Elsevier

Metabolic Engineering

Volume 69, January 2022, Pages 59-72
Metabolic Engineering

Adaptive laboratory evolution of Escherichia coli W enhances gamma-aminobutyric acid production using glycerol as the carbon source

https://doi.org/10.1016/j.ymben.2021.11.004Get rights and content

Highlights

  • E. coli W evolution under glycerol limitation improved fitness and glycerol utilization.

  • Whole-genome and transcriptome sequencing and enzyme kinetic assays to understand the molecular basis of adaptation.

  • Glycerol-adapted E. coli W produced GABA by using glycerol as the feedstock carbon.

  • Use of evolutionary and metabolite engineering improved the final GABA titer (3.9-fold) and productivity (4.3-fold).

Abstract

The microbial conversion of glycerol into value-added commodity products has emerged as an attractive means to meet the demands of biosustainability. However, glycerol is a non-preferential carbon source for productive fermentation because of its low energy density. We employed evolutionary and metabolic engineering in tandem to construct an Escherichia coli strain with improved GABA production using glycerol as the feedstock carbon. Adaptive evolution of E. coli W under glycerol-limited conditions for 1300 generations harnessed an adapted strain with a metabolic system optimized for glycerol utilization. Mutation profiling, enzyme kinetic assays, and transcriptome analysis of the adapted strain allowed us to decipher the basis of glycerol adaptation at the molecular level. Importantly, increased substrate influx mediated by the mutant glpK and modulation of intracellular cAMP levels were the key drivers of improved fitness in the glycerol-limited condition. Leveraging the enhanced capability of glycerol utilization in the strain, we constructed a GABA-producing E. coli W-derivative with superior GABA production compared to the wild-type. Furthermore, rationally designed inactivation of the non-essential metabolic genes, including ackA, mgsA, and gabT, in the glycerol-adapted strain improved the final GABA titer and specific productivity by 3.9- and 4.3-fold, respectively, compared with the wild-type.

Introduction

In parallel with the rapid growth of the biofuel market, increasing amounts of glycerol, which accounts for nearly 10% (v/v) of the total biodiesel products, are generated as byproducts (Da Silva et al., 2009; Mu et al., 2006). The excess glycerol made available from biodiesel transesterification has led to a dramatic decrease in the market cost of glycerol (Dharmadi et al., 2006), making it an economically attractive feedstock carbon. As many microbes utilize glycerol as their sole carbon source, numerous studies have focused on the microbial conversion of this renewable feedstock into value-added products (Da Silva et al., 2009).

Escherichia coli W is one of the many microbes that utilize glycerol as their sole carbon source. It is also an emerging strain for industrial-scale production owing to its high tolerance to environmental stress and low acetate production without tight sugar control (Alterthum and Ingram, 1989; Monk et al., 2016). As one of the fastest-growing strains among commercial E. coli (Archer et al., 2011), it is thought to be an ideal platform strain for producing desired metabolites that are often generated in a growth-coupled manner. Previously, it was demonstrated that E. coli W, W3110, and BL21 (DE3) exhibited some of the highest production of γ-aminobutyric acid (GABA), using glycerol as the feedstock carbon (Hou and Kang, 2018). However, the absence of optimization schemes, such as metabolic engineering, to maximize GABA production requires additional engineering efforts to evaluate further the potential of GABA production by E. coli W.

GABA has attracted considerable interest because of its multifunctional role in pharmaceuticals, food additives, and the synthesis of biodegradable polymers (Abdou et al., 2006; Kanjee and Houry, 2013; Park et al., 2013). It is a natural, non-proteinogenic amino acid that can be naturally produced from L-glutamate by the enzyme glutamate decarboxylase (GAD). In bacterial cells, GABA is a byproduct of an acid-resistant system that plays an important role in buffering the cells against low pH (Kanjee and Houry, 2013). Considering the value-added nature of GABA (USD 9–20/kg; Pyson, Shaanxi, China) compared with food-grade (USD 0.6–0.9/kg) or crude-grade (USD 0.09–0.2/kg) glycerol (Abdul Raman et al., 2019), bacterial-based bioconversion of GABA using glycerol feedstock is thought of as an economically viable venture. However, the wild-type GAD is only active under low-pH; thus, an efficient microbial GABA production requires heterologous expression of a mutant of glutamate decarboxylase (mtGAD) catalytically functional at neutral pH (Luo et al., 2021; Valgepea et al., 2010).

