17.3 GLUTAMIC ACID AS A BUILDING BLOCK
17.3.1 Production of Chemicals from Glutamic Acid
Among the chemicals that can be produced from glutamic acid, the microbiological production of GABA, PGA, and ALA have been investigated. GABA is originally produced by microorganisms, especially lactic acid bacteria (LAB) in fermented foods. GABA is also known as a neurotransmitter in humans and can be used in pharmaceuticals and foods. Recently, GABA was determined to be a building block for the polymer nylon 4 (polyamide 4). PGA is produced byBacillussp. and increases the tolerance of the microorganism toward environmental changes. PGA can be used both industrially and medically as drug carriers, biological adhesives, water- absorbing polymers and metal-absorbing materials. ALA is a precursor to porphyrin biosynthesis from tRNA-charged glutamic acid (known as C5 pathway) that is used as a pharmaceutical and biodegradable herbicide.
17.3.1.1 Production of 𝛄-Aminobutyric Acid from Glutamic Acid by Microorganisms GABA can be synthesized chemically but alternative produc- tion strategies are necessary to reduce the utilization of fossil resources. GABA is synthesized from glutamic acid by glutamate decarboxylase (GAD) and produced by some LAB in fermented foods. Therefore, purified GAD should be able to con- vert glutamic acid to GABA. The direct conversion of glutamic acid and sugars to GABA by natural and recombinant microorganisms has been performed. The micro- bial production of GABA from glutamic acid and sugars, and polymer production from GABA is reviewed in this section.
Plokhov et al. (2000) reported the production of GABA using recombinantE. coli expressing thegadAgene, which encodes GAD. In this study, recombinantE. coli
γ
γ
FIGURE 17.4 Derivatives of glutamic acid that can be used as possible targets for bio-based productions. From US DOE Report (Werpy and Petersen, 2004).
was used as whole-cell biocatalyst. As a result, heat-treated recombinant E. coli converted glutamic acid to GABA with a productivity of 23–25 g/g of wet cells.
Le Vo et al. (2012) demonstrated that the overexpression ofE. coli gadBandgadC genes encoding GAD and the glutamic acid/GABA antiporter, respectively, in the E. colimutant strain ofgabTencoding GABA aminotransferase yielded GABA from approximately 90% MSG.
GABA is produced by some LAB such asLactobacillus paracasei,Lactobacil- lus brevis,Lactobacillus plantarum,andStreptococcus thermophilus. For example, enhanced GABA production inBifidobacterium longum was achieved by overex- pressing GAD from rice (Park et al., 2005). GABA production by growing and the resting cells ofL. breviswere also reported (Zhang et al., 2012b). The overexpression of thegadBgene fromS. thermophilusin a non-GABA-producingS. thermophilus produced GABA (Somkuti et al., 2012). In addition, the overexpression ofE. coli GAD inL. brevisresulted in enhanced GABA production (Kim et al., 2007). Aside from LAB, Kato et al. (2002) demonstrated that the overexpression of thegadAgene fromAspergillus oryzaeconverted glutamic acid into GABA.
Recently, the utilization of a glutamic-acid-producing microorganismC. glutam- icumwas examined for GABA production. Sugars without the addition of glutamic acid can produce GABA using a glutamic acid-producing microorganism. Shi and Li (2011) reported that the expression of GAD genes fromL. brevisinC. glutamicum produced GABA from glucose without any triggering treatments. In addition, Taka- hashi et al. (2012) reported that the C. glutamicumrecombinant strain expressing E. coli gadBgene could produce GABA from glucose without requiring the addition of Tween 40.
GABA is a building block in the synthesis of nylon 4 (Figure 17.5). Studies on nylon 4 production from biologically produced GABA have been conducted.
