Production of glutaric acid from 5-aminovaleric acid using Escherichia coli whole cell bio-catalyst overexpressing GabTD from Bacillus subtilis
Introduction
As an alternative approach to producing platform chemicals to replace petroleum based production, biological techniques through fermentation or biotransformation from renewable sources have been extensively studied for several decades as a sustainable technology [[1], [2], [3], [4], [5]]. Consequently, many studies have been carried out on the use of platform chemicals such as 3-hydroxypropionic acid [6], succinic acid [7], itaconic acid [8], putrescine [9], cadaverine [10], pipecolic acid [11], and 5-aminovaleric acid [[12], [13], [14], [15], [16]]. Among these, 5-aminovaleric acid (5-AVA) and glutaric acid are basic 5-carbon platform chemicals used for the production of 5-nylon. Similar to cadaverine, they can be manufactured from L-lysine, which can be produced in quantities of up to 2.2 million tons per year [17]. 5-aminovaleric acid is a precursor of 5-hydroxyvaleric acid, glutaric acid, and 1,5-pentanediol. Glutaric acid is a precursor of a plasticizer, 1,5-pentanediol, and glutaric acid itself can be involved in polymerization reaction, generating polyols and polyamides [12]. The production of 5-aminovaleric acid and glutaric acid from model organisms such as Corynebacterium glutamicum and Escherichia coli has been reported in previous works [12,14,[18], [19], [20], [21], [22]]. Most of the metabolically engineered strains were designed to utilize a 5-aminovaleric acid degradation pathway (AMV pathway), which naturally exists in Pseudomonas putida [13,16,23,24].
Conversion of lysine to glutaric acid via the AMV pathway is catalyzed by four enzymes. Enzymes involved in the native glutaric acid production pathway of P. putida include lysine monooxygease (DavB), 5-aminovaleramidase (DavA), 5-aminovaleric acid aminotransferase (DavT), and glutarate semialdehyde dehydrogenase (DavD) [23,24]. Previous studies demonstrated that 4-aminobutyrate aminotransferase (GabT) and succinate semialdehyde dehydrogenase (GabD) can also be utilized in the conversion of 5-AVA and glutarate semialdehyde (GSA), respectively [14]. The conversion of lysine to 5-AVA is a relatively simple step compared to that of 5-AVA to glutaric acid. The two reactions of DavB and DavA yielding 5-AVA do not require costly co-factors, except for oxygen for oxygenation of L-lysine by DavB. Otherwise, the conversion of 5-AVA to glutaric acid by DavT and DavD requires expensive co-factors such as PLP and NAD(P)+, respectively. In addition, DavT reaction requires another substrate, α-ketoglutaric acid as an amine acceptor which is conferred one amine group during the reaction from 5-AVA. (Fig. 1).
The first two steps in this pathway involve davB and davA encoding of lysine monooxygenase and 5-aminovaleramidase, respectively. This results in a high concentration of 5-AVA production of up to 773 mM and 2134 mM by fermentation and whole cell conversion, respectively [18,25]. In contrast, the fermentative production of glutaric acid in engineered strains via the AMV pathway using various aminotransferases (DavT or GabT) and diacid semialdehyde dehydrogenases (DavD or GabD) catalyzing 5-AVA to glutaric acid have produced only low concentrations of glutaric acid, up to 103 mM with 0.656 mM/h of productivity [12,14,20,26]. Although some genuine pathways have been studied to produce glutaric acid via the AMA (2-aminoadipate) pathway or a new synthetic pathway using E. coli as the host strain, these pathways yielded even lower concentration of glutaric acid than the AMV pathway (Table 1) [21,22,27]. The reason for the low conversion rate from 5-AVA to glutaric acid is not clearly known.
Previous studies have suggested that the potent 5-aminovaleric acid transporter or permeases might export 5-aminovaleric acid to outside of the cell, yielding low glutaric acid production by fermentation [12,13,16]. Adkins et al. highlighted the importance of α-ketoglutaric acid to improve glutaric acid production, indicating that the regeneration of this co-substrate using glutamic acid dehydrogenase may lead to an improvement in productivity [12]. However, no previous studies have been carried out to determine the influence of co-factors such as pyridoxal 5′-phosphate (PLP) and NAD(P)+, which are co-factors of 5-AVA aminotransferase and glutarate semialdehyde dehydrogenase, respectively. Additionally, previous studies have not identified which of the reaction steps is a rate limiting step in 5-AVA to glutaric acid conversion reaction among the transamination of 5-AVA and dehydrogenation of glutaric acid semialdehyde.
As a solution to determine the important nodes and to increase the production yield, whole cell biotransformation can be considered [8,10,[28], [29], [30], [31], [32]]. A whole cell bioconversion system has advantages such as relatively easy control of each enzyme, supply of cofactors in a short period of time, and higher robustness to harsh environments than purified enzymes. In addition, our previous results showed that, with sufficient starting materials such as lysine and citric acid, the whole cell system could improve yield and productivity after the optimization of reaction parameters [8,10].
In this paper, gabTD from Bacillus subtilis was cloned into E. coli and applied to 5-AVA degradation for glutaric acid production. The E. coli whole cell biocatalyst was applied to determine the detailed reaction conditions and optimum concentration of 5-AVA, α-ketoglutaric acid, PLP, and NAD(P)+, which are required for glutaric acid production (Fig. 1). Through this study, the effect of PLP and NAD(P)+, which are co-factors of GabT and GabD, respectively, on reaction was observed. Although we could not clarify all the reasons for the low glutaric acid productivity, several ways to improve glutaric acid conversion were revealed.
Section snippets
Chemicals
3-aminopropanoic acid (>99%), 5-aminovaleric acid (>97%), 6-aminocaproic acid (>99%), glutaric acid (>99%), glutamic acid (>99%), pyridoxal-5-phosphate hydrate (>98%), and diethylethoxymethylene malonate (>99%) were purchased from Sigma-Aldrich Co. (USA). 4-aminobutyric acid (>98%) and α-ketoglutaric acid (>99%) were obtained from Tokyo Chemical Industry Co. (Japan). β-nicotinamide adenine dinucleotide sodium salt (>99%), β-nicotinamide adenine dinucleotide phosphate sodium salt (>99%), Tris
Introduction of new gabTD from B. subtilis for whole cell reaction of glutaric acid production
To determine the different sources of gabTD system other than the known strains such as P. putida and C. glutamicum, we searched several strains through bibliographic and bioinformatic approaches and found gabTD derived from B. subtilis (BsgabTD). In nature, the gabTD operon plays a role in the utilization of glutamate as a nitrogen source correlated with glutamate decarboxylase (Gad) enzymes in many microorganisms [33,34]. Especially for the Bacillus species, which forms spores at the end
Conclusion
To date, 5-AVA and glutaric acid have been produced by fermentation, introducing davB, davA, davT, and davD genes from P. putida KT2440. High conversion from lysine to 5-aminovaleric acid was achieved up to 773 mM by fermentation and 2134 mM by whole cells with high productivity of 8.05 mM/h and 76.2 mM/h, respectively. However, until now, low concentrations (103 mM) with low productivity (0.656 mM/h) have been recorded for the conversion of 5-aminovaleric acid to glutaric acid via
Acknowledgements
This study was supported by the National Research Foundation (NRF) of Korea (NRF-2015M1A5A1037196, NRF-2016R1D1A1B03932301), the Research Program initiated to address social issues highlighted by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3A9E4077234), and the R & D Program of MOTIE/KEIT (10067772, 10049674). This work was also supported by polar academic program (PAP,PE18900). The consulting service of the Microbial Carbohydrate Resource
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