Construction of deoxyriboaldolase-overexpressing Escherichia coli and its application to 2-deoxyribose 5-phosphate synthesis from glucose and acetaldehyde for 2'-deoxyribonucleoside production

The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2'-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.     

One-pot Microbial Synthesis of 2′-deoxyribonucleoside from Glucose, Acetaldehyde, and a Nucleobase

A one-pot enzymatic synthesis of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker’s yeast (Saccharomyces cerevisiae) generated ATP which was used to produce d-glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The d-glyceraldehyde 3-phosphate produced was transformed to 2′-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2′-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2′-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.     

Biochemical retrosynthesis of 2′-deoxyribonucleosides from glucose, acetaldehyde, and a nucleobase

2′-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2′-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2′-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2′-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker’s yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5′-triphosphate generated from adenosine 5′-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via d-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2′-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2′-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2′-deoxyribonucleoside synthesis. 2′-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.     

Amino acid-mediated aldolase immobilisation for enhanced catalysis and thermostability

To improve the properties of the immobilised 2-deoxy-d-ribose-5-phosphate aldolase (DERA), unreacted functional groups on support surface were blocked with amino acids. The relative activities of the immobilised enzyme were 144.7 and 141.9% when the post-immobilisation modification was done with Arg and Phe, respectively. The residual activity of immobilised DERA after heating at 60 °C for 120 min was 65.1% when Phe and Val were used as the blocking amino acids, a 2.0- and 2.87-fold increase over that of the immobilised (no post-immobilisation blocking) and free DERA. Immobilised DERA maintained maximal activity in 2-deoxyribose-5-phosphate (DR5P) synthesis up to 600 mM of acetaldehyde, which was much higher than the amount of acetaldehyde tolerated by free enzyme (300 mM). This superior resistance to high acetaldehyde concentrations would accelerate the DR5P reaction by shifting the reaction equilibrium towards the product. The results from this study suggest that the novel immobilised DERA may be useful for industrial applications.     

Efficient synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose by using new 2-deoxy-d-ribose-5-phosphate aldolase from Streptococcus suis with moderate activity and aldehyde tolerance

The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg⁻¹, K_M of 0.8 mM, and V_max of 32.9 μmol min⁻¹ mg⁻¹ toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N backbone and sidechain resonance assignments of a monomeric variant of <Emphasis Type="Italic">E. coli</Emphasis> deoxyribose-5-phosphate aldolase

Deoxyribose-5-phosphate aldolase (DERA) catalyses the reversible conversion of 2-deoxyribose-5-phosphate (dR5P) into glyceraldehyde-3-phosphate (G3P) and acetaldehyde. For industrial applications, this enzyme is used in organic synthesis for aldol reactions between acetaldehyde as a donor and a wide range of aldehydes as acceptors. Here, we present a near complete set of sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments of a 28 kDa monomeric variant of the Escherichia coli DERA. These assignments provide the basis for ongoing structural and dynamic analysis of DERA substrate specificity.     

Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-d-ribose-5-phosphate aldolase

Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25 degrees C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25 degrees C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 A. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.     

Cloning, expression, and characterization of a new deoxyribose 5-phosphate aldolase from Yersinia sp. EA015

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The specific activity was 137 μmol/min/mg. The Michaelis constant (k_m value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 °C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.     

Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system

A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na₂, 0.5 mM ATP, 5 mM Mg²⁺, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.     

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli

Glutathione (GSH) is one of the most ubiquitous non-protein thiols that is involved in numerous cellular activities. The gene coding for a novel bifunctional enzyme catalyzing the reaction for glutathione synthesis, gshF, was cloned from Streptococcus thermophilus SIIM B218 and expressed in Escherichia coli JM109. In the presence of the precursor amino acids and ATP, the induced cells of E. coli JM109 (pTrc99A-gshF) could accumulate 10.3 mM GSH in 5 h. The S. thermophilus GshF was insensitive to feedback inhibition caused by GSH even at 20 mM. At elevated concentrations of the precursor amino acids and ATP, E. coli JM109 (pTrc99A-gshF) produced 36 mM GSH with a molar yield of 0.9 mol/mol based on added cysteine and of 0.45 mol/mol based on added ATP. When ATP was replaced with glucose, E. coli JM109 (pTrc99A-gshF) produced 7 mM in 3 h. Saccharomyces cerevisiae was used to generate ATP for GSH production. In the presence of glucose and the pmr1 mutant of S. cerevisiae BY4742, JM109 (pTrc99A-gshF) produced 33.9 mM GSH in 12 h with a yield of 0.85 mol/mol based on added l-cysteine. It is shown that the S. thermophilus GshF can be successfully used for GSH production.     

Antibacterial Activity of l-Lysine α-Oxidase against rec+ and rec− Strains of Bacillus subtilis

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Production of d-ribose by metabolically engineered Escherichia coli

Escherichia coli was metabolically engineered for the production of d-ribose, a functional five-carbon sugar, from xylose. For the accumulation of d-ribose, two genes of transketolase catalyzing the conversion of d-ribose-5-phosphate to sedoheptulose-7-phosphate in pentose phosphate pathway were disrupted to create a transketolase-deficient E. coli SGK013. In batch fermentation, E. coli SGK013 grew by utilizing glucose and then started to produce d-ribose from xylose after glucose depletion. E. coli SGK013 produced 0.75 g/L of d-ribose, which was identical to the standard d-ribose as confirmed by HPLC and LC/MS analyses. To improve D-ribose production, the ptsG gene encoding the glucose-specific IICB component was disrupted additionally, resulting in the construction of E. coli SGK015. The carbon catabolite repression-negative E. coli SGK015 utilized xylose and glucose simultaneously and produced up to 3.75 g/L of d-ribose, which is a 5-fold improvement compared to that of E. coli SGK013.     

