Application of Glyceric Acid to Bio-related Functional Materials and Improvement of Microbial Production

: Glyceric acid (GA) is an oxidative product of glycerol, and its d -isomer is obtained as a phytochemical from tobacco leaves and fruits of some plants. However, the production and applications of GA have not yet been fully investigated. In this review, recent developments in the microbial production of GA and its application to bio-related materials are summarized. The sodium salt of diacylated GA showed superior surface tension-lowering activity and antitrypsin activity. GA and its glucosyl derivative had positive effects on the viability and collagen production of skin cells in vitro , respectively. Glucosyl derivatives of GA showed protective effects against heat-induced protein aggregation. In addition, the microbial production of GA using raw glycerol as the starting material was investigated. The effect of methanol, a major impurity in raw glycerol, on GA production was investigated, and mutant strains to tolerate methanol in the culture were constructed. Enantioselective production of GA using newly isolated microbial strains has also been developed.

cohols, organic acids, and polymers. However, the rapid increase in the production of biodiesel fuels resulted in a surplus of impure glycerol, called raw glycerol. Raw glycerol contains alkali metals and methanol as impurities Table  1 1 5 , making it difficult to use as a starting material for chemical production.
Glyceric acid GA , i.e., 2,3-dihydroxypropanoic acid, is a simple but attractive derivative of glycerol because of its structure with two kinds of functional groups and chiral isomers Fig. 1 . GA was originally found as a constituent of tobacco leaves in 1956, and its structure was reported as a D-R isomer 6 . Subsequent studies using D-GA showed some biological activities, such as liver stimulation 7 . In addition, both enantiomers of GA are recognized as markers of symptoms of D-glyceric academia/acidurias or hyperoxaluria type 2 8 . Although some applications have been investigated, there have been few industrial and commercial applications of GA. Thus, to expand the use of GA, more attractive functions of GA and its derivatives are necessary. GA can be obtained by the oxidation of glycerol via chemical and biological processes. Chemical oxidation by metal catalysts such as Au and Pt has been widely investigated 9 . However, the products from chemical oxidation usually contain GA as well as other oxidative products, such as tartronic acid and mesooxalic acid. In addition, many metal-catalyzing reactions produce a racemic mixture of GA. In contrast, the biological process to generate GA was developed by Habe et al. It was demonstrated that some acetic acid bacteria can convert glycerol to GA efficiently 10,11 . With the selection of species and strains of acetic acid bacteria, the enantioselectivity of GA was varied. However, in both methods of GA production, it was revealed that raw glycerol from biodiesel production could not be used because impurities in raw glycerol inhibited the oxidative reaction of glycerol 12 . The refining process of raw glycerol is expensive, resulting in waste or burning of raw glycerol. Thus, the use of raw glycerol without a refining process for GA production needs to be developed.
In this review, recent research progress on the application of GA, including derivatization and functional analysis of GA and its derivatives, are summarized. Improvement in microbial production of GA by bioengineering of GA-producing and GA-assimilating bacteria was also reviewed.

Application of GA and GA-derivatives to Advanced
Bio-related Functional Materials 2.1 Chemical and biological properties of GA GA has one carboxyl group and two hydroxy groups with a chiral carbon atom at the C-2 position Fig. 1 . It dissolves well in polar solvents such as water, short-chain alcohols, and acetone, but not in other organic solvents, such as diethyl ether, n-hexane, and chloroform. In contrast, the calcium salts of GA with high purity have been commercialized for research. However, it is difficult to dissolve the calcium salts in conventional organic solvents. Therefore, its use as a starting material in organic synthesis is limited.
Some biological effects of D-GA on ethanol metabolism have been reported. Erikson et al. reported that ethanol metabolism in rat liver was accelerated by D-GA calcium salt 13 . Habe et al. investigated the effect of GA calcium salts on gastric cells in vitro and found that the viability of ethanol-treated cells increased 14 . These properties are favorable for the use of GA as a supplement for alcohol treatment. Additionally, effects of GA on other human cells in vitro were investigated 15 . It was revealed that the viability of normal human dermal fibroblasts NHDF was enhanced by 45 in the presence of 0.78 mM GA sodium salt. Another aspect of GA is the protection of biological macromolecules. In the same report, radical scission of DNA was protected in the presence of 200 mM GA sodium salt. Protection against heat aggregation of egg white was also observed in the presence of 1 wt GA sodium salt. These features will be useful for the application of GA salts in skincare, cosmetic, and other personal care products.

Synthesis and properties of acyl GAs
GA has two hydroxy groups; thus, their acylated compounds, namely diacyl GA or monoacyl GA, are expected to have amphiphilic properties. In addition, metabolites from Penicillium funiculosum, OR-1, contained mixtures of diacyl GAs with acyl chain lengths of C14 to C18 Fig.  2A , which showed a strong inhibitory effect on trypsin digestion of casein 16 . Habe et al. reported the synthesis and evaluation of dioleoyl GA, a main component of OR-1, from GA calcium salt 17 . The dioleoyl GA showed a relatively lower inhibitory effect than OR-1, suggesting that another potent substance in OR-1 was responsible for trypsin inhibition. Other diacyl GAs with long acyl chains C16 or C18:2 were synthesized via the benzyl ester derivative of GA Fig. 2B 18,19 . The long acyl chains in the compounds resulted in low water solubility. They showed no toxic effects on NHDFs and normal human dermal microvascular endothelial cells up to 34 μM. Table 1 Examples of compositions of raw glycerol derived from various biodiesel fuel manufacturing processes.