To synthesize nylon 4, GABA has to be converted into the nylon 4 precursor, 2- pyrrolidone. Yamano et al. (2012) reported the production of 2-pyrrolidone from biologically produced glutamic acid usingE. coli. The authors prepared a mixture of E. coli, glutamic acid, and water, resulting in the release of 303.7 g GABA into water from 560 g of glutamic acid 14 times every 24 hour. The resulting GABA (without purification) was converted to 2-pyrrolidone by removing the water, heating the mixture to 200–240◦C, and distilling the product under reduced pressure. Moreover, Park et al. (2013) reported the synthesis of nylon 4 from biologically produced GABA using an E. coli recombinant strain. GABA was produced from glutamic acid usingE. coliexpressing theLactococcus lactis gadBgene encoding GAD as a
γ
FIGURE 17.5 Synthesis of nylon 4 from glutamic acid via GABA and 2-pyrrolidone.
whole-cell biocatalyst. The resulting GABA was converted to 2-pyrrolidone in a reaction using Al2O3as a catalyst in organic solvent, and nylon 4 could be synthesized by bulk polymerization from obtained 2-pyrrolidone. The molecular weight of nylon 4 was 200,000–300,000, which varied based on the polymerization conditions used.
The chemical properties of nylon 4 synthesized from 2-pyrrolidone converted from biologically produced GABA were similar to those nylon 4 chemically synthesized from 2-pyrrolidone. The biologically-produced 2-pyrrolidone has the potential to be used as an alternative raw material to fossil-resources-based materials in order to produce nylon 4.
In addition to nylon 4, the microbial production of polyhydroxyalkanoates (PHA) from GABA has also been studied. Valentin et al., reported the introduction of metabolic reactions to supply 4-hydroxybutyryl-CoA to the recombinant E. coli strain expressingRalstonia eutropha phaA,phaB, andphaCgenes, which encode the enzymes that convert 3-hydroxybutyryl-CoA to poly(3-hydroxybutyrate-co-4- hydroxybutyrate) during the conversion of GABA to PHA. To introduce metabolic reactions that produce 4-hydroxybutyryl-CoA, the following four genes were intro- duced: acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri (orfZ), 4-hydroxybutyrate dehydrogenase fromR. eutropha(gbd), glutamate:succinic semialdehyde transaminase fromE. coli(gabT), and GAD fromArabidopsis thaliana or E. coli (gad) (Valentin et al., 2000). Using glutamic acid as a carbon source, the resulting recombinant strain produced PHA, which included 0.7 mol% 4- hydroxybutyrate units. Moreover, PHA could be produced from glutamic acid and 2-oxoglutarate, and the 4-hydroxybutyrate units increased to 1.2 mol%.
17.3.1.2 Production of Poly(𝛄-Glutamic Acid) by Microorganisms PGA is generally obtained by polymerizing d- and/or l-glutamic acids with the linkage betweenα-amino andγ-carboxyl groups of two glutamic acid molecules. The synthe- sis of PGA is ribosome-independent and produced mainly byBacillussp., including B. licheniformis,B. subtilis, and B. anthracis. The molecular weight of PGA pro- duced byBacillussp. is in the range of 105–106. Initially during PGA biosynthesis (Figure 17.6), d- and l-glutamic acids are synthesized. d-Glutamic acid is produced by d-glutamic acid–pyruvic acid aminotransferase and glutamate racemase, and the resulting glutamic acid is polymerized by PGA synthetase encoded bypgsBCAgenes.
