ATP-regenerating System Research Articles

Use of endogenous acetate kinase for ATP regeneration system of whole-cell L-theanine production catalyzed by γ-glutamylmethylamide synthetase

Use of endogenous acetate kinase for ATP regeneration system of whole-cell L-theanine production catalyzed by γ-glutamylmethylamide synthetase

Enhanced L-theanine production through semi-rational design of γ-glutamylmethylamide synthetase from Methylovorus mays

The thermal instability of γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays has imposed limitations on its industrial applications, affecting both stability and activity at reaction temperatures. In this study, disulfide bridges were introduced through a combination of directed evolution and rational design to enhance GMAS stability. Among the variants that we generated, M12 exhibited a 1.46-fold improvement in relative enzyme activity and a 6.23-fold increase in half-life at 40℃ compared to the wild-type GMAS. Employing variant M12 under optimal conditions, we achieved the production of 645.7 mM (112.49 g/L) L-theanine with a productivity of 29.3 mM/h, from 800 mM substrate in an ATP regeneration system. Our strategy significantly enhances the biosynthesis efficiency of L-theanine by preserving the structural stability of the enzyme during the catalysis process.

Enhancing L-asparagine Production Through In Vivo ATP Regeneration System Utilizing Glucose Metabolism of Escherichia coli.

L-asparaginase synthetase, an ATP-dependent enzyme, necessitates ATP for its catalytic activity. However, the integration of L-asparaginase synthetase into industrial processes is curtailed by the prohibitive cost of ATP. To address this limitation, this study explores the construction of an efficient ATP regeneration system using the glucose metabolism of Escherichia coli, synergistically coupled with L-asparaginase synthetase catalysis. The optimal conditions for L-asparagine yield were determined in shake flasks. A total of 2.7 g/L was the highest yield achieved under specific parameters, including 0.1 mol/L of substrate, 0.2 mol/L glucose, 0.01 mol/L MgCl2 at pH 7.5, a temperature of 37 °C, and agitation at 300 r/min over 12 h. The process was then scaled to a 3-L fermenter, optimizing the addition rates of the substrate and magnesium chloride, and employing a constant glucose feed of 10 g/L/h. The scale-up process led to a significant enhancement in the production of L-asparagine. The yield of L-asparagine was increased to 38.49 g/L after 20 h of conversion, and the molar conversion rate reached 29.16%. This strategy has proven to be effective in improving the efficiency of L-asparagine production. When compared to in vitro ATP regeneration methods, this in vivo approach showcased superior efficiency and reduced costs. These findings furnish pivotal insights that may propel the enzymatic synthesis of L-asparagine toward viable industrial application.

Enhancing L-Asparagine Bioproduction Efficiency Through L-Asparagine Synthetase and Polyphosphate Kinase-Coupled Conversion and ATP Regeneration.

L-Asparagine, a crucial amino acid widely used in both food and medicine, presents pollution-related and side reaction challenges when prepared using chemical synthesis method. Although biotransformation methods offer significant advantages such as high efficiency and mild reaction conditions, they also entail increased costs due to the need for ATP supplementation. This study aimed to address the challenges associated with biopreparation of L-asparagine. Firstly, the functionality and characteristics of recombinant L-asparagine synthetase enzymes derived from Escherichia coli and Lactobacillus salivarius were evaluated to determine their practical applicability. Subsequently, recombinant expression of polyphosphate kinase from Erysipelotrichaceae bacterium was conducted. A reaction system for L-asparagine synthesis was established using a dual enzyme-coupled conversion approach. Under the optimal reaction conditions, a maximum yield of 11.67 g/L of L-asparagine was achieved, with an 88.43% conversion rate, representing a 5.03-fold increase compared to the initial conversion conditions. Notably, the initial addition of ATP was reduced to only 5.66% of the theoretical demand, indicating the effectiveness of our ATP regeneration system. These findings highlight the potential of our approach in enhancing the efficiency of L-asparagine preparation, offering promising prospects for the food and medical industries.

Engineering the thermal stability of a polyphosphate kinase by ancestral sequence reconstruction to expand the temperature boundary for an industrially applicable ATP regeneration system.