Sample
Organic materials (wt%) Alkali metals (wt%) Ash (wt%) Water (wt%) Reference Glycerol Methanol Fatty acid salts Others In contrast, diacyl GAs with medium acyl chains C6 to C12 were synthesized by direct acylation of GA Fig. 2B 20,21 . Although the acid form of diacyl GAs with medium acyl chains showed little solubility in water, their sodium salts had good water solubility. Among them, dioctanoyl GA sodium salt showed great surface-tension lowering properties in water Table 2 20 . This also showed a greater inhibitory effect on trypsin activity than dioleoyl GA. This is probably due to the difference in water solubility. Another water-soluble sodium salt of diacyl GAs, namely didecanoyl GA sodium salt, also had inhibitory effects on trypsin activity comparable to that of dioctanoyl GA sodium salt 21 . Additionally, sodium salts of dioctanoyl GA and didecanoyl GA form large associates with vesicle-like structures at high concentration 21 . The understanding of this unique property of diacyl GAs will lead to new applications of GA diacyl surfactants.
Monoacyl GAs were also synthesized from D-GA 22 . Conventional acylation with acyl chlorides provided a mixture of regioisomers, 2-O-acyl GA and 3-O-acyl GA. These could be separated via column chromatography, and their physicochemical properties were investigated. In contrast to diacyl GAs, monoacyl GAs showed solubility in water without saponification. The 2-O-acyl GAs showed great surface-tension lowering properties Table 2 ; therefore, they are considered as a new class of green surfactants.

Biological features of glucosyl derivatives
Some microbes are known to possess glycosides with a GA moiety inside the cells 23 . The glycosides, called compatible solutes or osmolytes, have some attractive biological applications. For example, osmolytes are produced in response to external stimuli such as heat, drought, and ultraviolet light to protect biological molecules, such as DNA and proteins 24 . To expand the application of GA, glucosylglyceric acid GGA was synthesized from the calcium salt of GA and sucrose by sucrose phosphorylase Fig. 3 , and its functions were investigated 15 . The synthesized GGA showed protection against DNA scission by hydroxy radicals and heat-induced aggregation of egg white in vitro. In addition, the effect of GGA on NHDFs in vitro was investigated. No positive effect on cell viability of NHDF was observed; however, the production of type I collagen by NHDF increased by 1.4-fold in the presence of 34 mM GGA. Increased collagen production was not observed in the presence of GA. These biological functions of GGA are key features for its use in skincare products and cosmetics.
To expand its application, GGA was further derivatized with an alkyl chain to add amphiphilic property 25 . The car-  boxylic acid in GGA was amidated by N-dodecylamine in the presence of a condensing reagent of 4-4,6-dimethoxy-1,3,5-triazin-2-yl -4-methylmorpholinium chloride, resulting in the synthesis of N-dodecyl glucosylglyceric acid amide Fig. 3 . This compound showed good solubility in some organic solvents such as ethanol, acetone, dimethylformamide as well as water. The water solution showed a good surface tension-lowering property of 27.8 mN/m at a critical micelle concentration of 0.157 mM. This compound had a great protective property against heat-induced aggregation of egg white at low concentrations. Commercialized alkanoyl-N-methylglucamides for example, MEGA-8 and MEGA-10 are solubilizing reagents for membrane proteins. Considering the structural similarity, N-dodecyl glucosylglyceric acid amide could also have similar property.

Oxidative fermentation of GA from glycerol
Acetic acid bacteria are widely used for vinegar production from ethanol. This process is called oxidative fermentation because it consumes oxygen not to completely oxidize ethanol to carbon dioxide, but to accumulate acetic acid in the culture media. This oxidative fermentation is also used to produce sorbose, dihydroxyacetone, 2-ketogluconate, etc. Habe et al. developed a microbial method to produce GA through oxidative fermentation Fig. 4 10,11 . By selecting microbial strains, the enantioselectivity of GA was varied from 70 to 99 enantiomeric excess ee . Acetobacter tropicalis NBRC16470 produced D-GA with 99 ee, whereas Gluconobacter frateurii NBRC103465 produced D-GA with 72 ee Fig. 5 . The accumulation of GA in the culture broth of G. frateurii reached 136.5 g/L in 7 d upon fed-batch cultivation in a 5 L jar fermenter.

