Microbial Production of Amino Acids and its Processes | Industrial Microbiology

In this article we will discuss about the production of various amino acids by microorganisms. Learn about:- 1. Microbial Production of Amino Acids 2. Amino Acid Production by Microorganisms 3. Amino Acid Production Processes 4. Production of Essential Amino Acids 5. Production of Amino Acids by Fermentation 6. Amino Acid Production.

L-Tryptophan and Other Aromatic Amino Acids:

L-Tryptophan:

Microbial production of L-tryptophan using precursors has been reviewed by Terui (1972). With Hansenula anomala, L-tryptophan production from anthranilic acid reached 5.7 g per liter. By feeding anthranilic acid to a derepressed anthranilic acid auxotroph of Bacillus subtilis, 5.5 g of L- tryptophan per liter was produced from 5 g of anthranilic acid per liter. Candida utilis (synonym Torulopsis utilis) 295-t pro­duced 6.4 g of L-tryptophan per liter from 4.2 g of anthranilic acid per liter in 36 hr.

By feeding indole, a 5-methyltryptophan-resistant mutant of B. subtilis ATCC 21336 produced 10.4 g of L-tryptophan per liter in 96 hr with a medium containing 7% glucose. A strain carrying an F Try-episome in addition to the chromosomal try operon was obtained by sexduction from a feedback-resistant and derepressed mutant of E. coli K12 which is resistant to 5-methyltryptophan (5-MT) and 5-fluorotryptophan (5-FT). This strain produced 5 g of L- tryptophan per liter by feeding indole (1.5 g per liter) and L-serine (7 g per liter).

Tryptophanase, which catalyzes synthesis of L-tryptophan by reversal of the α, β-elimination reaction at rates similar to the forward reaction, was utilized for production of l-tryptophan and related compounds such as 5-hydroxytryptophan.

The culture broth of Proteus rettgeri (AJ 2770), was used as the enzyme for the reaction. For the synthesis of L-tryptophan, a reaction mixture contained 6.0 g of indole in 10 ml of methanol, 8.0 g of sodium pyruvate, 8.0 g of ammonium acetate, 0.001 g of pyridoxal phosphate, 0.1 g of Na₂S0₄, and 100 ml of the cultured broth in a total volume of 120 ml.

After the pH value of the mixture was adjusted to 8.8 with 6 N KOH, it was incubated at 34°C for 48 hr. Under these conditions, 7.5 g of L-tryptophan were synthesized. Similarly, 5-hydroxy-L-tryptophan was synthesized from 5-hydroxyindole, pyruvate, and ammonia.

The preceding methods using precursors are pot advantageous since the precursor compounds are expensive at present. Recently, direct production of L-tryptophan from carbohydrate by fermentation has been developed to the practical level.

Figure 17.12 shows the genealogy of L-tryptophan-producing mutants of Corynebacterium glutamicum. Mutants producing a large amount of L-tryptophan were derived from a phenylalanine and tyrosine double auxotroph of C. glutamicum KY 9456 which produced only a trace amount of L-tryptophan and anthranilate.

A mutant (4MT- 11), which stepwise acquired resistance to 5-MT, tryptophan hydroxamate (TrpHx), 6-fluorotryptophan (6-FTF), and 4-methyltryptophan (4-MT), pro­duced L-tryptophan to a concentration of 4.9 g per liter in a cane molasses medium containing 10% reducing sugar as invert. L-Tryptophan production with this mutant was inhibited by L-phenylalanine and L-tyrosine.

Accord­ingly, mutants resistant to phenylalanine and tyrosine analogs such as p-fluorophenylalanine (PFP), p-aminophenylalanine (PAP), tyrosine hydroxamate (TyrHx), and phenylalanine hydroxamate (PheHx), were de­rived from this mutant. One of the mutants thus obtained (Px-115-97) produced 12 g of L-tryptophan per liter in the molasses medium.

The me­dium used had the following composition- 10% reducing sugars as invert (as cane molasses), 0.05% KH₂PO₄, 0.05% K₂HPO₄, 0.025% MgSO₄.7H₂O, 2% (NH₄)₂SO₄, 1% corn-steep liquor, 2% CaCO₃ (pH 7.2). Production of L- tryptophan with the mutant was still sensitive to L-phenylalanine and L-tyrosine. Hence, further genetic improvement of the strain may be pos­sible.

