Putrescine apparently has a specific role in skin phys­iology and neuroprotection (Janne et al., 2005). Fermen­tative production of putrescine can be achieved by manipulating arginine decarboxylase (ADC) pathway or ornithine decarboxylase (ODC) pathway in E. coli (Figure 19.4(a)) and C. glutamicum (Figure 19.4(b)) and of which, the ODC pathway is preferable as it comprises only a single reaction compared to two or three reactions of the ADC pathway. To increase L-ornithine formation, its conversion to L-arginine may be blocked; however, this results in unfavorable auxotrophy for L-arginine. Thus, the maintenance of prototrophy with concomitant high L-ornithine supply is a focus in strain construction. The pathway for biosynthesis of L-arginine and L-orni- thine, the substrates of the initial decarboxylase reac­tions in the ADC and ODC pathway, respectively, are similar in E. coli and C. glutamicum. There is some differ­ence in ornithine synthesis between them; C. glutamicum has a cyclic pathway while E. coli has a linear pathway (Glansdorff and Xu, 2007). The cyclic pathway is economical in terms of metabolic cost for ornithine syn­thesis when compared to linear pathway, because in linear pathway there is a concomitant hydrolysis of acetyl-CoA to acetic acid. The L-ornithine was then con­verted to citrulline by ornithine carbamoyl phosphate transferase ArgF (EC 2.1.3.3). The synthesis of all en­zymes in the pathway is subject to repression by L-argi — nine, which is mediated by the repressor ArgR in E. coli and C. glutamicum (Glansdorff and Xu, 2007). In order to use a microorganism in industrial fermentations or bio­transformations the organism should possess high toler­ance to the desired product. Concentrations of up to 66 g/l putrescine reduced the growth rate of C. glutami — cum by 34% and that of E. coli by 78% (Schneider and Wendisch, 2010).

In order to overproduce putrescine in E. coli, several attempts have been done so far. The ADC pathway is completed by agmatinase SpeB, which hydrolyzes agmatine to putrescine and urea. While urea cannot be reused by E. coli, putrescine can be utilized by E. coli as a sole carbon source. The overexpression of ODC genes speC (b2965) and of speF (b0693) in the wild — type genetic background led to comparable results as 0.72 or 0.87 g/l of putrescine accumulated in batch cul­tures (Eppelmann, 2006). The simultaneous overexpres­sion of speF and speAB, the ADC encoding gene speA as well as speB coding for the agmatinase of E. coli (b2938, b2937) increased putrescine accumulation up to 1.03 g/l (Eppelmann et al., 2006). In order to increase the putres — cine production a base strain was constructed, by inacti­vating the putrescine degradation and utilization pathways, and the engineered E. coli strain was able to produce 1.68 g/l of putrescine with a yield of 0.168 g/

FIGURE 19.4 (a) Engineered putrescine and cadaverine production pathways used in E. coli. GdH, glutamic acid dehydrogenase (EC1.4.1.4);

ArgA, amino acid N-acetyltransferase (EC 2.3.1.1); ArgB, acetylglutamic acid kinase (EC 2.7.2.8); ArgC, N-acetylglutamylphosphate reductase (EC 1.2.1.38); ArgD, acetylornithine aminotransferase (EC 2.6.1.11); ArgE, acetylornithinase (EC 3.5.1.16); ArgF, ornithine carbamoyltransferase (EC 2.1.3.3); ArgG, argininosuccinic acid synthetase (EC 6.3.4.5);ArgH, argininosuccinic acid lyase (EC 4.3.2.1); Pepck, phosphoenolpyruvic acid carboxykinase (EC 4.1.1.32); Ppc, phosphoenolpyruvic acid carboxylase (EC 4.1.1.31); Pyc, pyruvic acid carboxylase (EC 6.4.1.1); AspC, aspartic acid aminotransferase (EC 2.6.1.1); LysC, aspartokinase (EC 2.7.2.4); Asd, aspartic acid semialdehyde dehydrogenase (EC 1.2.1.11); MetL, ThrA bifunctional aspartokinase/ homoserine dehydrogenase (EC 2.7.2.4/1.1.1.3); DapA, dihydrodipicolinic acid synthase (EC 4.2.1.52); DapB, dihydrodipicolinic acid reductase (EC 1.3.1.26); DdH, meso-diaminopimelic acid dehydrogenase (EC 1.4.1.16); LysA, diaminopimelic acid decarboxylase (EC 4.1.1.20); ODC, ornithine decarboxylase (EC 4.1.1.17); ADC, arginine decarboxylase (EC 3.5.3.1).

