The Catalytic Role of RuBisCO for in situ CO2 Recycling in Escherichia coli

Ju Jiun Pang; Jong Shik Shin; Si Yu Li

doi:10.3389/fbioe.2020.543807

The Catalytic Role of RuBisCO for in situ CO₂ Recycling in Escherichia coli

Ju Jiun Pang, Jong Shik Shin, Si Yu Li

Research output: Contribution to journal › Article › peer-review

4 Citations (Scopus)

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme responsible for biological CO₂ assimilation. RuBisCO can be heterologously expressed in Escherichia coli so that glucose and CO₂ are co-metabolized to achieve high mixotrophic metabolite production, where the theoretical yield of mixotrophic metabolite production is 2.4 mol_(ethanol₊_acetate₊_pyruvate)/mol_glucose. However, RuBisCO is known for its low k_cat and for forming inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates, yet the inhibited form of RuBisCO can be reversed by RuBisCO activase (Rca). In this study, RuBisCO forms I and II were cloned and expressed in Escherichia coli for in situ CO₂ recycling, where CO₂ produced during glucose fermentation was recycled and co-metabolized with the glucose. In addition, forms I and II RuBisCO activases were co-expressed with RuBisCO in E. coli to determine their in vivo effects on in situ CO₂ recycling. Form I RuBisCO activase (Rca1) was co-expressed with form I RuBisCO and form II RuBisCO activase (Rca2) was co-expressed with form II RuBisCO. The results showed that both form I and form II RuBisCO exhibit comparable activities in E. coli and generated similar levels of in situ CO₂ recycling. A significant increase in the total metabolite yield from 1.5 ± 0.1 to 2.2 ± 0.1 mol_(ethanol₊_acetate₊_pyruvate)/mol_glucose occurred when Rca2 was co-expressed with form II RuBisCO. Meanwhile, the total metabolite yield increased from 1.7 ± 0.1 to 2.0 ± 0.1 mol_(ethanol₊_acetate₊_pyruvate)/mol_glucose when Rca1 was co-expressed with form I RuBisCO. This data suggests that both forms I and II RuBisCO are subject to in vivo RuBP inhibition yet can be relieved by the co-expression of Rca. Interestingly, it is suggested that the in vivo RuBP inhibition of form II RuBisCO can be more easily reversed compared to form I. When the catalytic power of RuBisCO is maintained by Rca, the high activity of phosphoribulokinase (Prk) plays an important role in directing glucose to the RuBisCO-based engineered pathway and fermentation yields of 2.1–2.3 mol_(ethanol₊_acetate₊_pyruvate)/mol_glucose can be obtained. This study is the first to demonstrate that in vivo RuBP inhibition of RuBisCO can be a bottleneck for in situ CO₂ recycling in E. coli.

Original language	English
Article number	543807
Journal	Frontiers in Bioengineering and Biotechnology
Volume	8
DOIs	https://doi.org/10.3389/fbioe.2020.543807
Publication status	Published - 2020 Nov 30

Bibliographical note

Funding Information:
The authors thank Prof. Oliver Mueller-Cajar of Biological Sciences School at Nanyang Technological University, Singapore for his generous gifts of pET30bAfcbbLS, pHueAfcbbM, pHueAfcbbQ1, pHueAfcbbQ2, pBAD33UbAfcbbO1, and pBAD33UbAfcbbO2. The authors thank Prof. Richard Parnas of Chemical & Biomolecular Engineering at the University of Connecticut, United States for proofreading the manuscript. Funding. This work was funded by the Ministry of Science and Technology Taiwan, MOST-106-2221-E-005-058-MY3, MOST-108-2628-E-005-002-MY3, and the Ministry of Education, Taiwan, R.O.C. under the Higher Education Sprout Project.

Funding Information:
This work was funded by the Ministry of Science and Technology Taiwan, MOST-106-2221-E-005-058-MY3, MOST-108-2628-E-005-002-MY3, and the Ministry of Education, Taiwan, R.O.C. under the Higher Education Sprout Project.

Publisher Copyright:
© Copyright © 2020 Pang, Shin and Li.