A 5-FT-resistant mutant derived from B. subtilis produced 4 g of L-typtophan and L-phenylalanine per liter. A leucine auxotroph derived from the mutant produced 6.15 g of L-tryptophan per liter a medium containing 300 μg of L-leucine per ml. Suppressor prototrophic revertants from No. 149 rec histidine auxotroph of B. subtilis produced L-tryptophan in the medium. Elimination of rec mutation from the gene using genetic transformation resulted in an increase in tryptophan productivity by 60%. This strain produced 5-6 g of L-tryptophan per liter.

A 5-FT-resistant histidine auxotrophic mutant of Brevibacterium flavum produced 2.4 g of L-tryptophan per liter. Production was increased to 3.8 g per liter using an m-fluorophenylalanine (MFP) resistant mutant derived from the 5-MT resistant one. A phenylalanine auxotrophic mutant from the latter mutant produced 6.2 g of L-tryptophan.

Flavobacterium aminogenes nov. sp. has an intracellular enzyme system which degrades aromatic amino acid hydantoins into corresponding L- amino acids. Molar conversion of dl-tryptophan hydantoin into l-trypto­phan was 100% when 5% each of DL-tryptophan hydantoin and inosine were treated at 40°C for 100 hr with cells of a mutant of F. aminogenes in which the activity to break down tryptophan had been reduced. In this system inosine removes L-tryptophan from the reaction system by forming a complex with it. Spontaneous racemization of the sub­strate allows the conversion of the D-form hydantoin into L-tryptophan.

L-phenylalanine:

L-Phenylalanine production has been much improved using a regulatory mutant after the review of Oishi (1972). A prototrophic mutant resistant to p-fluorophenylalanine produced 5.5 g of L-phenylalanine per liter and trace amounts of L-tyrosine in a medium containing 10% reducing sugars as invert (as cane molasses).

A tyrosine auxo­trophic mutant, resistant to PFP and PAP, produced 9.5 g of L-phenylala­nine per liter in the molasses-containing medium. The medium used had the following composition – 10% reducing sugars as invert (as cane molas­ses), 2% (NH₄)₂SO₄, 0.05% KH₂PO₄, 0.05% K₂HPO₄, 0.025% MgSO₄.7H₂O, 0.25% NZ-amine (enzymic digest of casein), and 2% CaCO₃ (pH 7.2). L- Phenylalanine production in these mutants was inhibited by L-tyrosine and was stimulated by L-tryptophan.

A mutant of B. subtilis resistant to 5-FT produced 6.0 g of L-phenylalanine per liter in addition to 4.0 g of L-tryptophan per liter. Maximum L-phenylalanine production by a mutant of Brevibac­terium lactofermentum was attained at an oxygen deficiency degree be­tween 0.45 and 0.65.

This oxygen deficiency degree was attained at the redox potential of the medium (Eh, mv) between -200 mv and -275 mv. A tyrosine auxotroph of a Corynebacterium species, an, n-paraffin-utilizing glutamic acid producer, has also been reported to pro­duce L-phenylalanine.

L-Tyrosine and L-DOPA:

The tyrosine productivity of a phenylalanine auxotroph was improved by endowing a certain analog resistance. Figure 17.13 shows the genealogy of the finally selected L-tyrosine producers. A combination of auxotrophy and multiple resistances to analogs of aromatic amino acids were necessary to yield a large amount of L-tyrosine.

A phenyl­alanine auxotroph (91-1-x-71), which became multiply resistant to the analogs of phenylalanine and tyrosine [PFP, PAP, 3-aminotyrosine (3-AT), and tyrosine hydroxamate (TyrHx)], produced 13.5 g of L-tyrosine per liter in a cane molasses medium containing 10% reducing sugars as invert. This strain is a so-called leaky mutant. It has some L-phenylalanine synthesiz­ing activity similar to the original strain; in fact, it sometimes excreted trace amounts of L-phenylalanine.