(b) Engineered putrescine and cadaverine production pathways used in C. glutamicum. GdH, glutamic acid dehydrogenase (EC1.4.1.4); ArgJ, bifunctional ornithine acetyltransferase/N-acetylglutamic acid synthase (EC 2.3.1.35/2.3.1.1); ArgB, acetylglutamic acid kinase (EC 2.7.2.8); ArgC, N- acetylglutamylphosphate reductase (EC 1.2.1.38); ArgD, acetylornithine aminotransferase (EC 2.6.1.11); ArgE, acetylornithinase (EC 3.5.1.16); ArgF, ornithine carbamoyltransferase (EC 2.1.3.3); ArgG, argininosuccinic acid synthetase (EC 6.3.4.5); ArgH, argininosuccinic acid lyase (EC 4.3.2.1); Pepck, phosphoenolpyruvic acid carboxykinase (EC 4.1.1.32); Ppc, phosphoenolpyruvic acid carboxylase (EC 4.1.1.31); Pyc, pyruvic acid carboxylase (EC 6.4.1.1); AspC, aspartic acid aminotransferase (EC 2.6.1.1); LysC, aspartokinase (EC 2.7.2.4); Asd, aspartic acid semialdehyde dehydrogenase (EC 1.2.1.11); MetL, ThrA, bifunctional aspartokinase/homoserine dehydrogenase (EC 2.7.2.4/1.1.1.3); DapA, dihydrodipicolinic acid synthase (EC 4.2.1.52); DapB, dihydrodipicolinic acid reductase (EC 1.3.1.26); DdH, meso-diaminopimelic acid dehydrogenase (EC 1.4.1.16); LysA, dia­minopimelic acid decarboxylase (EC 4.1.1.20); ODC, ornithine decarboxylase (EC 4.1.1.17); ADC, arginine decarboxylase (EC 3.5.3.1).

g glucose. A further optimization by 25% was achieved by promoter exchange of genes encoding the enzymes converting L-glutamic acid into L-ornithine, as well as the exchange of speF—potE promoter (potE encodes the ornithine—putrescine antiporter) (Qian et al., 2009).

In contrast to E. coli, C. glutamicum is unable to degrade and utilize putrescine as a carbon source. The expression of genes of the ADC and ODC pathway from E. coli in the wild-type background of C. glutami — cum only led to minor amounts of putrescine. The dele­tion of argR and argF led to accumulation of L-ornithine but rendered the resulting strain arginine auxotrophic. When speC and speF from E. coli were expressed in the argR—argF deletion strain of C. glutamicum, produc­tion of 5 g/l putrescine resulted, which was about 50 times higher than strains endowed with the ADC pathway. To avoid costly supplementation with L-arginine and the strong feedback inhibition of the key enzyme N-acetylglutamate kinase (ArgB) by L-argi — nine, a plasmid addiction system for low-level argF expression was developed. This strain resulted in pu — trescine yields on glucose from less than 0.001 up to 0.26 g/g, the highest yield in bacteria reported to date and was named as PUT21. In fed-batch cultivation with C. glutamicum PUT21, a putrescine titer of 19 g/l at a volumetric productivity of 0.55 g/l h and a yield
of 0.16 g/g glucose was achieved (Schneider et al.,

2012) . Moreover, while plasmid segregation of the initial strain required antibiotic selection, plasmid segregation in C. glutamicum PUT21 was fully stable for more than 60 generations without antibiotic selection even in the pres­ence of L-arginine.

