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A kinetic model of the central carbon metabolism for acrylic acid
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production in Escherichia coli
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Alexandre Oliveira
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, Joana Rodrigues
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, Eugénio Ferreira
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, Lígia Rodrigues
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, Oscar Dias
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¶
*
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Centre of Biological Engineering, University of Minho, Braga, Portugal
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* Corresponding Author:
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E-mail: odias@deb.uminho.pt (OD)
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¶
These authors contributed equally to this work
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&
These authors also contributed equally to this work
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Abstract
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Acrylic acid is a value-added chemical used in industry to produce diapers, coatings, paints, and
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adhesives, among many others. Due to its economic importance, there is currently a need for new and
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sustainable ways to synthesise it. Recently, the focus has been laid in the use of Escherichia coli to
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express the full bio-based pathway using 3-hydroxypropionate as an intermediary through three distinct
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pathways (glycerol, malonyl-CoA, and β-alanine). Hence, the goals of this work were to use COPASI
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software to assess which of the three pathways has a higher potential for industrial-scale production,
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from either glucose or glycerol, and identify potential targets to improve the biosynthetic pathways
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yields.
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When compared to the available literature, the models developed during this work successfully
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predict the production of 3-hydroxypropionate, using glycerol as carbon source in the glycerol
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pathway, and using glucose as a carbon source in the malonyl-CoA and β-alanine pathways. Finally,
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this work allowed to identify four potential over-expression targets (glycerol-3-phosphate
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dehydrogenase (G3pD), acetyl-CoA carboxylase (AccC), aspartate aminotransferase (AspAT), and
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aspartate carboxylase (AspC)) that should, theoretically, result in higher AA yields.
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Author summary
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Acrylic acid is an economically important chemical compound due to its high market value.
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Nevertheless, the majority of acrylic acid consumed worldwide its produced from petroleum
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derivatives by a purely chemical process, which is not only expensive, but it also contributes towards
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environment deterioration. Hence, justifying the current need for sustainable novel production methods
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that allow higher profit margins. Ideally, to minimise production cust, the pathway should consist in
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the direct bio-based production from microbial feedstocks, such as Escherichia coli, but the current
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yields achieved are still to low to compete with conventional method. In this work, even though the
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glycerol pathway presented higher yields, we identified the malonyl-CoA route, when using glucose
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as carbon source, as having the most potential for industrial-scale production, since it is cheaper to
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implement. Furthermore, we also identified potential optimisation targets for all the tested pathways,
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that can help the bio-based method to compete with the conventional process.
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Introduction
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Acrylic acid (AA) (C
3
H
4
O
2
) is an important chemical compound that is one of the key
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components of superabsorbent polymers [1–3]. According to the Allied Market Research, in 2015, the
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global market for AA was valued at 12,500 million US dollars, and is expected to reach 19,500 million
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US dollars until 2022 [4]. Despite its economic importance, the vast majority of AA is still produced
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by the oxidation of propylene or propane in a purely chemical process [1,5,6]. Ergo, the principal
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method for AA production was found to be expensive, with a high energy demand, thus contributing
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to the planet’s environment decay. Hence, the development of an innovative and sustainable biological
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production method has been attracting the attention of the scientific community [1,2,7]. In the last
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decade, several semi-biological methods have emerged and were optimised. These methods consist of
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the bio-based production of 3-hydroxypropionate (3-HP) and its subsequent chemical conversion to
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AA. Despite the substantial improvements obtained with these methods, this process involves a
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catalytic step that increases the production costs and environmental impact due to high energy demands
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[1–3,5]. Hence, the AA’s production method should, ideally, be a bio-based direct route as, in theory,
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microbial feedstocks are less expensive, allowing a higher profit margin [1]. Moreover, a more
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sustainable bioprocess allows to decrease non-renewable resources dependence and CO
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emissions.