In this study, we employed adaptive laboratory evolution (ALE) and metabolic engineering for GABA production in E. coli W. ALE is a robust approach that facilitates the optimization of cellular regulatory and metabolic networks in microbes to attain better fitness under user-defined selection pressures. The fitness attributes of causative mutations acquired during adaptive evolution can manifest in different ways, including the increase in resistance against specific stressors, utilization of non-natural substrates, and more efficient substrate utilization (Applebee et al., 2011; Goodarzi et al., 2010; Guzman et al., 2019; Kang et al., 2019). In this respect, ALE holds relevance in bioconversion of waste which is often low in energy content, difficult for microbes to utilize, and contains inhibitory substances that hinder bacteria growth. A notable example includes ALE of Lactobacillus delbrueckii in sugarcane molasses, which is a low-cost, agro-industrial waste, to select for the mutant that efficiently utilizes the renewable waste material with improved productivity of target chemical D-lactic acid (Liang et al., 2020). Recently, ALE of an obligate anaerobe Eubacterium limosum on carbon monoxide not only improved strain fitness, but also increased the production of acetate up to 8-folds (Kang et al., 2020). Here, E. coli W underwent 1300 generations of ALE in a minimal medium supplemented with glycerol, a carbon that is sub-optimally utilized by E. coli (Ibarra et al., 2002; Weikert et al., 1997). Whole-genome resequencing of the glycerol-evolved progeny revealed key causative mutations that conferred a fitness advantage in glycerol-limited culture conditions. Additionally, transcriptome sequencing (RNA-Seq) revealed genome-wide gene expression changes that reflected rewired metabolic networks. Finally, we leveraged the glycerol-adapted traits of the evolved E. coli W to convert low-cost, low-value carbon glycerol into a value-added product, GABA. This study highlights that adaptive evolution provides a robust method to harness microbes that effectively valorize low-value chemicals.

Section snippets

Adaptive laboratory evolution of E. coli W in a minimal glycerol medium

E. coli W is one of the fastest-growing E. coli strains with a high tolerance to environmental stresses (Alterthum and Ingram, 1989; Archer et al., 2011; Monk et al., 2016), making it ideal for the production of commodity chemicals from non-preferential carbon sources, such as glycerol. Previous studies have shown that the fitness of E. coli strains remains suboptimal in minimal glycerol media (Ibarra et al., 2002; Weikert et al., 1997), requiring specific interventions, such as ALE, for the

Discussion

It has been previously shown that the metabolic network in wild-type E. coli strains is not optimized for glycerol utilization and that strain fitness under glycerol limitation can be further improved through adaptive evolution experiments (Cheng et al., 2014; Herring et al., 2006; Ibarra et al., 2002; Kang et al., 2019). ALE is a robust top-down approach that harnesses microbes with desired characteristics. This approach exploits the innate ability of microbes to rapidly adapt to new

Bacterial strains, culture conditions and primers

Cells were grown in M9 minimal medium (BD, Franklin Lakes, NJ, USA) supplemented with 0.2% (v/v) glycerol (Samchun Chemicals, Seoul, South Korea) or Luria Bertani (LB) medium unless stated otherwise. The growth profile in Fig. 1, Fig. 5 was measured in 50 mL culture in 250 mL Erlenmeyer flasks at 37 °C, 200 rpm for every 2 h intervals. The growth profile in Fig. S7 was performed using the EPOCH2 plate reader (BioTek, Winnoski, VT, United States) at 37 °C with shaking (807 rpm double orbital)

Author contributions

Kangsan Kim: Data curation, Investigation, Formal Analysis, Writing- Original draft preparation, Visualization, Methodology, Software. Chen Yuan Hou: Data curation, Investigation, Visualization, Formal Analysis, Methodology. Donghui Choe: Data curation, Investigation, Visualization, Formal Analysis, Methodology, Writing-Reviewing and Editing. Minjeong Kang: Data curation, Formal Analysis, Visualization, Methodology. Suhyung Cho: Data curation, Formal Analysis, Visualization, Methodology. Bong

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

Not applicable.

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