Engineered,highly productive biosynthesis of artificial,lactonized statin side-chain building blocks: The hidden potential of Escherichia coli unleashed

We have recently reported the development of an efficient, whole-cell process for chemoenzymatic production of key chiral intermediates in statin synthesis by employing high-density Escherichia coli culture with the overexpressed deoxyribose-5-phosphate aldolase (DERA). The optically pure, 6-substituted cyclic hemiacetals can be used for the synthesis of atorvastatin, rosuvastatin and pitavastatin using further chemical steps. All of the synthetic routes established to date begin with a regiospecific oxidation of these lactol intermediates into the corresponding lactones, followed by several steps yielding 6-substituted, open-chain or lactonized derivatives which can be coupled by various approaches with the heterocyclic part of the statin molecule. Here we report for the first time the use of PQQ-dependent glucose dehydrogenases for a highly efficient, regioselective oxidation of artificial, derivatized aldohexoses, more specifically, the statin lactol intermediates. First, PQQ-dependent dehydrogenases of both soluble and membrane-bound type were characterized for their activity toward various DERA-derived lactols. Further, we describe a highly productive whole-cell system for oxidation of these 2,4-dideoxyaldopyranoses using a PQQ-dependent glucose dehydrogenase (Gcd) overexpressed in E. coli while taking advantage of the respiratory chain as the mediator of the electron transfer to oxygen. Finally, a two-step artificial biosynthetic pathway was developed by unleashing the intrinsic genetic potential of E. coli. The combined overexpression of the endogenous DERA and the membrane-bound, PQQ-dependent glucose dehydrogenase, the latter being coupled to the respiratory chain, allows direct biosynthesis of 6-substituted lactones in a highly productive, high-yield, cost-effective and industrially scalable process.     

2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.

     

Microbial production of 2-deoxyribose 5-phosphate from acetaldehyde and triosephosphate for the synthesis of 2'-deoxyribonucleosides

2-Deoxyribose 5-phosphate was produced from acetaldehyde and dihydroxyacetone phosphate via D-glyceraldehyde 3-phosphate by Klebsiella pneumoniae B-4-4 through deoxyriboaldolase- and triosephosphate isomerase-catalyzing reactions. Under the optimum conditions, 98.7 mM 2-deoxyribose 5-phosphate was produced from 200 mM acetaldehyde and 117 mM dihydroxyacetone phosphate in 2 h with a molar yield of 84%. The 2-deoxyriobse 5-phosphate produced was directly transformed to 2'-deoxyribonucleoside by phosphopentomutase- and nucleoside phosphorylase-catalyzing reactions.     

Cloning and characterisation of a new 2-deoxy-D-ribose-5-phosphate aldolase from Rhodococcus erythropolis

A new 2-deoxy-D-ribose-5-phoshate aldolase (DERA) gene was cloned from Rhodococcus erythropolis strain DSM 311, recombinantly expressed in Escherichia coli, and purified via affinity chromatography which yielded a homo-dimeric enzyme of 44.3 kDa as apparent by size exclusion chromatography. To characterise the enzyme, investigations about pH and temperature tolerance, stability, as well as analyses on resistance to organic solvents and acetaldehyde were performed. In addition, kinetic constants of the new DERA(RE) were compared to respective values of the DERA from E. coli (DERA(EC)). Stability of DERA(RE) turned out to be a crucial factor: The pH for optimal DERA(RE) activity was determined to be 7.0, whereas the highest stability was achieved at pH 9.0 with a half-life of approximately 20 days. The optimal temperature for DERA(RE) activity was 65 °C, but coupled with a rather low stability (half-life of 2 min). The highest stability was achieved at 25 °C. The new enzyme exhibits high resistance to organic solvents and acetaldehyde with a half-life being 2.5× higher compared to DERA(EC) under the exposure of 300 mM acetaldehyde. Hence it has the potential as a new promising biocatalyst with applications in organic synthesis.     

Distinct Promoters Affect Pyrroloquinoline Quinone Production in Recombinant Escherichia coli and Klebsiella pneumoniae

Pyrroloquinoline quinone (PQQ) is a versatile quinone cofactor participating in numerous biological processes. Klebsiella pneumoniae can naturally synthesize PQQ for harboring intact PQQ synthesis genes. Previous metabolic engineering of K. pneumoniae failed to overproduce PQQ due to the employment of strong promoter in expression vector. Here we report that a moderate rather than strong promoter is efficient for PQQ production. To screen an appropriate promoter, a total of four distinct promoters—lac promoter, pk promoter of glycerol dehydratase gene (dhaB1), promoter of kanamycin resistance gene, and T7 promoter (as the control)—were individually used for overexpressing the endogenous PQQ genes in K. pneumoniae along with heterologous expression in Escherichia coli. We found that all recombinant K. pneumoniae strains produced more PQQ than recombinant E. coli strains that carried corresponding vectors, indicating that K. pneumoniae is superior to E. coli for the production of PQQ. Particularly, the recombinant K. pneumoniae recruiting the promoter of kanamycin resistance gene produced the highest PQQ (1,700 nmol), revealing that a moderate rather than strong promoter is efficient for PQQ production. Furthermore, PQQ production was roughly proportional to glucose concentration increasing from 0.5 to 1.5 g/L, implying the synergism between PQQ biosynthesis and glucose utilization. This study not only provides a feasible strategy for production of PQQ in K. pneumoniae, but also reveals the exquisite synchronization among PQQ biosynthesis, glucose metabolism, and cell proliferation.     

A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg²⁺ for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li⁺ (50% inhibitory concentration [IC₅₀] = 3.6 mM) but only slightly sensitive to Na⁺ (IC₅₀ = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li⁺ and Na⁺, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li⁺ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.