ATP-dependent energy-consuming enzymatic reactions are widely used in cell-free biocatalysis. However, the direct addition of large amounts of expensive ATP can greatly increase cost, and enzymatic production is often difficult to achieve as a result. Although a polyphosphate kinase (PPK)-polyphosphate-based ATP regeneration system has the potential to solve this challenge, the generally poor thermal stability of PPKs limits the widespread use of this method. In this paper, we evaluated the thermal stability of a PPK from Sulfurovum lithotrophicum (SlPPK2). After directed evolution and computation-supported design, we found that SlPPK2 is very recalcitrant and cannot acquire beneficial mutations. Inspired by the usually outstanding stability of ancestral enzymes, we reconstructed the ancestral sequence of the PPK family and used it as a guide to construct three heat-stable variants of SlPPK2, of which the L35F/T144S variant has a half-life of more than 14 h at 60°C. Molecular dynamics simulations were performed on all enzymes to analyze the reasons for the increased thermal stability. The results showed that mutations at these two positions act synergistically from the interior and surface of the protein, leading to a more compact structure. Finally, the robustness of the L35F/T144S variant was verified in the synthesis of nucleotides at high temperature. In practice, the use of this high-temperature ATP regeneration system can effectively avoid byproduct accumulation. Our work extends the temperature boundary of ATP regeneration and has great potential for industrial applications.IMPORTANCEATP regeneration is an important basic applied study in the field of cell-free biocatalysis. Polyphosphate kinase (PPK) is an enzyme tool widely used for energy regeneration during enzymatic reactions. However, the thermal stability of the PPKs reported to date that can efficiently regenerate ATP is usually poor, which greatly limits their application. In this study, the thermal stability of a difficult-to-engineer PPK from Sulfurovum lithotrophicum was improved, guided by an ancestral sequence reconstruction strategy. The optimal variant has a 4.5-fold longer half-life at 60°C than the wild-type enzyme, thus enabling the extension of the temperature boundary for ATP regeneration. The ability of this variant to regenerate ATP was well demonstrated during high-temperature enzymatic production of nucleotides.

Enhanced theanine production with reduced ATP supply by alginate entrapped Escherichia coli co-expressing γ-glutamylmethylamide synthetase and polyphosphate kinase

L-theanine is an amino acid with a unique flavor and many therapeutic effects. Its enzymatic synthesis has been actively studied and γ-Glutamylmethylamide synthetase (GMAS) is one of the promising enzymes in the biological synthesis of theanine. However, the theanine biosynthetic pathway with GMAS is highly ATP-dependent and the supply of external ATP was needed to achieve high concentration of theanine production. As a result, this study aimed to investigate polyphosphate kinase 2 (PPK2) as ATP regeneration system with hexametaphosphate. Furthermore, the alginate entrapment method was employed to immobilize whole cells containing both gmas and ppk2 together resulting in enhanced reusability of the theanine production system with reduced supply of ATP. After immobilization, theanine production was increased to 239 mM (41.6 g/L) with a conversion rate of 79.7% using 15 mM ATP and the reusability was enhanced, maintaining a 100% conversion rate up to the fifth cycles and 60% of conversion up to eighth cycles. It could increase long-term storage property for future uses up to 35 days with 75% activity of initial activity. Overall, immobilization of both production and cofactor regeneration system could increase the stability and reusability of theanine production system.

Novel In Vitro Multienzyme Cascade for Efficient Synthesis of d-Tagatose from Sucrose

d-Tagatose is a rare sugar with low calories, and is extensively used in food, beverage, and drug additives. In this study, an in vitro multienzyme cascade route for d-tagatose synthesis from sucrose (MCTS) was designed, which contains five enzymes (sucrose phosphorylase, fructokinase, d-fructose 6-phosphate 4-epimerase, d-tagatose 6-phosphate phosphatase, and polyphosphate kinase). The whole MCTS route comprised a sucrose phosphorylation reaction, and a phosphorylation–dephosphorylation reaction coupled with an ATP regeneration system. After optimization, the conversion of d-tagatose from 10 mM sucrose reached 82.3%. At an elevated sucrose concentration of 50 mM, 72.4% of d-tagatose conversion and 0.27 g·L–1·h−1 of space–time yield were obtained. Furthermore, ADP consumption decreased to 1% of the sucrose concentration after introducing the ATP regeneration system. The MCTS strategy is an efficient and cost-effective approach for d-tagatose production.