The L-phenylalanine pool of this mutant may reach a value high enough to inhibit synthesis of L-tyrosine due to deviation of its regulation for L-phenylalanine synthesis, in addition to its deviation of the regulation of L-tyrosine synthesis. Therefore, mutants which are definitely defective in L-phenylalanine synthesis were expected to produce higher amounts of L-tyrosine. Such a mutant may be selected as an L-tyrosine-sensitive strain.

It grows slowly in the minimal medium supplemented with excess L-tyrosine because L-tyrosine antagonizes the entrance of L-phenylalanine into the cells and inhibits growth of a phenyl­alanine auxotroph in proportion to the degree of its requirement for L-phenylalanine. Colonies grown slowly in the presence of tyrosine were selected as mutants sensitive to L-tyrosine.

Some mutants thus obtained produced larger amounts of L-tyrosine than the parent strain, notably strains Pr-20 and Pr-102, which produced L-tyrosine at a concentration of 17.6 g and 17.3 g per liter, respectively. The medium had the following composition – 10% reducing sugars as invert (as cane molasses), 2% (NH₄)₂SO₄, 0.05% K₂HPO₄, 0.05% KH₂PO₄, 0.025% MgSO₄.7H₂O, and 2% CaCO₃ (pH 7.2).

An increase in L-tyrosine production was also noted in many auxotrophic mutants derived from a phenylalanine auxotroph of C. glutamicum. Among them, LM-96, a phenylalanine and purine double auxotrophic strain, produced L-tyrosine at a concentration of 15.1 g per liter in a medium containing 20% sucrose.

A reversion of the α, β-elimination reaction catalyzed by β-tyrosinase was utilized for preparation of L-tyrosine and L-DOPA.

Cells of Erwinia herbicola prepared by growing at 28°C for 28 hr in an appropriate medium were used as a source of enzyme. A reaction mixture (100 ml) containing 0.5 g sodium pyruvate, 1.0 g phenol or 0.8 g pyrocate­chol, 5 g ammonium acetate, and cells were incubated. At intervals, sodium pyruvate and phenol or pyrocatechol were added. Under these conditions, 6.05 g of L-tyrosine or 5.85 g L-DOPA were synthesized.

A 3-AT-resistant mutant of Pseudomonas maltophila (synonym P. melanogenum) produced 14-15 g of L-DOPA per liter from 26 g of L-tyrosine per liter (68% molar conversion ratio). The high L-DOPA productivity of the improved mutants was found to be due to the increased tyrosinase activity of the mutants.

Mechanism of Overproduction of Aromatic Amino Acids:

Regulatory properties of the enzyme involved in aromatic amino acid biosynthesis in C. glutamicum wild and mutant strains were investigated. The overall control pattern (Fig. 17.14) is a new addition to the list of control patterns in aromatic amino acid biosynthesis in microorganisms.

A phenylalanine and tyrosine double auxotrophic L- tryptophan producer, Px-115-97, has anthranilate synthetase partially re­leased from the inhibition by L-tryptophan and DAHP synthetase of a wild type, L-Tryptophan production by the mutant appeared to be caused by the release from the feedback inhibition of anthranilate synthetase by L- tryptophan and blockage of chorismate mutase.

Deregulation for L-tyrosine overproduction can be understood by Fig. 17.15 which shows the control of aromatic amino acid biosynthesis in C. glutamicum mutant Pr-20.

L-Arginine, L-Ornithine and L-Citrulline:

L-Arginine:

Efficient production of L-arginine was obtained by using regulatory mu­tants of Bacillus subtilis, Corynebacterium glutamicum, Brevibacterium flavum, and Serratia marcescens. An L-arginine hydroxamate-resistant mutant of B. subtilis produced 4.5 g of L-arginine per liter in shaken culture. This mutant formed N^δ-acetylornithine as a by-product and its ornithine carbamoyltransferase was strongly derepressed.

Therefore, a shortage of carbamoyltransferase was thought to be the cause of N-acetylornithine formation. A 6-azauracil-resistant mutant derived from the arginine and hydroxamate-resistant mutant produced 28 g of L-arginine per liter without forming N^δ-acetylornithine in a medium containing 8% glucose and 3.5% glutamic acid.

