coli, yeast, or fungi. 

Li, R. and Townsend, C.A. (2006) Rational strain improvement for enhanced clavulanic acid production by genetic engineering of the glycolytic pathway in Streptomyces clavuligerus. Metabolic Engineering, 8, 240-252. 

Demain, A.L. and Adrio, J.L. (2008) Strain improvement for production of pharmaceuticals and other microbial metabolites by fermentation. Progress in Drug Research, 65, 251, 253-289. 

G. N. Stephanopoulos, H. Alper, and J. Moxley, Exploiting biological complexity for strain improvement through systems biology. Nat. Biotechnol. 22, 1261 1267 (2004). 

Whether a biocatalyst consists either of an isolate from a natural environment, of a classically improved strain or of a strain improved by various types of genetic engineering, the biocatalyst that is obtained after a screening effort will eventually be applied in a process. In order to obtain sufficient amounts of active biocatalyst, the cells have to be produced by fermentation. 

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