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Fortunately, in recent years, it has been proven that it is possible to use engineered Escherichia
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coli to convert glucose or glycerol into AA. Like in the semi-biological methods, the bioprocess is also
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divided into two main parts, the production of 3-HP and its subsequent conversion to AA. This part of
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the pathway, from 3-HP to AA, has not been extensively studied. So far, there are only three studies
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that successfully converted glucose or glycerol to AA in E. coli [1,2,7]. However, the synthesis of 3-
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HP is well reported, and three distinct pathways have been identified, namely the glycerol route, the
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malonyl-CoA route, and the β-alanine route. From these pathways, it is well established that the
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glycerol pathway is associated with the highest yields. However, one of the reactions of this route
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requires the supplementation of vitamin B
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(Fig 1), which is an expensive practice at an industrial-
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scale production, hence a significant disadvantage of this route [8,9].
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Fig 1. Biosynthetic pathways for acrylic acid (AA) production from Glucose using 3-
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hydroxypropionate (3-HP) as an intermediary.
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3-HP can be produced from Glucose through three distinct pathways: glycerol (red arrows), malonyl-
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CoA (green arrows), and β-alanine (blue arrows). Furthermore, E. coli can also direct Glycerol towards
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the central carbon metabolism, allowing it to be used as a carbon source.
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The bio-based method is currently considered a promising alternative to the conventional process
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as the production of 3-HP increased considerably in the last few years. Recently, studies reported
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productions of up to 8.10 g/L with the glycerol pathway [1], 3.60 g/L with the malonyl-CoA pathway
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[10], and 0.09 g/L with the β-alanine pathway [11]. However, the AA yields obtained by Tong et al.
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(2016) [2] (0.0377 g/L) and Chu et al. (2015) [1] (0.12 g/L) for the glycerol pathway, and Liu and Liu
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(2016) [7] (0.013 g/L) for the malonyl-CoA pathway, established that this process still needs to be
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optimised to compete with the currently used methods.
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Taking these considerations into account, the main goals of this work are to identify the reactions
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of the known routes for AA production (glycerol, malonyl-CoA, and β-alanine pathways) and to
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determine which pathway have a higher potential for industrial-scale production. E. coli’s central
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carbon metabolism (CCM) kinetic models will be used to analyse the three pathways using either
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glucose or glycerol as carbon source. Finally, novel optimisation strategies to improve the AA yields
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of the three biosynthetic pathways will also be sought.
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Results and Discussion
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Time Course Simulations
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During the simulations, several issues arose, leading to changes in parameters before the analysis
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of the 3-HP and AA production. These changes are explained in detail in the S1 Appendix, section 1.1.
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Regarding the production of 3-HP in the glycerol pathway, simulations with models set to use
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either glucose (Glu-Gly) or glycerol as carbon source (Gly-Gly), predicted, the production of 0.19 g/L
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(after three hours), and 8.30 g/L (after six-hours), respectively (Fig 3). Whereas, concerning the
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production of AA, the Glu-Gly and Gly-Gly models predicted 0.16 g/L and 6.71 g/L, respectively (Fig
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4). From these results, glycerol seems to be associated with higher yields, which is in good agreement
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with the available literature [1]. Moreover, regarding the production of AA, the intracellular
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concentration of 3-HP showed that there is no accumulation (Fig 4), meaning that most 3-HP is
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converted into AA. These results are most likely associated with the use of excessive enzyme
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concentration to calculate the V
max
for the heterologous pathway, which led to a state in which the main
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limiting factor in the synthesis of AA was the CCM’s flux distribution. However, this is not the case
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in vivo, as the studies that tested the full bio-based pathway show that 3-HP and other intermediates
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indeed accumulate during this process [1,2].
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Fig 2. Simulation results for 3-hydroxypropionate (3-HP) production via the glycerol pathway.
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(A) Glucose (GLCx) consumption and variation of extracellular 3-HP (3-HPx) over time; (B) Glycerol
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(GLYx) consumption and variation of 3-HPx over time.
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Fig 3. Simulation results for acrylic acid (AA) production via the glycerol pathway.
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(A) Glucose (GLC) consumption and variation of extracellular AA (AAx) over time; (B) Variation of
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3-hydroxypropionate (3-HP) concentration over time when using Glucose as carbon source; (C)
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Glycerol (GLYx) consumption and variation of extracellular AAx over time; (D) Variation of 3-HP
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concentration over time when using Glycerol as carbon source.
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