Corynebacterium glutamicum DSS8, isolated as a D-serine-sensitive mu­tant from an isoleucine auxotroph KY 10150, was found to be sensitive to D-arginine and arginine hydroxamate. Furthermore, strain DSS8 produced L-arginine in a culture medium.

Most of the L-arginine analog-resistant mutants derived from DSS8 produced large amounts of L-arginine. An isoleucine revertant from one of these mutants produced 19.6 g of L-arginine per liter in a medium containing 15% reducing sugars as invert (as cane molasses). Strain DSS8 seems to be a mutant with increased permeability to D- and L-arginine.

An efficient l-arginine producer was obtained through several mutation and selection steps from strain No. 33038, a guanine auxotroph of B. flavum (Fig. 17.16). Strain No. 352, a final, isolated mutant, produced 25.3 g of L-arginine per liter. Maximum L-arginine production of 28.4 g per liter was found at a concentration of 0.1 mg of guanine per ml which was a suboptimal concentration for growth of the mutant.

Increasing the concentration of (NH₄)₂SO4 in the medium increased L-arginine production. Maximum pro­duction of 29.4 g per liter was attained with 7% (NH₄)₂SO₄. L-Histidine markedly retarded growth of strain No. 352 and also that of the parent strain ATCC 14067. The time course of l-arginine production is presented in Fig. 17.17.

Arginine-producing strains of Serratia marcescens were obtained by a rather complex process owing to the insensitivity of the bacterium to argi­nine analogs. The process is shown in Fig. 17.18. First, a mutant (ArD^–) which does not utilize arginine as a sole nitrogen source was isolated from the wild strain by mutagenic treatment.

A lysine auxo­troph, PA 2028 derived from the mutant (ArD^–) can grow on acetyllysine owing to the substrate specificity of acetylornithinase which allows degra­dation of acetyllysine to lysine. But it (ArD^– lysA^–) cannot grow on acetyllysine in the presence of arginine because arginine represses the formation of acetylornithinase. By mutagenic treatment of PA 2028, a mutant strain which is able to grow in the presence of both acetyllysine and arginine is obtained. This mutant, PA 3179 (ArD^– lysA argR), produced 3.2 g of L-arginine per liter of the medium.

On the other hand, a revertant of proA/B was found to be sensitive to arginine, and inhibition by arginine was reversed by proline. This phenom­enon is due to indirect suppression. N-Acetylglutamate-γ-semialdehyde accumulated due to argD mutation and was transformed to glutamate-γ- semialdehyde by acetylornithinase and finally to proline (Fig. 17.19). Thus it grows without proline but is sensitive to arginine because arginine represses acetylornithinase.

A mutant resistant to arginine derived from the mutant (proA/B, argD) excreted proline because of derepression of the arginine pathway including acetylornithinase. But proline excretion was inhibited by arginine because of the inhibition of N-acetylglutamate synthetase by arginine.

A regulatory mutant in which N-acetylglutamate synthetase became desensitized to the inhibition by arginine was isolated as one which grows in the presence of arginine and 3,4-dehydro-DL-proline. The latter compound competes with proline and inhibits the growth of wild type strains and proA/B, argD, argR. The desensitized mutant (RA 4240) can grow by overcoming the inhibitory action of 3, 4-dehydro-DL-proline by overproduction of proline.

Finally, AT404 (ArD^–, argR, arg A) was obtained by cotransduction of argA with lysA⁺ form RA 4240 into PA 3179 (ArD^–, lysA, argR). AT404 produced 25.2 g of L-arginine per liter in a medium containing 10% glucose.

L-Ornithine:

L-Ornithine production from carbohydrate is now known among arginine or citrulline auxotrophs of many microorganisms such as species of Coryne- bacterium, Brevibacterium, Arthrobacter, Bacillus, and Escherichia and of Streptomyces. L-Ornithine production from hydrocarbons by the same type of auxotroph of C. hydrocarboclastus and Arthrobacter paraffineus is also known.

Among these, a C. glutamicum mutant is the first reported efficient L-ornithine producer. The molar yield was as high as 36%. The fermentation conditions are similar to those for glutamic acid production except that the medium contained an appropriate concentration of arginine and a large concentration of biotin.

