Improvement of the phosphoenolpyruvate carboxylase activity of Phaeodactylum tricornutum PEPCase 1 through protein engineering
Introduction
Because of the continuously increasing use of fossil fuels and levels of exhaust emissions since the Industrial Revolution, greenhouse gas levels in the atmosphere, especially CO2, have been increasing and contributing to global warming and climate change. Therefore, worldwide scientific efforts have attempted to mitigate CO2 accumulation. One useful solution among the various schemes for CO2 abatement is the capture and utilization of CO2 (CCU), in which waste CO2 is recycled and converted into valuable chemicals through biocatalysis [1], [2], [3].
Photosynthetic plants and microalgae have developed a physiological CO2 fixation process for a wide range of CO2 concentrations [4], [5]. Importantly, marine microalgae in particular have the capacity to fix CO2 that produces organic compounds under conditions of lower CO2 concentration. In order to fix a lower concentration of CO2 around the unicellular microalgae efficiently, these organisms have developed a carbon dioxide concentration mechanism (CCM). Carbonic anhydrase (CA; EC 4.2.1.1) is an important biocatalyst in the CCM process via its stimulation of the CO2 hydration reaction that produces bicarbonate, as well as the reverse dehydration reaction from bicarbonate to CO2 in a concentration-dependent manner [6]. Another significant biocatalysis, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; EC 4.1.1.39) is a fundamental enzyme in the carbon fixation reaction of the Calvin cycle. However, this enzyme has a relatively slower reaction rate and reacts with CO2 and O2 as substrates in a concentration-dependent manner. The oxygenase reaction rate of Rubisco increases as temperature increases, as the solubility of O2 is relatively greater than that of CO2 at higher temperatures [7]. This photorespiration reaction of Rubisco reduces the carbon fixation rate and also attenuates the efficiency and effectiveness of photosynthetic energy [8]. Therefore, Rubisco would appear to be a poor candidate for a biocatalyst contributing to CO2 abatement.
Another biocatalyst specific to CO2 fixation is phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31). PEPCase catalyzes the conversion of phosphoenolpyruvate (PEP) to oxaloacetate (OAA) using bicarbonate. Unlike Rubisco, PEPCase is not affected by, nor does it react with O2 [9]. Additionally, it has a higher affinity for CO2 than Rubisco [9]. The lower Km value of PEPCase as compared to Rubisco reflects a higher CO2 fixation rate [10]. PEPCase is also an anaplerotic enzyme that provides OAA and/or malate to replenish tricarboxylic acid (TCA) cycle intermediates in all organisms with the exception of animals and fungi [11]. The PEPCase product OAA can also be converted into succinate, a very valuable C4 chemical. Thus, PEPCase could be an important enzyme in the production of C4 chemicals and in the mitigation of CO2.
There have been many attempts to reduce CO2 levels via conversion to useful chemical feedstocks. Among these strategies, we studied the CO2-utilizing bioconversion system of CA and PEPCase purified from marine microalgae [12]. The heterogeneously expressed and purified recombinant CA and PEPCase proteins have demonstrated the possibility of CO2 capture and utilization for conversion to the C4 chemical compound OAA using integrated sequential CA-PEPCase enzyme systems. This previous study utilized the expression and purification of diatom Phaeodactylum tricornutum PEPCase 1 (PtPEPCase 1) for the CA-PEPCase bioconversion system [12]. In order to further study the CA-PEPCase system, it was imperative to prepare a large quantity of high quality PEPCase proteins via modification of the expression or purification methods.
Plants and microalgae acquired plastid via the evolutionary process of endosymbiosis. Most organellar proteins are encoded by the host genome and the precursor proteins of plastid proteins are translocated to their target organelle. These precursor proteins have an N-terminal extension sequence referred to a transit peptide. These N-terminal presequences are classified based on their targeting either to mitochondria or to plastid such as chloroplast. Despite the number and variety of transit peptides, they all share a characteristic α-helix structure as well as amphipathic sequences [13], [14]. The current study was performed to analyze the amino acid sequence of PtPEPCase 1 and to develop a technical approach that could increase protein expression levels from the heterogeneous expression system of Escherichia coli by eliminating the N-terminal presequence in the protein and purify PEPCase proteins with enhanced activity via the expression of mutated PtPEPCase 1.
