Efficient one-step production of (S)-1-phenyl-1,2-ethanediol from (R)-enantiomer plus NAD+–NADPH in-situ regeneration using engineered Escherichia coli

Background

Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli.

Results

Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell.

Conclusions

In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.     

Cysteine desulphydrase activity in higher plants: Evidence for the action of L- and D-cysteine specific enzymes

Emission of hydrogen sulphide in response to D-cysteine by leaf discs of cucurbit plants or cultured tobacco cells was considerably smaller than in response to L-cysteine. Whereas hydrogen sulphide emission from L-cysteine was inhibited by 100 μM aminooxyacetic acid (AOA), emission from D-cysteine was unaffected. These results from in vivo studies were found to be inconsistant with the L- and D-cysteine desulphydrase activities measured in crude homogenates. In vitro, D-cysteine desulphydrase activity was more than one order of magnitude higher than L-cysteine desulphydrase activity; L-cysteine desulphydrase was inhibited by 100 μM AOA to a smaller, D-cysteine desulphydrase to a higher extent than in vivo. Cystine lyase activity, which may interfere in the cysteine desulphydrase assay, was not found. In cucurbit leaves, the differences between in vivo and in vitro experiments can partially be explained by differences in the influx of L- and D-cysteine into the leaf discs. Influx of L-cysteine proceeded at a rate about four times higher than the influx of D-cysteine; it was inhibited by 100 μM AOA, whereas influx of D-cysteine was unaffected. Subcellular distribution of L- and D-cysteine desulphydrase was analysed in cultured tobacco cells. Both enzyme activities were found to be soluble. The D-cysteine activity was predominantly localized in the cytoplasm whereas L-cysteine activities were also found in chloroplasts and mitochondria. The L-cysteine desulphydrase in the cytoplasmic fraction may entirely be due to broken chloroplasts and mitochondria. Inhibitor studies with ammonium, pyruvate, AOA and O-acetylserine revealed considerable differences between L- and D-cysteine desulphydrase activity and between L-cysteine desulphydrase activity in chloroplasts and mitochondria. Therefore, the present data suggest that degradation of L- and D-cysteine are catalysed by different enzymes in different compartments of the cell.     

Kinetics of Inhibition of Green Crab (Scylla serrata) Alkaline Phosphatase by L-Cysteine

The inhibition of alkaline phosphatase from green crab (Scylla serrata) by L-cysteine has been studied. The results show that L-cysteine gives a mixed-type inhibition. The progress-of-substrate-reaction method previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 391–436] was used to study the inactivation kinetics of the enzyme by L-cysteine. The microscopic rate constants were determined for reaction of the inhibitor with the free enzyme and the enzyme–substrate complex (ES) The results show that inactivation of the enzyme by L-cysteine is a slow, reversible reaction. Comparison of the inactivation rate constants of free enzyme and ES suggests that the presence of the substrate offers marked protection of this enzyme against inactivation by L-cysteine.     

Nitrilase-Catalyzed Production of Nicotinic Acid from 3-Cyanopyridine in Rhodococcus rhodochrous J1

The nitrilase which occurs abundantly in cells of Rhodococcus rhodochrous J1 catalyzes the direct hydrolysis of 3-cyanopyridine to nicotinic acid without forming nicotinamide. By using resting cells, the reaction conditions for nicotinic acid production were optimized. Under the optimum conditions, 100% of the added 3-cyanopyridine could be converted to nicotinic acid, the highest yield achieved being 172 mg of nicotinic acid per 1.0 ml of reaction mixture containing 2.89 mg (dry weight) of cells in 26 h.     