Reaction Engineering and Comparison of Electroenzymatic and Enzymatic ATP Regeneration Systems

AbstractInvited for this issue's Front Cover are the group of Bioprocess Engineering at TU Dortmund University, Department of Chemistry at Johannes Gutenberg University Mainz, and the Biotechnology Group at Constructor University. The front cover illustrates the electroenzymatic regeneration of ATP. The electrons of an enzymatically catalyzed redox reaction necessary for the ATP regeneration cascade are transferred to an electrode mediated by ferrocenyl‐methanol. Regenerative and green energy can therefore be used to drive the reaction. Cover design by Tobias Prenzel and Finn Moeller. Read the full text of the Research Article at 10.1002/celc.202300332.

Front Cover: Reaction Engineering and Comparison of Electroenzymatic and Enzymatic ATP Regeneration Systems (ChemElectroChem 22/2023)

Front Cover: Reaction Engineering and Comparison of Electroenzymatic and Enzymatic ATP Regeneration Systems (ChemElectroChem 22/2023)

Novel multienzyme cascade for efficient synthesis of d-allulose from inexpensive sucrose

d-Allulose is one of the most common rare sugars with low calorie and is extensively applied in the food, cosmetic, and pharmaceutical fields. Here, a novel in vitro multienzyme cascade route (MCAS) was designed to produce d-allulose from sucrose. The MCAS route comprised three modules (sucrose phosphorolysis, phosphorylation–dephosphorylation, and ATP regeneration), including five enzymes (sucrose phosphorylase, fructokinase, d-allulose 6-phosphate 3-epimerase, d-allulose 6-phosphatephosphatase, and polyphosphate kinase). As a rate-limiting enzyme, d-allulose 6-phosphate 3-epimerase from Pantoea sp. was semi-rationally engineered, and variant Y143F led to 88.8% yield of d-allulose from 10 mM sucrose using one-pot strategy. ATP consumption was reduced to 5% of the sucrose concentration after introducing ATP-regeneration system. At elevated sucrose concentration of 100 mM by two-step strategy, a d-allulose yield of 70.4% was achieved with a space-time yield of 1.06 g L−1·h−1. This study provides an efficient and cost-effective approach for producing d-allulose from sucrose.

Reaction Engineering and Comparison of Electroenzymatic and Enzymatic ATP Regeneration Systems

AbstractAdenosine‐5’‐triphosphate (ATP) plays a crucial role in many biocatalytic reactions and its regeneration can influence the performance of a related enzymatic reaction significantly. Here, we established electrochemically coupled ATP regeneration by pyruvate oxidase and acetate kinase (ACK) for the phosphorylation of mevalonate catalyzed by mevalonate kinase. A yield of 84 % for the product mevalonate phosphate was reached and a total turnover number for ADP of 68. These metrics are promising for the development of an economic feasible bioprocess and surpass many other enzymatic ATP regeneration systems. A comparison was made to polyphosphate kinases (PPKs), ACK, pyruvate kinase, and creatine kinase in terms of the phosphate donor properties and biocatalytic metrics of exemplary reactions. Furthermore, our system was expanded by a PPK that enables the phosphorylation of AMP, which can broaden the spectrum of applications even further.

Fermentative Production of L-Theanine in Escherichia coli via the Construction of an Adenosine Triphosphate Regeneration System

Theanine is a non-protein amino acid that is highly represented in tea plants and is one of the delicious ingredients in tea. In recent years, the global market demand for theanine has continued to rise, and the industry has developed rapidly. Here, we designed and constructed a promising pathway in Escherichia coli to produce L-theanine. This biosynthesis pathway employs four enzymes to achieve the production of L-theanine. This route involves the co-expression of four functional enzymes: γ-glutamylmethylamide synthetase (GMAS) from Methyloversatilis universalis, polyphosphate kinase (PPK) from E. coli, alanine transaminase from Bacillus subtilis (BsAld), and alanine decarboxylase from Camellia sinensis (CsAlaDC). Polyphosphate kinase from Escherichia coli was overexpressed in E. coli FD02, constructing an ATP regeneration system that increased the titer of L-theanine by 13.4% compared to E. coli FD01. A titer of 334 mg/L of L-theanine was produced via engineering strain FD03 in shake flasks. Moreover, glutamine permease from Saccharomyces cereviside (GNP1) was overexpressed in E. coli FD04, and the L-theanine titer increased by 14.7%. Finally, 2.9 g/L of L-theanine was obtained via FD04 in a 1 L bioreactor. In addition, the molecular docking results indicated that L-glutamate could bind to the hydrophobic cavity of GMAS due to the formation of hydrogen bonds and hydrophobic interactions with the surrounding amino acid residues.

Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPKK81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPKK81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

A high-throughput dual system to screen polyphosphate kinase mutants for efficient ATP regeneration in L-theanine biocatalysis

ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from Cytophaga hutchinsonii (ChPPK) had the best catalytic activity for the substrates ADP and polyP6. An extracellular–intracellular dual system was constructed to high-throughput screen for better catalytic activity of ChPPK mutants. Finally, the specific activity of ChPPKD82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL ChPPKD82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL ChPPKD82N-K103E, which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably.

Improved Synthesis of Deoxyadenosine Triphosphate by Saccharomyces cerevisiae Using an Efficient ATP Regeneration System: Optimization of Response Surface Analysis.

Deoxyadenosine triphosphate (dATP) is an important biochemical molecule. In this paper, the synthesis of dATP from deoxyadenosine monophosphate (dAMP), catalyzed by Saccharomyces cerevisiae, was studied. By adding chemical effectors, an efficient ATP regeneration and coupling system was constructed to achieve efficient synthesis of dATP. Factorial and response surface designs were used to optimize process conditions. Optimal reaction conditions were as follows: dAMP 1.40 g/L, glucose 40.97 g/L, MgCl2·6H2O 4.00 g/L, KCl 2.00 g/L, NaH2PO4 31.20 g/L, yeast 300.00 g/L, ammonium chloride 0.67 g/L, acetaldehyde 11.64 mL/L, pH 7.0, temperature 29.6 °C. Under these conditions, the substrate conversion was 93.80% and the concentration of dATP in the reaction system was 2.10 g/L, which was 63.10% higher than before optimization, and the concentration of product was 4 times higher than before optimization. The effects of glucose, acetaldehyde, and temperature on the accumulation of dATP were analyzed.

Irreversible biosynthesis of D-Allulose from D-glucose in Escherichia coli through fine-tuning of carbon flux and cofactor regeneration engineering.

As a rare hexose with low calories and various physiological functions, D-allulose has drawn increasing attention. The current industrial production of D-allulose from D-fructose or D-glucose is achieved via epimerization based on the Izumoring strategy; however, the inherent reaction equilibrium during reversible reaction limits its high conversion yield. Although the conversion of D-fructose to D-allulose could be enhanced via phosphorylation-dephosphorylation mediated by metabolic engineering, biomass reduction and byproduct accumulation remain a largely unresolved issue. After modifying the glycolytic pathway of Escherichia coli and optimizing the whole-cell reaction condition, the engineered strain produced 7.57 ± 0.61 g L-1 D-allulose from 30 g L-1 D-glucose after 24 h of catalysis. By developing an ATP regeneration system for enhanced substrate phosphorylation, the cell growth inhibition was alleviated and D-allulose production increased by 55.3% to 11.76 ± 0.58 g L-1 (0.53 g g-1 ). Fine-tuning of carbon flux caused a 48% reduction in D-fructose accumulation to 1.47 ± 0.15 g L-1 . After implementing fed-batch co-substrate strategy, the D-allulose titer reached 15.80 ± 0.31 g L-1 (0.62 g g-1 ) with a D-glucose conversion rate of 84.8%. This study presented a novel strategy for high-yield D-allulose production from low-cost substrate. This article is protected by copyright. All rights reserved.