All Science Journal Classification (ASJC) codes

Biotechnology
Bioengineering
Histology
Biomedical Engineering

Access to Document

10.3389/fbioe.2020.543807

Cite this

@article{61e754866bd24002b35e2af84db97fd6,

title = "The Catalytic Role of RuBisCO for in situ CO2 Recycling in Escherichia coli",

abstract = "Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme responsible for biological CO2 assimilation. RuBisCO can be heterologously expressed in Escherichia coli so that glucose and CO2 are co-metabolized to achieve high mixotrophic metabolite production, where the theoretical yield of mixotrophic metabolite production is 2.4 mol(ethanol+acetate+pyruvate)/molglucose. However, RuBisCO is known for its low kcat and for forming inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates, yet the inhibited form of RuBisCO can be reversed by RuBisCO activase (Rca). In this study, RuBisCO forms I and II were cloned and expressed in Escherichia coli for in situ CO2 recycling, where CO2 produced during glucose fermentation was recycled and co-metabolized with the glucose. In addition, forms I and II RuBisCO activases were co-expressed with RuBisCO in E. coli to determine their in vivo effects on in situ CO2 recycling. Form I RuBisCO activase (Rca1) was co-expressed with form I RuBisCO and form II RuBisCO activase (Rca2) was co-expressed with form II RuBisCO. The results showed that both form I and form II RuBisCO exhibit comparable activities in E. coli and generated similar levels of in situ CO2 recycling. A significant increase in the total metabolite yield from 1.5 ± 0.1 to 2.2 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose occurred when Rca2 was co-expressed with form II RuBisCO. Meanwhile, the total metabolite yield increased from 1.7 ± 0.1 to 2.0 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose when Rca1 was co-expressed with form I RuBisCO. This data suggests that both forms I and II RuBisCO are subject to in vivo RuBP inhibition yet can be relieved by the co-expression of Rca. Interestingly, it is suggested that the in vivo RuBP inhibition of form II RuBisCO can be more easily reversed compared to form I. When the catalytic power of RuBisCO is maintained by Rca, the high activity of phosphoribulokinase (Prk) plays an important role in directing glucose to the RuBisCO-based engineered pathway and fermentation yields of 2.1–2.3 mol(ethanol+acetate+pyruvate)/molglucose can be obtained. This study is the first to demonstrate that in vivo RuBP inhibition of RuBisCO can be a bottleneck for in situ CO2 recycling in E. coli.",

author = "Pang, {Ju Jiun} and Shin, {Jong Shik} and Li, {Si Yu}",

note = "Funding Information: The authors thank Prof. Oliver Mueller-Cajar of Biological Sciences School at Nanyang Technological University, Singapore for his generous gifts of pET30bAfcbbLS, pHueAfcbbM, pHueAfcbbQ1, pHueAfcbbQ2, pBAD33UbAfcbbO1, and pBAD33UbAfcbbO2. The authors thank Prof. Richard Parnas of Chemical & Biomolecular Engineering at the University of Connecticut, United States for proofreading the manuscript. Funding. This work was funded by the Ministry of Science and Technology Taiwan, MOST-106-2221-E-005-058-MY3, MOST-108-2628-E-005-002-MY3, and the Ministry of Education, Taiwan, R.O.C. under the Higher Education Sprout Project. Funding Information: This work was funded by the Ministry of Science and Technology Taiwan, MOST-106-2221-E-005-058-MY3, MOST-108-2628-E-005-002-MY3, and the Ministry of Education, Taiwan, R.O.C. under the Higher Education Sprout Project. Publisher Copyright: {\textcopyright} Copyright {\textcopyright} 2020 Pang, Shin and Li.",

year = "2020",

month = nov,

day = "30",

doi = "10.3389/fbioe.2020.543807",

language = "English",

volume = "8",

journal = "Frontiers in Bioengineering and Biotechnology",

issn = "2296-4185",

publisher = "Frontiers Media S. A.",

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TY - JOUR

T1 - The Catalytic Role of RuBisCO for in situ CO2 Recycling in Escherichia coli

AU - Pang, Ju Jiun

AU - Shin, Jong Shik

AU - Li, Si Yu

N1 - Funding Information: The authors thank Prof. Oliver Mueller-Cajar of Biological Sciences School at Nanyang Technological University, Singapore for his generous gifts of pET30bAfcbbLS, pHueAfcbbM, pHueAfcbbQ1, pHueAfcbbQ2, pBAD33UbAfcbbO1, and pBAD33UbAfcbbO2. The authors thank Prof. Richard Parnas of Chemical & Biomolecular Engineering at the University of Connecticut, United States for proofreading the manuscript. Funding. This work was funded by the Ministry of Science and Technology Taiwan, MOST-106-2221-E-005-058-MY3, MOST-108-2628-E-005-002-MY3, and the Ministry of Education, Taiwan, R.O.C. under the Higher Education Sprout Project. Funding Information: This work was funded by the Ministry of Science and Technology Taiwan, MOST-106-2221-E-005-058-MY3, MOST-108-2628-E-005-002-MY3, and the Ministry of Education, Taiwan, R.O.C. under the Higher Education Sprout Project. Publisher Copyright: © Copyright © 2020 Pang, Shin and Li.