Development of Production and Application of Glyceric Acid
Habe et al. also developed a recovery process for GA from culture broth using the electrodialysis method 26,27 . Although the production process of GA from glycerol has been developed, the use of raw glycerol for GA production had to be developed. For example, it was demonstrated that GA production from raw glycerol by Gluconobacter spp. requires pretreatment of raw glycerol to remove impurities 12 .
3.2 Investigation and improvement of GA production from methanol-containing glycerol When using raw glycerol in microbial production of GA, methanol, a major impurity of raw glycerol, could have a negative effect on microbial growth. Indeed, addition of 1 vol methanol in the culture of Gluconobacter strains decreased GA accumulation level by 30-51 compared to no addition of methanol 28 . Therefore, the effect of methanol on GA production by G. frateurii was investigated in detail. The draft genome sequence of G. frateurii NBRC103465 strain was first analyzed 29 . This strain possesses putative genes for methanol detoxification as well as those for GA production. The DNA microarray was designed based on the draft sequence, followed by transcriptome analysis to investigate the responses of G. frateurii cells to methanol in the culture for GA production 30 . In comparison to methanol-free conditions, the cells upregulated a homolog gene of class III alcohol dehydrogenase adhC Gf in the presence of methanol. This enzyme is known to catalyze oxidation of formaldehyde to formic acid. The adhC Gf was heterologously expressed in Escherichia coli and the gene product was confirmed to have dehydrogenase activity toward formaldehyde. Therefore, G. frateurii could detoxify formaldehyde arising from methanol oxidation by upregulating the class III alcohol dehydrogenase gene. Interestingly, no changes in the expression of genes responsible for GA production were observed. The effect of methanol on the enzymatic reaction of glycerol oxidation to glyceraldehyde was investigated 28 . This reaction is catalyzed by membrane-bound alcohol dehydrogenase mADH 10 , and therefore the activity of purified mADH from G. oxydans toward glycerol was evaluated in the presence of methanol. With 0.3 vol methanol in the reaction, the dehydrogenase activity of mADH toward glycerol decreased by half; apparently, methanol caused a decrease in GA production. Therefore, it will be useful to develop mutant mADHs with decreased affinity toward methanol.
An approach for improving GA production was attempted by chemical mutagenesis of G. frateurii to produce methanol-resistant mutant strains 31 . Using N-methyl-Nnitro-N-nitrosoguanidine, a methanol-resistant mutant library of G. frateurii NBRC103465 was constructed, followed by screening of these mutants by GA production level with a pH indicator. Upon evaluating 1000 colonies, Gf398 strain was found to exhibit superior methanol-resistant ability compared to that of parental strain. The Gf398 strain also produced more GA in the presence of 3-5 vol methanol than the parental strain. The jar fermenter experiment of this mutant strain demonstrated GA production by fed-batch cultivation from methanol-containing glycerol, which was two-fold greater than the parental strain unpublished data . Investigation of mutation points of the Gf398 strain will elucidate how the strain tolerates external methanol and detoxifies methanol and formaldehyde, and will lead to further development of useful microbial strains to use methanol-containing raw glycerol.
Another study of GA production by methylotrophic acetic acid bacteria, Acidomonas methanolica, provided a new approach for GA production from methanol-containing glycerol 32 . A. methanolica is a unique acetic acid bacterium that assimilates methanol; therefore, GA production from methanol-containing glycerol by this bacterium was expected. GA production was first confirmed, and subsequent experiments with methanol-containing glycerol revealed that A. methanolica produced more GA from glycerol with 1 vol methanol than that without methanol. Interestingly, the enantioselectivity of the GA produced by A. methanolica, 44 ee of D-GA, was lower than those of other acetic acid bacteria such as G. frateurii and Ace. tropicalis Fig. 5 . This strain broadened the insight into GA production from methanol-containing glycerol as well as the stereoselectivity of GA by acetic acid bacteria.
3.3 Enantioselective production of GA by microbial resolution Although D-GA can be utilized for various applications, L-GA is expected to be used as the starting material for generating L-sugars for biological and pharmacological applications 33 . Although conventional conversion of glycerol to GA by metal catalysts often yields a racemic mixture, oxidative fermentation of GA by acetic acid bacteria produces mainly D-GA Fig. 5 . Therefore, separation methods to obtain pure L-GA from racemic mixtures are necessary for L-GA production. Microbial resolution is an effective method to obtain chiral isomers from racemic mixture. Therefore, screening experiments for obtaining D-GA-assimilating bacteria were conducted to construct a microbial resolution method for L-GA production 34 . Accumulation of L-GA from a racemic mixture of GA was achieved by screening strains of Serratia sp. GA3R and Pseudomonas spp. GA72P, with ≥ 89 ee of L-GA Fig. 5 . This demonstrated that enantioselective production of both D-GA and L-GA could be achieved by microbial fermentation by selecting GA-producing and GA-assimilating bacteria.

Summary
In this review, recent achievements in microbial production of GA and its application were summarized to promote the utilization of raw glycerol and to apply GAs to bio-related functional materials. GA itself as well as its acyl and glucosyl derivatives showed various bio-related functions, expanding the applications of GA to skincare and cosmetic products. Microbial production of both D-and L-GA was developed by GA-producing Gluconobacter strains, methylotroph acetic acid bacteria, and newly isolated GA-assimilating bacterial strains. These developments will promote the use of GA in chemical and biological industries, leading to expanded use of raw glycerol in oleochemical and energy industries.