Engineering Streptomyces albulus to enhance ε-poly-L-lysine production by introducing a polyphosphate kinase-mediated ATP regeneration system

Backgroundε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL biosynthesis, a large amount of ATP is used for the polymerization of L-lysine. A shortage of intracellular ATP is one of the major factors limiting the increase in ε-PL production. In previous studies, researchers have mainly tried to increase the oxygen supply to enhance intracellular ATP levels to improve ε-PL production, which can be achieved through the use of two-stage dissolved oxygen control, oxygen carriers, heterologous expression of hemoglobin, and supplementation with auxiliary energy substrates. However, the enhancement of the intracellular ATP supply by constructing an ATP regeneration system has not yet been considered.ResultsIn this study, a polyphosphate kinase (PPK)-mediated ATP regeneration system was developed and introduced into S. albulus to successfully improve ε-PL production. First, polyP:AMP phosphotransferase (PAP) from Acinetobacter johnsonii was selected for catalyzing the conversion of AMP into ADP through an in vivo test. Moreover, three PPKs from different microbes were compared by in vitro and in vivo studies with respect to catalytic activity and polyphosphate (polyP) preference, and PPK2Bcg from Corynebacterium glutamicum was used for catalyzing the conversion of ADP into ATP. As a result, a recombinant strain PL05 carrying coexpressed pap and ppk2Bcg for catalyzing the conversion of AMP into ATP was constructed. ε-PL production of 2.34 g/L was achieved in shake-flask fermentation, which was an increase of 21.24% compared with S. albulus WG608; intracellular ATP was also increased by 71.56%. In addition, we attempted to develop a dynamic ATP regulation route, but the result was not as expected. Finally, the conditions of polyP6 addition were optimized in batch and fed-batch fermentations, and the maximum ε-PL production of strain PL05 in a 5-L fermenter was 59.25 g/L by fed-batch fermentation, which is the highest ε-PL production reported in genetically engineered strains.ConclusionsIn this study, we proposed and developed a PPK-mediated ATP regeneration system in S. albulus for the first time and significantly enhanced ε-PL production. The study provides an efficient approach to improve the production of not only ε-PL but also other ATP-driven metabolites.

Efficient synthesis of Ala-Tyr by L-amino acid ligase coupled with ATP regeneration system.

The multi-enzyme coupling reaction system has become a promising biomanufacturing platform for biochemical production. Tyr is an essential amino acid, but the limited solubility restricts its use. Tyrosyl dipeptide has been paid more attention due to its higher solubility. In this study, an efficient enzymatic cascade of Ala-Tyr synthesis was developed by a L-amino acid ligase together with polyphosphate kinase (PPK). Two L-amino acid ligases from Bacillus subtilis and Bacillus pumilus were selected and applied for Ala-Tyr synthesis. The L-amino acid ligase from B. subtilis (Bs) was selected and coupled with the PPK from Sulfurovum lithotrophicum (PPKSL) for regenerating ATP to produce Ala-Tyr in one pot. In the optimization system, 40.1mM Ala-Tyr was produced within 3h due to efficient ATP regeneration with hexametaphosphate (PolyP(6)) as the phosphate donor. The molar yield was 0.89mol/mol based on the substrates added, while the productivity of Ala-Tyr achieved 13.4mM/h, which were the highest yield and productivity ever reported about Ala-Tyr synthesis with L-amino acid ligase.

Expression of polyphosphate kinase from Sphingobacterium siyangensis and its application in ATP regeneration system

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg2+), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.

Evolutionary approach for improved proton pumping activity of heterologous rhodopsin expressed in Escherichia coli

A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established. In this study, we attempted to develop an evolutionary engineering method to improve the proton-pumping activity of rhodopsins. We first introduced random mutations into delta-rhodopsin (dR) from Haloterrigena turkmenica using error-prone PCR to generate approximately 7000 Escherichia coli strains carrying the mutant dR genes. Rhodopsin-expressing E.coli with enhanced proton pumping activity have significantly increased survival rates in prolonged saline water. Considering this, we enriched the mutant E.coli cells with higher proton pumping rates by selecting populations able to survive starvation under 50μmolm-2s-1at 37°C. As a result, we successfully identified two strains, in which proton pumping activity was enhanced two-fold by heterologous expression in E.coli in comparison to wild-type strains. The combined approach of survival testing using saline water and evolutionary engineering methods used in this study will contribute greatly to the discovery of a novel rhodopsin with improved proton pumping activity. This will facilitate the utilization of rhodopsin in industrial applications.