Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli
Introduction
The introduction of heterologous metabolic pathways into naive microbial hosts can result in metabolite or product toxicity (Ling et al., 2014). Although natural metabolic pathways have evolved intricate regulation to prevent the buildup of toxic metabolites, heterologous pathways, which frequently consist of non-native reactions from different organisms, typically lack such sophistication (Chubukov et al., 2016). Without regulation, imbalances in assembled pathways frequently lead to metabolite accumulation and toxicity (Jones et al., 2015). Typical metabolic engineering and synthetic biology workflows for improved pathway expression (Boyle and Silver, 2012, Jullesson et al., 2015, Keasling, 2012) may amplify this issue by causing massive fluctuations in metabolite concentrations. Even if a pathway is well-balanced, many chemicals targeted for microbial production are toxic at the concentrations that are necessary for economical, industrial-scale production (Nicolaou et al., 2010). The practical importance of addressing this toxicity has resulted in numerous studies aimed at interrogating and alleviating product toxicity in common industrial hosts such as Escherichia coli and Saccharomyces cerevisiae (Mukhopadhyay, 2015).
Assessment of product or intermediate toxicity is challenging, particularly in an engineered host that produces the molecules in vivo. In engineered microbes harboring heterologous pathways, increased metabolic burden (Wu et al., 2016) and unexpected impacts on endogenous metabolism make the isolated study of metabolite toxicity difficult. To avoid this complexity, most studies have relied on well-controlled systems where the toxic product is exogenously added to the wild-type host and systems analyses are employed to study the stress response (Brynildsen and Liao, 2009). Though multiple studies have successfully used these approaches, there are several limitations. For example, exogenous addition is only successful when the compound of interest crosses the cell membrane through active transport or diffusion (Mukhopadhyay, 2015). While this is not an issue for solvents such as isobutanol (Atsumi et al., 2010), many toxic intermediates or products contain functional groups that prevent membrane transport. Even if the compound is transported, it is unclear if the stress response associated with exogenous addition reflects the response associated with in vivo production. This is particularly true for a wild type host, which likely has different metabolism and overall fitness compared to an extensively engineered production strain (Wu et al., 2016). Finally, exogenous addition can introduce unintended artifacts or impurities to the experimental system. In the case of limonene, for instance, extended chemical storage forms hydroperoxide derivatives that are significantly more toxic than limonene itself (Chubukov et al., 2015).
The S. cerevisiae mevalonate pathway for isoprenoid biosynthesis has been heavily engineered to produce valuable compounds including the anti-malarial drug artemisinin (Paddon and Keasling, 2014) and several candidate biofuels (George et al., 2015a). Considerable progress has been made in the production of isoprenol, a C5 alcohol derived from isopentenyl pyrophosphate (IPP), which is one of two universal precursors for isoprenoid compounds (George et al., 2015b, Zheng et al., 2013). In E. coli, the toxicity of both isoprenol (Foo et al., 2014) and IPP (George et al., 2014) has been suggested to impact cell growth and limit product yields. Although the toxicity of IPP (and its longer chain derivatives GPP and FPP) was observed over a decade ago (Martin et al., 2003), a systematic assessment of this stress and its impact on host physiology has yet to be conducted. Metabolic engineering strategies have instead focused on avoiding its accumulation, primarily through careful pathway “balancing”, improved expression of terminal enzymes, and, most recently, through the assembly of alternative pathways (Kang et al., 2016). Since IPP is a universal precursor for isoprenoid compounds, understanding its mode of toxicity could benefit engineering in a variety of hosts and pathway variants. More broadly, uncovering the mechanism of IPP toxicity could hold biological relevance for the study of isoprenoid pathway regulation in a variety of organisms.
In this work, we developed a three-strain experimental system to study IPP toxicity in vivo in isoprenol-producing E. coli. First, we assessed the impact of IPP accumulation on host physiology by measuring growth characteristics, morphology, and viability. Informed by these data, we next collected comprehensive metabolomics and proteomics measurements over an 18-h fermentation time course. We used principal component analysis (PCA) to direct our investigation, which linked IPP accumulation to reduced nutrient uptake, a “pause” in metabolism, and reduced ATP levels. Finally, we performed RNA-seq at critical time points corresponding to the onset and alleviation of IPP-induced toxicity. We use these complementary data to propose a possible mechanism for IPP toxicity involving the formation of an isoprenyl-ATP analog, ApppI.
Section snippets
Strains and culture conditions
Derivatives of E. coli DH1 WT EV (DH1 harboring pBbA5c and pTrc99A), 3A (DH1 harboring pJBEI-6829 and pJBEI-6833), and 3Amk (DH1 harboring pJBEI-6829 and pJBEI-6834) were used in this study. Overnight cultures were grown in 10 mL of LB media supplemented with 30 µM chloramphenicol (Cm30) and 100 µM carbenicillin (Carb100). Cultures were inoculated directly from frozen glycerol stocks and incubating overnight at 37 °C. Production experiments were carried out in 200 mL of EZ-Rich defined medium
Developing a platform to selectively assess in vivo IPP toxicity
The heterologous pathway for isoprenol production in E. coli consists of 8 reactions, 7 of which are catalyzed by plasmid-borne genes (Fig. 1A). It was previously demonstrated through proteomics analysis that the level of mevalonate kinase (MK) is the primary determinant of downstream pathway flux to IPP (George et al., 2014). We took advantage of this relationship to develop an experimental platform to isolate the impact of IPP toxicity from heterologous pathway induction (e.g., metabolic
Discussion
In this work, we show that IPP toxicity is a complex stress with severe physiological consequences. Elevated levels of IPP caused growth inhibition, reduced cell viability as measured by live/dead staining and CFU counts, and plasmid instability. Several results suggested that membrane function or integrity were compromised in the presence of IPP, including the observation of cell elongation, increased levels of osmoprotectants such as proline and betaine, and differential expression of genes
Conclusion
In this work, we design a three-strain experimental platform to isolate the impact of IPP toxicity from the stress associated with heterologous pathway expression. Using this platform, we assess the physiological consequences of IPP accumulation with complementary multi-omics data. We show that IPP accumulation is linked to reduced cell viability, pathway “breakage” at PMK, reduced nutrient uptake and ATP levels, and perturbed nucleotide metabolism. We also observe the extracellular
Acknowledgments
This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable,
Financial interest
Jay Keasling has a financial interest in Amyris, Lygos, Demetrix, Constructive Biology and Napigen.
Author contributions
Conceptualization, K.W.G., M.G.T, and T.S.L.; Methodology, K.W.G., M.G.T., J.K., E.E.K.B., and C.J.P.; Investigation, K.W.G., M.G.T., J.K., E.E.K.B., G.W., V.T.B., L.J.G.C., V.B., and S.Y.; Writing – Original Draft, K.W.G., M.G.T., J.K., J.D.K., and T.S.L.; Writing – Review & Editing, K.W.G., M.G.T., J.K., J.D.K., and T.S.L.; Resources, T.S.L., J.D.K., C.J.P., P.D.A., and P.T.; Supervision, T.S.L.
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These authors contributed equally to this work.