Some amino acids have been produced in Japan by bacterial fermentation since 1950. Their biosynthesis is now possible by genetically manipulating and physiologically altering the microorganism.
Corynebacterium glutamicum and Brevibacterium flavum are involved in the synthesis of L-lysine and L-threonine from a common intermediate, aspartic acid. The regulatory mutants of bacteria produce lysine in sufficient amount. Such bacteria eliminate the dependence of enzyme formation on inducer addition (Fig. 20.22).
The second aspartokinase is repressed by 1 -methionine as well as by 1-threonine and isoleucine in addition to repression by 1-lysine. The dihydrodipicolinate synthase is also inhibited by end product 1-lysine.
1. L-Lysine Production:
Generally, Escherichia coli and Enterobacter aero-genes are used for the formation of diaminopalmelic acid and for the decarboxylation of the diaminopalmelic acid by an enzyme DAP decarboxylase (E. aero-genes origin) respectively. E. aero-genes is an auxotroph requires L- homoserine or a mixture of L-threonine and L-methionine.
The lysine-histidine, double auxotrophic mutant of E.coli (ATCC 13002) produces diaminopalmelic acid on a molasses medium with a yield of 19-24 g/litre. The entire fermentation solution including the cell material is subsequently incubated with E. aero-genes (ATCC 12409) at 35°C. After 20h, the DAP is quantitatively decarboxylated to L-lysine (C6H14N2O2).
For industrial production of lysine, the seed culture is to be prepared by using glucose (20g) peptone (10g), meat extract (5g), sod. chloride (2.5g) in 1 litre of tap water. Culture obtained is re-inoculated for second seed culture in media containing sugarcane molasses (200g), soyprotein hydrolysate (18g) mixed in tap water (1 litre), inoculated by Brevibacterium flavum, double auxotrophic mutant.
The acetate is used as C source. After preparation of first, second and main culture, the production medium is used. The fermentation media consist of glycerol, com steep liquor, ammonium sulphate. In addition, calcium carbonate is employed in the production medium. The pH is left neutral and incubation is carried out for 72h at 28°C with high aeration.
The yield of lysine is as high as 75g/litre Lysine, earlier was produced by two stage processes using two different organisms. But now a days single stage process using mutants of Conynebacterium, Brevibacterium etc. are grown on a synthetic medium containing glucose, an inorganic nitrogen source and a small concentration of either homoserine, methionine, etc. in addition to a small concentration of biotin.
Although, lysine is bound in the cell but due to mutations in the producing strains, it is secreted out and recovered.
(iii) Strain Improvement for Lysine Production:
The biosynthesis of lysine is depicted in Fig. 20.22. Certain analogs of end products act as false feedback effectors and inhibit microbial growth. To resist the feedback inhibition of aspartate kinase by lysine and threonine, mutants resistant to 5- (2- amino-ethyl)-L- cystine (AEC) a lysine analog, been reported in Brevibacterium flavum. In such cases, lysine production may be increased to the extent of 57mg/ ml.
The strain does not accumulate threonine in the medium because normal feedback inhibition of homoserine dehydrogenase still operates. Some other amino acids, arginine, histidine, proline, valine, leucine, citrulline etc. are also improved by isolating analog-resistant mutants (regulatory mutants).
2. L-Glutamic Acid:
Kinosita et al. (1957) observed that L-glutamic acid (C5H9O4N) is produced by using bacterial isolate, Micrococcus glutamicus (syn: Corynebacterium glutamicum). Although, this organism is not an auxotroph but requires biotin in the medium for growth.
This amino acid is present both intracellularly as well as leaked out in the medium subjected to optimum biotin level available for fermentative production of L-glutamic acid. If excess of biotin is present in the medium, there is heavy cell growth, but lactic acid production starts.
Some microorganisms such as Corynebacterium herculis, C. lilium, Arthrobacter globiformis, Micro bacterium salicinovorum, Brevibacterium divaricatum, B. amino-genes, B.flavum, Bacillus megaterium are other glutamic acid producing species.
Modifications of the permeability of a microorganism is provided by the glutamic acid fermentation. The permeability of Corynebacterium glutamicum may be controlled by the composition of the culture medium if supplemented with biotin (Fig. 20.23).
The medium contains glucose (121g), ammonium acetate (5g), molasses (6g), potassium hydrogen phosphate (1.2 g), potassium sulphate (6 µg), manganese sulphate (6 µg), and antifoam agent (0.1 ml) in one litre distilled water. The size of the inoculum remain 6 percent. The fermentative organism is Brevibacterium divaricatum (NRRL B-231).
The incubation was carried out for 16 hours at 35°C. At the beginning of the fermentations 0.65 ml per litre of olive oil is added. The pH is set at 8.5 with ammonia and is automatically maintained at 7.8 during fermentation. After growth of the culture (about 14 h), the temperature is increased from 32-33°C to 38°C. The glucose feeding is done until the fermentation is completed.
The glutamic acid content is analysed hourly. The fermentation is stopped after 30-35 hours with a yield of 100 g per litre. If molasses from starch saccharification is substituted for glucose, the glutamic acid yield is 94 g per litre after 36 hours.
(iii) Strain Improvement for Glutamic Acid Production:
Sometimes elimination of the permeability barrier in the membrane proved effective or improved. A natural biotin auxotroph that has altered membrane permeability found suitable for the hyper-production of glutamic acid.
The oleic acid auxotroph of Br. thiogenitalis showed good amount of glutamic acid production. Oleic acid auxotrophy is not applicable for production of glutamic acid from n-paraffin because the biosynthetic pathway of fatty acids from n-paraffin is different from that of glucose.
Mutants with altered permeability in the membrane to glutamic acid led to the success in obtaining a glycerol auxotroph from Corynebacterium alkanolyticum. Protease production by kabacidin resistant strains of Fusarium sp is another example of participation of permeability barrier in production.
Sometimes conditional mutants that behave as lethals under one set of conditions termed restricted and as wild type under, permissive conditions help in improvement of the primary metabolites as in case of Br. lactofermentun.
It is used as a flavor enhancer in the form of monosodium glutamate. It also helps in meat tenderization.