Enzymatic synthesis of l-cysteine by O-acetylserine sulfhydrylase of 3-chloro-l-alanine resistant Bacillus sphaericus L-118

The regulatory properties of serine-O-transacetylase and O-acetylserine sulfhydrylase have been investigated with 3-chloro-l-alanine resistant Bacillus sphaericus L-118. The enhancement of O-acetylserine sulfhydrylase formation by 3-chloro-l-alanine was observed and this effect was counteracted by corepressor l-cysteine. O-Acetylserine sulfhydrylase occurring in B. sphaericus L-118 can catalyse β-replacement reaction of 3-chloro-l-alanine in the presence of a high concentration of sodium hydrosulfide to form l-cysteine. The optimal reaction conditions for l-cysteine production were studied using resting cells. Under optimal conditions, about 80% of the added 3-chloro-l-alanine could be converted to l-cysteine. The highest yield achieved was 70 mg of l-cysteine per 1.0 ml of the reaction mixture.     

酶法转化DL-ATC合成L-半胱氨酸的酶促反应条件研究

目的:考察酶源保存方式、酶促反应时间、底物pH值、底物浓度、酶浓度、金属离子等因素对酶活力的影响。方法:以假单胞菌(Pseudomonassp.)TS1138为供试菌株,采用酸式茚三酮法测定L-半胱氨酸含量,研究了酶法转化DL-ATC合成L-半胱氨酸的酶促反应条件。结果:TS1138菌株中L-半胱氨酸脱巯基酶具有较高的活性,而且Mg2 、Mn2 、Fe2 、Zn2 、Cu2 等5种金属离子对DL-ATC水解酶酶系有不同程度的抑制,其中Cu2 对该酶系的抑制作用很大。结论:确定了TS1138菌株酶法转化DL-ATC合成L-半胱氨酸的最适酶促反应条件,为酶促反应动力学的研究奠定了基础。     

Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli

L-cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of L-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in L-cysteine degradation, increased L-cysteine productivity in E. coli. The use of an L-cysteine efflux system could be promising for breeding L-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as L-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on L-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by L-cysteine. We also found that overexpression of these eight genes reduces intracellular L-cysteine levels after cultivation in the presence of L-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes L-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more L-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in L-cysteine export and overproduction in genetically engineered E. coli cells.     

Desulfuration of l-cysteine through transamination and transsulfuration in rat liver

Desulfuration of L-cysteine by rat liver via a pathway of transamination followed by transsulfuration was studied using cyanide as a sulfur acceptor. More than a 5-fold increase in formation of thiocyanate from L-cysteine and cyanide was observed in the presence of 2-oxoglutarate and pyruvate. L-Cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase activities in the same preparations were also determined. It was concluded that L-cysteine was desulfurated through transamination and transsulfuration of the resulting 3-mercaptopyruvate, and that the rate-limiting step appears to be the transamination reaction.     

恶臭假单胞菌TS1138转化生产L-胱氨酸的工艺研究

对以DL-2-氨基-?2-噻唑啉-4-羧酸(DL-2-amino-?2-thiazoline-4-carboxylic acid, DL-ATC)为底物原料, 经微生物酶法催化合成L-半胱氨酸, 并进一步氧化和分离纯化产物L-胱氨酸的生产工艺和条件进行了研究。建立了以恶臭假单胞菌TS1138 (Pseudomonas putida TS1138)全细胞为酶源, 反复多次催化底物合成L-半胱氨酸, 并以2.0%二甲基亚砜(DMSO)为氧化剂氧化生成L-胱氨酸, 进而通过001×7型阳离子交换树脂纯化胱氨酸的新工艺。采用高效液相色谱法考察该方法L-胱氨酸的总收率可以达到78.55%, 纯度为99.12%。该方法简单高效, 解决了酶稳定性差不能重复使用, 而固定化酶方法繁琐成本高的问题, 为我国L-半胱氨酸和L-胱氨酸的生产开辟一条新途径。     

