Applied Microbiology and Biotechnology

, Volume 102, Issue 8, pp 3623–3633 | Cite as

Enhanced productivity of gamma-amino butyric acid by cascade modifications of a whole-cell biocatalyst

  • Xinwei Yang
  • Chongrong Ke
  • Jiangming Zhu
  • Yan Wang
  • Wenchao Zeng
  • Jianzhong Huang
Biotechnological products and process engineering

Abstract

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.

Keywords

Gamma-aminobutyric acid GABA L-glutamic acid L-Glu Escherichia coli 

Notes

Acknowledgements

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.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8881_MOESM1_ESM.pdf (149 kb)
ESM 1 (PDF 148 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.National Engineering Research Center of Industrial Microbiology and Fermentation Technology, College of Life SciencesFujian Normal UniversityFuzhouPeople’s Republic of China

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