L-Citrulline:

Arginine auxotrophs of C.glutamicum and B.subtilis produced 10.7 g and 16.5 g of L-citrulline per liter in a medium containing 10 and 13% glucose, respectively. An arginine auxotroph of Corynebacterium sp. produced 8 g of L-citrulline per liter in a medium containing 10% (v/v) n-paraffin.

Production of L-citrulline by an arginine auxotroph resistant to arginine hydroxamate was not influenced by the concentration of arginine in the medium. L-Citrulline production reached 26 g per liter using an arginine auxotroph derived from a mutant resistant to both arginine hydroxamate and 6-azauracil.

Mechanism of Overproduction of Arginine, Ornithine, and Citrulline:

Arginine is synthesized from glutamic acid via ornithine and citrulline in microorganisms as shown in Fig. 17.20. Eight different enzymes participate in the chain of reactions. The first and fifth step reactions are different depending on the microorganism. In the bacteria of the Enterobacteriaceae and species of Bacillus, ornithine is formed by hydrolytic cleavage of acetyl- ornithine with an acylase. In this group of microorganisms, end product inhibition acts on the first enzyme in the pathway.

On the other hand, in species of Pseudomonas, Corynebacterium, Streptomyces, and in yeast, transacetylase catalyzes the transfer of an acetyl residue from acetyl-ornithine to glutamic acid forming ornithine and acetylglutamic acid. In this group of microorganisms, arginine regulates the activity of the second enzyme, and probably the first enzyme as well.

If the second enzyme is not regulated, arginine synthesis proceeds without control because acetyl-glutamic acid will be formed by cyclic utilization of the acetyl residue regardless of the activity of the first enzyme. The end product (arginine) has the role of corepressor in some bacteria, i.e., enzyme formation is repressed only in the presence of arginine.

Other bacteria also have a repression mechanism since a much larger amount of enzyme is synthesized after a single mutation. Overproduction of ornithine becomes possible because of an increase in the amount of enzyme and the weak or nonexistent inhibition of enzyme activity by arginine when arginine is supplied in limited concen­trations to cultures of an arginine auxotroph.

A similar mechanism also explains citrulline production by arginine auxotrophs. In Brevibacterium flavum No. 352, N-acetylglutamokinase activity is 9 times greater than with the wild strain. It is not repressed by addition of 1% arginine, which represses the enzyme in the wild strain.

L- Histidine:

L-Histidine Production:

L-Histidine is an essential amino acid for some animals though not for humans.

Before the establishment of L-histidine production by a “direct fermenta­tion” method, histidinol production by a histidine auxotrophic mutant and its conversion to L-histidine had been studied.

A 1,2,4-triazole-3-alanine (TRA)-resistant mutant (KY 10260) and a L- thiazolealanine (TA)-resistant mutant derived from C. glutamicum ATCC 13761, a wild type strain, produced several grams of L-histidine per liter in a cane molasses medium. The histidine productivity of the TRA- resistant mutant could be improved stepwise by successively endowing resistance to 6-mercaptoguanine, 8-azaguanine, 4-thiouracil, and 6-mercaptopurine, and increased resistance to triazolealanine and 5-methyl- tryptophan resistance.

Improvement of L-histidine productivity in each step was rather minor but, as a total, the finally selected mutant strain, KY 10522, produced about twice the amount of L-histidine as the original L-histidine producer. Among the steps, insertion of 4-thiouracil resistance resulted in a most significant increase in L-histi­dine productivity. The rationale of the improvement was an increased supply of 5-phosphoribosyl pyrophosphate and adenine nucleotide for L-his­tidine biosynthesis by releasing the supposed feedback regulation of their biosynthesis.

This is based on the speculation that the regulatory mechan­ism of L-histidine biosynthesis and related biosynthesis known in some other microorganisms is applicable to this bacterium. An improvement caused by increased resistance to TRA could be explained by a further release of end product regulation of the histidine pathway. This could be the result of an additional mutation.

Strain KY 10522 produced 15 g of L-histidine per liter equivalent to 10% (w/w) of the initial sugar.