Section snippets
Cloning, construction, and transformation of the mutated PtPEPCase 1
The full-length cDNA of the open reading frame encoding P. tricornutum PEPCase 1 (PtPEPCase 1, XM_002180991) was cloned as Nde I and Pst I inserts into the pCold I DNA cold-shock expression system (Takara Bio Inc., Japan) as previously described [12]. Specific primers were designed for expression of the N-terminal truncated mutated isoforms of the PtPEPCase 1 protein. Forward primers used for the m-type mutant protein (mPtPEPCase 1) and the c-type mutant protein (cPtPEPCase 1) of PtPEPCase 1
Construction of the protein expression vectors for engineering P. tricornutum PEPCase 1
In the E. coli heterologous expression system, over-expressed proteins often form insoluble aggregates referred to as inclusion bodies [17]. Results from a previous study showed that sufficient enzyme activity of PEPCase itself could be purified from soluble lysate under native conditions when the recombinant full length PtPEPCase 1 was expressed using the cold shock expression system [12]. However, the amount of partially purified PtPEPCase 1 protein was relatively low because highly induced
Conclusion
In order to improve the CA-PEPCase system, we used PtPEPCase 1 that could be prepared with high yield and quality from the E. coli heterogeneous expression system by expressing N-terminal signal peptide deleted PtPEPCase 1 in E. coli BL21-CondonPlus (DE3)-RIPL harboring the pCold I vector. The recombinant cPtPEPCase 1 with the putative chloroplast transit peptide sequence deletion was produced in high yields and its higher enzymatic activity and increased stability under different temperature
Acknowledgements
This work was supported by the National Research Foundation of Korea Grant (NRF-CIABA001-2010-0020501) and also a Korea CCS R&D Center (KCRC) (NRF-2011-0031999) funded by the Korean Government (Ministry of Science, Ict & Future Planning).
References (37)
- et al.
On carbon dioxide storage based on biomineralization strategies
Micron
(2010) Chloroplast transit peptides: structure, function and evolution
Trends Cell Biol
(2000)- et al.
A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells
Biochem Biophys Res Commun
(1999) - et al.
Expression, purification, and characterization of a highly soluble N-terminal-truncated form of the neuron-specific membrane-associated phosphoprotein SCG10
Protein Expr Purif
(1997) - et al.
Enhancement of poplar glutaredoxin expression by optimization of the cDNA sequence
Protein Expr Purif
(2002) - et al.
Predicting subcellular localization of proteins based on their N-terminal amino acid sequence
J Mol Biol
(2000) - et al.
Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic and regulatory subunit of the novel class-2 PEPC complex of vascular plants
J Biol Chem
(2009) - et al.
Phosphoenolpyruvate carboxylase of Escherichia coli. Purification and some properties
J Biol Chem
(1972) - et al.
Estimation of phosphoenolpyruvate carboxylation mediated by phosphoenolpyruvate carboxykinase (PCK) in engineered Escherichia coli having high ATP
Enzyme Microb Technol
(2013) - et al.
Utilisation of CO2 as a chemical feedstock: opportunities and challenges
Dalton Trans
(2007)
Expression and characterization of codon-optimized carbonic anhydrase from Dunaliella species for CO2 sequestration application
Appl Biochem Biotechnol
CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution
Annu Rev Plant Biol
Insights into the evolution of CCMs from comparisons with other resource acquisition and assimilation processes
Physiol Plant
Recent progresses on the genetic basis of the regulation of CO2 acquisition systems in response to CO2 concentration
Photosynth Res
Oxygen inhibition of photosynthesis: I. Temperature dependence and relation to O2/CO2 solubility ratio
Plant Physiol
Photorespiration: metabolic pathways and their role in stress protection
Philos Trans R Soc Lond B Biol Sci
The diversity and coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO2-concentrating mechanisms in algae
Can J Bot
How do algae concentrate CO2 to increase the efficiency of photosynthetic carbon fixation
Plant Physiol
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2019, Studies in Natural Products ChemistryCitation Excerpt :Similar improvements in the contents of that oxycarotenoid, i.e., up to 2.4-fold higher, were detected in transformed lines of P. tricornutum with additional copies of integrated DXS DNA cloned genes [23]. Chang and collaborators were able to express one of the CCM proteins, PEPCase_1, which produced oxaloacetate (OAA) in vitro in the presence of recombinant carbonic anhydrase in E. coli [49,50]. In a later study, Seo and collaborators [51] expressed PEPCase_1 in P. tricornutum and confirmed its mitochondrial location, a finding previously predicted by in silico analysis [33,50,51].
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2018, Algal ResearchCitation Excerpt :However, the details of this CCM process are still not completely known. One of the CCM proteins, PtPEPC1, was produced in E. coli [23,33]. In our previous study, recombinant PtPEPCase1 clearly produced OAA in vitro in the presence of recombinant carbonic anhydrase (CA), which hydrates CO2 to produce bicarbonate.
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2016, Enzyme and Microbial TechnologyCitation Excerpt :10 mL of sample from the liquid culture assay were removed and the crude enzyme was prepared as the previously described with modifications [31]. PEPC activity was measured by spectrophotometer with minor modifications [32]. Reaction mixture (10 mL) containing 50 mmol L−1 Tris–HCl, pH 8.0, 10 mmol L−1 MgCl2, 10 mmol L−1 sodium bicarbonate, 0.15 mmol L−1 NADH, 4 mmol L−1 PEP, 20% (v/v) glycerol, 10 U of malic dehydrogenase, was initiated by the addition of 200 μL crude enzyme.