To improve PGA production, culture conditions, such as nutrition and aeration, were examined. PGA production byBacillussp. can be categorized into the following two groups: glutamic acid-dependent and glutamic acid-independent PGA produc- tion. For example, PGA production byB. licheniformisATCC 9945A is glutamic acid-dependent (Thorne et al., 1954).13C-nuclear magnetic resonance spectroscopy analysis of other glutamic acid-dependent PGA-producing species revealed that car- bon sources (e.g., glucose) are mainly used for cell growth and energy supply, such as ATP, whereas glutamic acid is used in PGA biosynthesis (Yao et al., 2010). On the other hand, PGA production by some strains ofB. subtilisandB. licheniformis do not require the addition of glutamic acid but is achieved from nitrogen and carbon sources (Ito et al., 1996; Cao et al., 2011). Moreover, the requirement of metal ions
FIGURE 17.6 PGA biosynthetic pathway inBacillussp. GS, glutamine synthase; GOGAT, glutamate:2-oxoglutarate aminotransferase; GDH, glutamate dehydrogenase; l-Glu:Pyr AT, l-glutamate:pyruvate aminotransferase; d-Glu:Pyr AT, d-glutamate:pyruvate aminotrans- ferase; GLR, glutamate racemase; ALR, alanine racemase; PGS, PGA synthetase.
and aeration for efficient PGA production has been investigated (for review, see Shih and Van (2001) and Bajaj and Singhal (2011)).
Metabolic engineering has been used to enhance PGA production by microorgan- isms.B. subtilisDB430 strain does not produce PGA even though it possesses genes related to PGA biosynthesis, such asywsC-ywtAB. An efficient synthetic expres- sion control sequence was introduced within the upstream region of PGA biosyn- thetic genes in the genome of this strain and PGA production was successfully achieved (Yeh et al., 2010). Yamashiro et al. (2011) showed that the contribution of PgsE protein with PGA production inB. subtilis. Furthermore, the induction of PgsE stimulated PGA production in the presence of zinc ions. PGA production by recombinant microorganisms other than Bacillussp. has also been examined. For example, the genes responsible for PGA biosynthesis inB. subtilisIFO 3336 (i.e., pgsBCA) were expressed inE. coli, and PGA production by the recombinant strain was observed (Ashiuchi et al., 1999). In addition, the enhanced expression of the gene-encoding glutamate racemase, which is involved in d-glutamic acid biosynthe- sis, was reported to enhance PGA production (Kada et al., 2004). Similarly, PGA production was also achieved by using a recombinantE. colistrain expressing the pgsBCA gene placed under a strong promoter from another B. subtilisstrain iso- lated from food (Jiang et al., 2006). Recently, PGA was produced by untreated cane molasses and MSG waste liquor byB. subtilisNX-2 (Zhang et al., 2012a). Metabolic flux analysis of PGA-producingB. subtilishas been used to develop culture conditions for improving PGA production (Wu et al., 2008; Zhang et al., 2011).
17.3.1.3 Microbiological Production of 5-Aminolevulinic Acid from Glu- tamic Acid Currently, ALA production has been achieved using photosynthetic bacteria and algae. There are two known biosynthetic pathways for ALA produc- tion in various organisms (Sasaki et al., 2002) (Figure 17.7). In some bacteria and
FIGURE17.7Biosynthesispathwaysofaminolevulinicacid(ALA).
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eukaryotes, ALA is synthesized from glycine and succinyl-CoA by ALA synthase via the C4 pathway. In most bacteria, plastids in plants and algae, ALA is synthe- sized from glutamic acid via glutamyl-tRNA and glutamate-1-semialdehyde via the C5 pathway. Recently, ALA production by recombinantE. colistrains was reported.
Kang et al. (2011) found that the rate-limiting step in ALA biosynthesis is the reaction of glutamyl-tRNA reductase, which converts glutamyl-tRNA to glutamate- 1-semialdehyde and is encoded by thehemAgene. Therefore, the overexpression of hemAwas performed and ALA production slightly increased. Because the stability of the HemA protein is known to be low under conditions with accumulated heme, the mutatedhemAgene ofSalmonella arizonawas introduced into theE. colistrain to increase the stability of mutated HemA protein and a significant increase in ALA production was observed. In addition, the overexpression of theE. coli rhtA gene encoding the threonine/homoserine exporter further enhanced the production of ALA.
Finally, approximately 4 g/L of ALA was produced from glucose without the addition of cofactors. The enhancement of glutamic acid production in this ALA-producing E. colistrain may improve ALA production.