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A kinetic model of the central carbon metabolism for acrylic acid production in Escherichia coli

15 May 2020-bioRxiv (Cold Spring Harbor Laboratory)-

TL;DR: This work identified the malonyl-CoA route, when using glucose as carbon source, as having the most potential for industrial-scale production, since it is cheaper to implement, and also identified potential optimisation targets for all the tested pathways that can help the bio-based method to compete with the conventional process.
Abstract: Acrylic acid (AA) is a value-added chemical used in industry to produce diapers, coatings, paints, and adhesives, among many others. Due to its economic importance, there is currently a need for new and sustainable ways to synthesize it. Recently, the focus has been laid in the use of Escherichia coli to express the full bio-based pathway using 3-hydroxypropionate (3-HP) as an intermediary through three distinct pathways (Glycerol, malonyl-CoA, and β-alanine). Hence, the goals of this work were to assess which of the three pathways has a higher potential for industrial-scale production from either Glucose or Glycerol, and identify potential targets to improve the biosynthetic pathways yields. The models developed during this work seem, when compared to the available literature, to successfully predict 3-HP production for the glycerol pathway, using Glycerol as carbon source, and for the malonyl-CoA and β-alanine pathways, using Glucose a carbon sources. Finally, this work allowed to identify four potential over-expression targets (G3pD, AccC, AspAT, and AspC) that should, theoretically, result in higher AA yields.

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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
1
, Joana Rodrigues
, Eugénio Ferreira
1&
, Lígia Rodrigues
1&
, Oscar Dias
1
*
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9
1
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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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 14, 2020. ; https://doi.org/10.1101/2020.05.13.093294doi: bioRxiv preprint

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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
18
adhesives, among many others. Due to its economic importance, there is currently a need for new and
19
sustainable ways to synthesise it. Recently, the focus has been laid in the use of Escherichia coli to
20
express the full bio-based pathway using 3-hydroxypropionate as an intermediary through three distinct
21
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,
23
from either glucose or glycerol, and identify potential targets to improve the biosynthetic pathways
24
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
40
as carbon source, as having the most potential for industrial-scale production, since it is cheaper to
41
implement. Furthermore, we also identified potential optimisation targets for all the tested pathways,
42
that can help the bio-based method to compete with the conventional process.
43
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 14, 2020. ; https://doi.org/10.1101/2020.05.13.093294doi: bioRxiv preprint

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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 [13]. 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 planets 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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[13,5]. Hence, the AAs 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
2
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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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 14, 2020. ; https://doi.org/10.1101/2020.05.13.093294doi: bioRxiv preprint

4
requires the supplementation of vitamin B
12
(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. colis 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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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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5
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 CCMs 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.
111
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 14, 2020. ; https://doi.org/10.1101/2020.05.13.093294doi: bioRxiv preprint

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