Cadaverine

Cadaverine can be overproduced by introduction of an overproduced lysine decarboxylase. The correspond­ing substrate, L-lysine, is synthesized in E. coli and C. glutamicum by similar pathways covering 10 enzy­matic steps initiating from the tricarboxylic acid cycle in­termediate oxaloacetate. The three initial steps in this pathway lead to aspartic acid semialdehyde, which is the branch point for biosynthesis of the amino acids, L-methionine, L-threonine, L-isoleucine and L-lysine (Figure 19.4). However, there were substantial differ­ences in the enzyme systems possessed by E. coli and C. glutamicum. When it is LysC from C. glutamicum that is additionally feedback inhibited by L-threonine, it was ThrA from E. coli that is subject to feedback inhibi­tion by L-threonine (Park and Lee, 2010). The tolerance of E. coli for cadaverine seems to be lower compared to pu — trescine. The biomass formed in the presence of 51 g/l

cadaverine was reduced by 30% in comparison to the same molar concentration of putrescine (Qian et al., 2011, 2009). Corynebacterium glutamicum was tested for growth on solid medium and grew even at concentra­tions of up to 31 g/l cadaverine (Mimitsuka et al., 2007).

Escherichia coli strains overexpressing the lysine decarboxylase gene cadA (b4131) in the wild-type ge­netic background led to accumulation of 0.8 g/l cadav — erine by growing cells. To avoid side reactions of enzymes active with putrescine toward cadaverine, a number of genes were deleted: the spermidine synthase gene speE, the spermidine acetyltransferase gene speG, the putrescine importer gene puuP, the putrescine aminotransferase gene puuA and ygjG, which encodes the initial enzyme of the second putrescine degradation pathway and is known to be active in vitro with cadav — erine. The resulting strain was able to accumulate 1.2 g/l cadaverine. Production of cadaverine was increased by 10% as a consequence of enhancing the flux of L-aspartic acid toward L-lysine by overexpression of dapA via pro­moter exchange. In fed-batch cultivation, this strain pro­duced 9.6 g/l cadaverine (Qian et al., 2011).

Cadaverine production in C. glutamicum was also achieved by insertional inactivation of homoserine de­hydrogenase gene, hom (cg1337, Figure 19.4, B-1) with cadA from E. coli. The resultant strain secretes 2.6 g/l cadaverine in the supernatant. The expression of cadA was driven by the strong kanamycin resistance gene promoter. But the strain was auxotrophic for L-methio — nine, L-threonine, and L-isoleucine (Mimitsuka et al., 2007). A different approach with biosynthetic lysine decarboxylase (LdcC) from E. coli led to 30% more cadaverine production than overexpression of cadA (Kind et al., 2010b). Later the C. glutamicum DAP-3c cadaverine-producing strain’s substrate spectrum was broadened for hemicellulose utilization by introducing xylA and xylB genes from E. coli (Buschke et al., 2011). Through various studies reasonable titers and produc­tivities were achieved for putrescine and cadaverine (Table 19.1).

CONCLUSION AND PERSPECTIVES

This chapter outlined the microbial production of amino acids, poly(amino acid)s and polyamines known so far. Biotechnological production of amino acids today serves a market with strong prospects of growth. In the foreground are the fermentation processes, which are now widely established in the production of proteino — genic amino acids; this can be extended to the produc­tion of other amino products like poly(amino acid)s and polyamines. The potential that will be leveraged in the future by modern methods and new findings in system biology will further stimulate and strengthen microbial production of amino products. Modern methods such as directed evolution will allow develop­ment of customized, highly selective, and stable en­zymes and whole cell biocatalysts, as well as efficient and ecologically sustainable production of the required products. It became a need to assess the feasibility of implementing, in addition to the established chemical processes, a biorefinery concept based on renewable raw materials. The poly(amino acid)s production does not have the luxury of background knowledge regarding the metabolic process leading to their synthe­sis when compared to the amino acids and polyamines. Even then, poly(amino acid)s was produced in recently good titers by using newly isolated strains and their genetically manipulated versions. However, the genetic engineering strategies were yet to attain maximal poten­tial in polyamine and poly(amino acid)s producing strains.