恶臭假单胞菌TS1138转化生产L-胱氨酸的工艺研究

对以DL-2-氨基-△2-噻唑啉-4-羧酸(DL-2-amino-△2-thiazoline-4-carboxylic acid,DL-ATC)为底物原料,经微生物酶法催化合成L-半胱氨酸,并进一步氧化和分离纯化产物L-胱氨酸的生产工艺和条件进行了研究.建立了以恶臭假单胞菌TS1138(Pseudomonas putida TS1138)全细胞为酶源,反复多次催化底物合成L-半胱氨酸,并以2.0%二甲基亚砜(DMSO)为氧化剂氧化生成L-胱氨酸,进而通过001×7型阳离子交换树脂纯化胱氨酸的新工艺.采用高效液相色谱法考察该方法L-胱氨酸的总收率可以达到78.55%,纯度为99.12%.该方法简单高效,解决了酶稳定性差不能重复使用,而固定化酶方法繁琐成本高的问题,为我国L-半胱氨酸和L-胱氨酸的生产开辟一条新途径.     

Cellular requirements for lymphocyte restimulation after a first challenge in vitro.

Human peripheral blood lymphocytes from donors who were sensitized in vivo to bacterial antigens were stimulated by these antigens in vitro. When the cells from these first cultures were challenged with irradiated allogeneic lymphocytes, a proliferative response was obtained, the kinetics of which resembled those of a primary mixed lymphocyte reaction (MLR). On the other hand, the addition, under these conditions, of bacterial antigens never led to any second proliferative response. It was shown that: (1) the addition of irradiated autologous mononuclear cells, together with the bacterial antigens, led to a reconstitution of a proliferative response in second culture; (2) the cells capable of reconstituting the reactivity to tetanus toxoid could also be obtained from donors whose own cells did not respond to that antigen in primary cultures, and (3) the reconstituting activity in the second culture could not be provided by monocytes alone.     

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli.

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.     

Evidence for an intracellular sulfur cycle in cucumber leaves

H₂S emission from cucumber (Cucumis sativus L.) leaf discs supplied with L-cysteine in the dark is inhibited 80–90% by aminooxyacetic acid (AOA), an inhibitor of pyridoxal-phosphate dependent enzymes. Exposure to L-cysteine in the light enhanced the emission of H₂S in response to this sulfur source. Turning off the light reduced the emission of H₂S to the rate observed in continuous dark; turning on the light enhanced the emission of H₂S to the rate observed in continuous light. Therefore, in the light H₂S emission in response to L-cysteine becomes a partially light-dependent process. Treatment with cyanazine, an inhibitor of photosynthetic electron transport, reduced H₂S emission in the light to the rate observed in continuous dark, but did not affect H₂S emission in the dark. In leaf discs pre-exposed to L-cysteine in the light, treatment with cyanazine+ AOA inhibited the emission of H₂S in response to L-cysteine completely. Therefore, only part of the H₂S emitted in response to this sulfur source is derived from a light-independent, but pyridoxal-phosphate-dependent process; the balance of the H₂S emitted is derived from a light-dependent process that can be inhibited by cyanazine. When cucumber leaf discs were supplied with a pulse of L-[^35S]cysteine, radioactively labeled H₂S was emitted in two waves, one during the first hour of exposure to L-cysteine, and a second after 3–4 h; unlabeled H₂S, however, was emitted continuously. The second wave of emission of labeled H₂S was not observed in pulse-chase experiments in which sulfate or cyanazine were added to the treatment solution after 3 h of exposure to L-cysteine, or when the lights was turned off. The labeling pattern of sulfur compounds inside cucumber cells supplied with a pulse of L-[³⁵S]cysteine showed that the labeled H₂S released from L-cysteine partially enters first the sulfite, then the sulfate pool of the cells. The radioactively labeled sulfate, however, is not incorporated into L-cysteine, but enters the H₂S pool of the cells again. These observations are consistent with the idea of an intracellular sulfur cycle in plant cells. The L-cysteine taken up by the leaf discs seems to be desulfhydrated in a light-independent, but pyridoxal-phosphate-dependent process. The H₂S synthesized this way may be partially released into the atmosphere; the other part of the H₂S produced in response to L-cysteine may be oxidized to sulfite, then to sulfate, which is subsequently reduced via the light-depent sulfate assimilation pathway. In the presence of excess L-cysteine, synthesis of additional cysteine may be inhibited, and the sulfide moiety may be split off carrier bound sulfide to enter the H₂S pool of the cells again. It is suggested that the function of this sulfur cycle may be regulation of the free cysteine pool.Abbreviation AOA aminooxyacetic acid     

Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

ABSTRACT: BACKGROUND: Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. RESULTS: Because the redox enzymes can reduce the disulfide that forms on proteins, wefirst tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coli L-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (cysI and cysJ) and the L-cysteine synthase gene (cysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (cysC or cysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coli L-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell . CONCLUSIONS: In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.     

A method for the synthesis of [14C]-kynurenine

A new method is described for the synthesis of [14C]-labelled L-kynurenine from [14C]-L-tryptophan, using extracts of tryptophan-adapted cells of Pseudomonas marginalis. It is based on the selective, rapid inactivation of kynureninase by a newly discovered inhibitor of this enzyme, 3-chloro-L-alanine. The yield of [14C]-kynurenine produced in this manner is 76% theoretical.     

Non-identity of cystine lyase with β-cystathionase in turnip roots

An active preparation of cystine lyase (EC 4.4.1.-) was prepared from turnip roots and its substrate specificity examined. Only L-cysteine, cysteine-S-SO₃, and the sulphoxides of L-djenkolic acid, S-methyl-and S-ethyl-L-cysteine were substrates. L-Cystathione, L-djenkolic acid, S-methyl-and S-ethyl-cysteines were not cleaved by this enzyme. The K_m for L-cystine was 1.3 mM and L-cystathionine acted as an effective competitive inhibitor with a K_i of 0.7 mM. After dialysis against 10 mM potassium phosphate buffer pH 7.5, added pyridoxal phosphate was absolutely necessary for activity. In addition a marked stimulation was observed in the presence of ammonium sulphate. The products of the reaction were cysteine persulphide, pyruvate and presumably ammonia. The persulphide was easily demonstrated by cleavage with CN^? to yield SCN^? under conditions in which elemental sulphur was unreactive.     

Conversion of L-cystine and L-cysteine to taurin by the enzyme systems of liver cells

The kinetics of conversion of sulfur-containing amino acids L-cystine and L-cysteine to taurin by the enzyme system of cattle liver cells was studied, and a mathematical model was developed. It was shown that L-cystine and L-cysteine conversion obeyed the Michaelis-Menten equations of serial-sequential conversions with regard to inhibition by the final product and inactivation. The yield of taurin under the optimized conditions of L-cystine and L-cysteine conversion (temperature, 40 degrees C; pH 1.5 and 3.0, respectively; and addition of enzyme preparations in five equal portions at 2-h intervals) was in the range 80-85% of the substrate weight.     

One-electron reduction of S-nitrosothiols in aqueous medium

One-electron reduction of S-nitrosothiols (RSNO) has been studied using radiolytically produced reducing entity, the hydrated electron (e(aq)(-)), in aqueous medium. Both kinetics of the reaction and the mechanistic aspects of the decomposition of S-nitroso derivatives of glutathione, L-cysteine, N-acetyl-L-cysteine, N-acetyl-D,L-penicillamine, N-acetylcysteamine, L-cysteine methyl ester, and D,L-penicillamine have been investigated at neutral and acidic pH. The second-order rate constants of the reaction of e(aq)(-) with RSNOs were determined using a pulse radiolysis technique and were found to be diffusion controlled (10(10) dm(3) mol(-1) s(-1)) at neutral pH. The product analysis using HPLC, fluorimetry, and MS revealed the formation of thiol and nitric oxide as the major end products. It is therefore proposed that one-electron reduction of RSNO leads to the liberation of NO. There is no intermediacy of a thiyl radical as in the case of oxidation reactions of RSNOs. The radical anion of RSNO (RSN(*)O(-)) is proposed as a possible intermediate. The overall reaction could be written as RSNO + e(aq)(-) --H+--> RSH + (*)NO.