The medium used had the following composition:

6% reducing sugars as invert (as cane molasses), 9% sucrose, 4% (NH₄)₂SO₄, 0.2% KH₂PO₄, 0.1% K₂HPO₄, 0.05% MgSO₄.7H₂O, 0.2% urea, 0.75% meat extract, 1 mg thiamin-HCl per liter, 80 μg biotin per liter, and 3% CaCO₃. Among the auxotrophic derivatives of strain KY10260, a leucine auxotroph produced L-histidine at a concentration of 11 g per liter equivalent to 5.8% (w/w) of the initial sugar.

L-Histidine-producing mutants were also isolated from Brevibacterium flavum. A TA-resistant mutant of B. flavum (No. 2247) produced 4 g of L-histidine per liter. Production was improved by adding the following markers successively; sulfa drug-resis­tance; resistance to α-amino-β-hydroxyvaleric acid, a threonine analog; and to 2-aminobenzothiazole.

An L-histidine-producing mutant was also found among sulfisoxazole-resistant mutants of B. flavum No. 2247. The produc­tivity was improved by successive insertion of the following markers- resis­tance to sulfadiazine; S-(β-aminoethyl)-L-cysteine, a lysine-analog; and ethionine. The mutant thus derived produced 9.7 g of L-histidine per liter.

An isoleucine-valine double auxotroph of Proteus rettgeri produced 3.8 g of L-histidine per liter in a medium containing 10% glucose. A deamination product of L-histidine, urocanic acid, has a “sun-screening” effect and is used in cosmetics. Hence an efficient enzymic process for urocanic acid production from L-histidine has been developed.

A 1,2,4-triazole-3-alanine-resistant mutant derived from histidase-deficient Serratia marcescens produced 13 g of L-histidine per liter. A strain producing urocanic acid was obtained by transducing the triazolealanine-resistance from the mutant into a urocanase-deficient mu­tant.

An urocanase-deficient mutant, KY-10550, of Brevibacterium ammoniagenes produced 7.2 g of urocanic acid per liter from 10 g of L-histidine per liter. A mutant resistant to 2-thiazole-DL-alanine derived from KY 10550 produced urocanic acid in a medium containing no histidine.

The productivity was improved by a sequence of mutations which endowed resistance to 2-fluoroadenine, 6-mercaptopurine, 8-azaguanine, and streptomycin. The mutant thus derived produced 7.2 g of urocanic acid per liter in a medium containing 15% glucose.

Mechanism of L-Histidine Production:

Genetic and enzymic studies on Salmonella typhimurium have provided much information on the biosynthetic pathway of histidine and control mechanisms of the pathway. The first step on the pathway is a condensation of 5-phosphoribosyI-1-pyrophosphate (PRPP) and ATP to form N-1-(5′- phosphoribosyl) adenosine triphosphate (phosphoribosyl-ATP) and pyro­phosphate.

The reaction is catalyzed by phosphoribosyl-ATP pyrophosphorylase and is subject to feedback inhibition by L-histidine. Certain mutants of S. typhimurium, resistant to TA (2-thiazolealanine) were found to have a pyrophosphorylase resistant to feedback inhibition.

This property offered an explanation for L-histidine excretion by TA-resistant mutants of this bacterium and E. coli. The histidine pathway is also under feedback repression control. Mutants of S. typhimurium resistant to TRA (1,2,4- triazole-3-alanine) have been found to be relieved of the repression control.

Phosphoribosyl-ATP pyrophosphorylases in 2 L-histidine producers of C. glutamicum, each selected as a TA-resistant and a TRA-resistant strain, were found to be 100-fold more resistant to L-histidine inhibition than the wild type enzyme.

It was also resistant to inhibition by TA, but was still as sensitive as the wild type enzyme to inhibition by α-methylhistidine. Formation of the pyrophosphorylase in these mutants was not significantly derepressed. However, 2-fold dere­pression was noted with a further improved L-histidine producer, namely, strain KY 10522.

Phosphoribosyl-ATP pyrophosphorylase of strain KY 10522 was found to be resistant to feedback inhibition like its parent strain. Thus, loss of feedback inhibition and derepression of phosphoribosyl-ATP pyrophosphorylase synthesis offer an explanation for L-histidine produc­tion in the above described mutants of C. glutamicum.