PY - 2020/11/30

Y1 - 2020/11/30

N2 - Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme responsible for biological CO2 assimilation. RuBisCO can be heterologously expressed in Escherichia coli so that glucose and CO2 are co-metabolized to achieve high mixotrophic metabolite production, where the theoretical yield of mixotrophic metabolite production is 2.4 mol(ethanol+acetate+pyruvate)/molglucose. However, RuBisCO is known for its low kcat and for forming inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates, yet the inhibited form of RuBisCO can be reversed by RuBisCO activase (Rca). In this study, RuBisCO forms I and II were cloned and expressed in Escherichia coli for in situ CO2 recycling, where CO2 produced during glucose fermentation was recycled and co-metabolized with the glucose. In addition, forms I and II RuBisCO activases were co-expressed with RuBisCO in E. coli to determine their in vivo effects on in situ CO2 recycling. Form I RuBisCO activase (Rca1) was co-expressed with form I RuBisCO and form II RuBisCO activase (Rca2) was co-expressed with form II RuBisCO. The results showed that both form I and form II RuBisCO exhibit comparable activities in E. coli and generated similar levels of in situ CO2 recycling. A significant increase in the total metabolite yield from 1.5 ± 0.1 to 2.2 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose occurred when Rca2 was co-expressed with form II RuBisCO. Meanwhile, the total metabolite yield increased from 1.7 ± 0.1 to 2.0 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose when Rca1 was co-expressed with form I RuBisCO. This data suggests that both forms I and II RuBisCO are subject to in vivo RuBP inhibition yet can be relieved by the co-expression of Rca. Interestingly, it is suggested that the in vivo RuBP inhibition of form II RuBisCO can be more easily reversed compared to form I. When the catalytic power of RuBisCO is maintained by Rca, the high activity of phosphoribulokinase (Prk) plays an important role in directing glucose to the RuBisCO-based engineered pathway and fermentation yields of 2.1–2.3 mol(ethanol+acetate+pyruvate)/molglucose can be obtained. This study is the first to demonstrate that in vivo RuBP inhibition of RuBisCO can be a bottleneck for in situ CO2 recycling in E. coli.

AB - Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme responsible for biological CO2 assimilation. RuBisCO can be heterologously expressed in Escherichia coli so that glucose and CO2 are co-metabolized to achieve high mixotrophic metabolite production, where the theoretical yield of mixotrophic metabolite production is 2.4 mol(ethanol+acetate+pyruvate)/molglucose. However, RuBisCO is known for its low kcat and for forming inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates, yet the inhibited form of RuBisCO can be reversed by RuBisCO activase (Rca). In this study, RuBisCO forms I and II were cloned and expressed in Escherichia coli for in situ CO2 recycling, where CO2 produced during glucose fermentation was recycled and co-metabolized with the glucose. In addition, forms I and II RuBisCO activases were co-expressed with RuBisCO in E. coli to determine their in vivo effects on in situ CO2 recycling. Form I RuBisCO activase (Rca1) was co-expressed with form I RuBisCO and form II RuBisCO activase (Rca2) was co-expressed with form II RuBisCO. The results showed that both form I and form II RuBisCO exhibit comparable activities in E. coli and generated similar levels of in situ CO2 recycling. A significant increase in the total metabolite yield from 1.5 ± 0.1 to 2.2 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose occurred when Rca2 was co-expressed with form II RuBisCO. Meanwhile, the total metabolite yield increased from 1.7 ± 0.1 to 2.0 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose when Rca1 was co-expressed with form I RuBisCO. This data suggests that both forms I and II RuBisCO are subject to in vivo RuBP inhibition yet can be relieved by the co-expression of Rca. Interestingly, it is suggested that the in vivo RuBP inhibition of form II RuBisCO can be more easily reversed compared to form I. When the catalytic power of RuBisCO is maintained by Rca, the high activity of phosphoribulokinase (Prk) plays an important role in directing glucose to the RuBisCO-based engineered pathway and fermentation yields of 2.1–2.3 mol(ethanol+acetate+pyruvate)/molglucose can be obtained. This study is the first to demonstrate that in vivo RuBP inhibition of RuBisCO can be a bottleneck for in situ CO2 recycling in E. coli.

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