Enhanced productivity of gamma-amino butyric acid by cascade modifications of a whole-cell biocatalyst
We previously developed a gamma-amino butyric acid (GABA)-producing strain of Escherichia coli, leading to production of 614.15 g/L GABA at 45 °C from L-glutamic acid (L-Glu) with a productivity of 40.94 g/L/h by three successive whole-cell conversion cycles. However, the increase in pH caused by the accumulation of GABA resulted in inactivation of the biocatalyst and consequently led to relatively lower productivity. In this study, by overcoming the major problem associated with the increase in pH during the production process, a more efficient biocatalyst was obtained through cascade modifications of the previously reported E. coli strain. First, we introduced four amino acid mutations to the codon-optimized GadB protein from Lactococcus lactis to shift its decarboxylation activity toward a neutral pH, resulting in 306.65 g/L of GABA with 99.14 mol% conversion yield and 69.8% increase in GABA productivity. Second, we promoted transportation of L-Glu and GABA by removing the genomic region encoding the C-plug of GadC (a glutamate/GABA antiporter) to allow its transport path to remain open at a neutral pH, which improved the GABA productivity by 16.8% with 99.3 mol% conversion of 3 M L-Glu. Third, we enhanced the expression of soluble GadB by introducing the GroESL molecular chaperones, leading to 20.2% improvement in GABA productivity, with 307.40 g/L of GABA and a 61.48 g/L/h productivity obtained in one cycle. Finally, we inhibited the degradation of GABA by inactivation of gadA and gadB from the E. coli genome, which resulted in almost no GABA degradation after 40 h. After the cascade system modifications, the engineered recombinant E. coli strain achieved a 44.04 g/L/h productivity with a 99.6 mol% conversion of 3 M L-Glu in a 5-L bioreactor, about twofold increase in productivity compared to the starting strain. This increase represents the highest GABA productivity by whole-cell bioconversion using L-Glu as a substrate in one cycle observed to date, even better than the productivity obtained from the three successive conversion cycles.
KeywordsGamma-aminobutyric acid GABA L-glutamic acid L-Glu Escherichia coli
Funding was provided by grants from the Natural Science Foundation of Fujian Province (2016J05074 and 2014J01037).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008. https://doi.org/10.1038/msb4100050 CrossRefPubMedPubMedCentralGoogle Scholar
- Georgopoulos C, Welch WJ (1993) Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol 9(1):601–634. https://doi.org/10.1146/annurev.cb.09.110193.003125 CrossRefPubMedGoogle Scholar
- Kanjee U, Houry WA (2013) Mechanisms of acid resistance in Escherichia coli. Annu Rev Microbiol 67:65–81. https://doi.org/10.1146/annurev-micro-092412-155708 CrossRefPubMedGoogle Scholar
- Lee S, Ahn J, Kim YG, Jung JK, Lee H, Lee EG (2013) Gamma-aminobutyric acid production using immobilized glutamate decarboxylase followed by downstream processing with cation exchange chromatography. Int J Mol Sci 14(1):1728–1739. https://doi.org/10.3390/ijms14011728 CrossRefPubMedPubMedCentralGoogle Scholar
- Pennacchietti E, Lammens TM, Capitani G, Franssen MC, John RA, Bossa F, De Biase D (2009) Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH. J Biol Chem 284(46):31587–31596. https://doi.org/10.1074/jbc.M109.049577 CrossRefPubMedPubMedCentralGoogle Scholar
- Pham VD, Lee SH, Park SJ, Hong SH (2015) Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli. J Biotechnol 207:52–57. https://doi.org/10.1016/j.jbiotec.2015.04.028 CrossRefPubMedGoogle Scholar
- Pham VD, Somasundaram S, Lee SH, Park SJ, Hong SH (2016a) Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter. J Ind Microbiol Biot 43(1):79–86. https://doi.org/10.1007/s10295-015-1712-8 CrossRefGoogle Scholar
- Pham VD, Somasundaram S, Park SJ, Lee SH, Hong SH (2016b) Co-localization of GABA shunt enzymes for the efficient production of gamma-aminobutyric acid via GABA shunt pathway in Escherichia coli. J Microbiol Biotechnol 26(4):710–716. https://doi.org/10.4014/jmb.1511.11037 CrossRefPubMedGoogle Scholar
- Shi F, Jiang JJ, Li YF, Li YX, Xie YL (2013) Enhancement of gamma-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis. J Ind Microbiol Biot 40(11):1285–1296. https://doi.org/10.1007/s10295-013-1316-0 CrossRefGoogle Scholar
- Shi F, Xie YL, Jiang JJ, Wang NN, Li YF, Wang XY (2014) Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near-neutral pH. Enzyme Microb Tech 61-62:35–43. https://doi.org/10.1016/j.enzmictec.2014.04.012 CrossRefGoogle Scholar
- Soma Y, Fujiwara Y, Nakagawa T, Tsuruno K, Hanai T (2017) Reconstruction of a metabolic regulatory network in Escherichia coli for purposeful switching from cell growth mode to production mode in direct GABA fermentation from glucose. Metab Eng 43:54–63. https://doi.org/10.1016/j.ymben.2017.08.002 CrossRefPubMedGoogle Scholar
- Tomas CA, Welker NE, Papoutsakis ET (2003) Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell’s transcriptional program. Appl Environ Microbiol 69(8):4951–4965. https://doi.org/10.1128/Aem.69.8.4951-4965.2003 CrossRefPubMedPubMedCentralGoogle Scholar
- Vo TDL, Ko JS, Park SJ, Lee SH, Hong SH (2013) Efficient gamma-aminobutyric acid bioconversion by employing synthetic complex between glutamate decarboxylase and glutamate/GABA antiporter in engineered Escherichia coli. J Ind Microbiol Biot 40(8):927–933. https://doi.org/10.1007/s10295-013-1289-z CrossRefGoogle Scholar
- Zhang C, Lu J, Chen L, Lu FX, Lu ZX (2014a) Biosynthesis of gamma-aminobutyric acid by a recombinant Bacillus subtilis strain expressing the glutamate decarboxylase gene derived from Streptococcus salivarius ssp. thermophilus Y2. Process Biochem 49(11):1851–1857. https://doi.org/10.1016/j.procbio.2014.08.007 CrossRefGoogle Scholar
- Zhang RZ, Yang TW, Rao ZM, Sun HM, Xu MJ, Zhang X, Xu ZH, Yang ST (2014b) Efficient one-step preparation of gamma-aminobutyric acid from glucose without an exogenous cofactor by the designed Corynebacterium glutamicum. Green Chem 16(9):4190–4197. https://doi.org/10.1039/c4gc00607k CrossRefGoogle Scholar
- Zhao AQ, Hu XQ, Pan L, Wang XY (2015) Isolation and characterization of a gamma-aminobutyric acid producing strain Lactobacillus buchneri WPZ001 that could efficiently utilize xylose and corncob hydrolysate. Appl Microbiol Biotechnol 99(7):3191–3200. https://doi.org/10.1007/s00253-014-6294-2 CrossRefPubMedGoogle Scholar