Cysteine synthase A overexpression in Corynebacterium glutamicum enhances l-isoleucine production.

Cysteine synthase A (CysK) catalyzes the last reaction of l-cysteine synthesis in bacteria, but its moonlighting functions have been revealed recently. In this study, CysK was overexpressed in Corynebacterium glutamicum IWJ001, an l-isoleucine producer. Compared with the control IWJ001/pDXW-8, IWJ001/pDXW-8-cysK cells grew fast during log phase, and produced 26.5% more l-isoleucine in flask fermentation and 23.5% more l-isoleucine in fed-batch fermentation. The key genes aspC, lysC, hom, thrB, ilvA, and ilvBN involved in l-isoleucine biosynthesis were all upregulated in IWJ001/pDXW-8-cysK, compared with IWJ001/pDXW-8. In addition, IWJ001/pDXW-8-cysK cells were longer and thicker than IWJ001/pDXW-8 cells. Compared with IWJ001/pDXW-8, the membrane permeability increased 15.8% and biofilm formation ability decreased 71.3% for IWJ001/pDXW-8-cysK cells. The results demonstrate that CysK overexpression in C. glutamicum is a good approach to enhance l-isoleucine production.

Metabolic engineering of Corynebacterium glutamicum for L-cysteine production.

L-cysteine, a valuable sulfur-containing amino acid, has been widely used in food, agriculture, and pharmaceutical industries. Due to the toxicity and complex regulation of L-cysteine, no efficient cell factory has yet been achieved for L-cysteine industrial production. In this study, the food-grade microorganism Corynebacterium glutamicum was engineered for L-cysteine production. Through deletion of the L-cysteine desulfhydrases (CD) and overexpression of the native serine acetyltransferase (CysE), the initial L-cysteine-producing strain CYS-2 was constructed to produce 58.2 ± 5.1 mg/L of L-cysteine. Subsequently, several metabolic engineering strategies were performed to further promote L-cysteine biosynthesis, including using strong promoter tac-M to enhance expression intensity of CysE, investigating the best candidate among several heterogeneous feedback-insensitive CysEs for L-cysteine biosynthesis, overexpressing L-cysteine synthase (CysK) to drive more metabolic flux, evaluating the efflux capacity of several heterogeneous L-cysteine transporters, engineering L-serine biosynthesis module to increase the precursor L-serine level and using thiosulfate as the sulfur source. Finally, the L-cysteine concentration of the engineered strain CYS-19 could produce 947.9 ± 46.5 mg/L with addition of 6 g/L Na2S2O3, approximately 14.1-fold higher than that of the initial strain CYS-2, which was the highest titer of L-cysteine ever reported in C. glutamicum. These results indicated that C. glutamicum was a promising platform for L-cysteine production.

Cytosolic Cysteine Synthase Switch Cysteine and Mimosine Production in Leucaena leucocephala.

In higher plants, multiple copies of the cysteine synthase gene are present for cysteine biosynthesis. Some of these genes also have the potential to produce various kinds of ß-substitute alanine. In the present study, we cloned a 1275-bp cDNA for cytosolic O-acetylserine(thiol)lyase (cysteine synthase) (Cy-OASTL) from Leucaena leucocephala. The purified protein product showed a dual function of cysteine and mimosine synthesis. Kinetics studies showed pH optima of 7.5 and 8.0, while temperature optima of 40 and 35 °C, respectively, for cysteine and mimosine synthesis. The kinetic parameters such as apparent Km, kcat were determined for both cysteine and mimosine synthesis with substrates O-acetylserine (OAS) and Na2S or 3-hydroxy-4-pyridone (3H4P). From the in vitro results with the common substrate OAS, the apparent kcat for Cys production is over sixfold higher than mimosine synthesis and the apparent Km is 3.7 times lower, suggesting Cys synthesis is the favored pathway.

Impact of overexpression of cytosolic isoform of O-acetylserine sulfhydrylase on soybean nodulation and nodule metabolome.

Nitrogen-fixing nodules, which are also major sites of sulfur assimilation, contribute significantly to the sulfur needs of whole soybean plants. Nodules are the predominant sites for cysteine accumulation and the activity of O-acetylserine(thiol)lyase (OASS) is central to the sulfur assimilation process in plants. Here, we examined the impact of overexpressing OASS on soybean nodulation and nodule metabolome. Overexpression of OASS did not affect the nodule number, but negatively impacted plant growth. HPLC measurement of antioxidant metabolites demonstrated that levels of cysteine, glutathione, and homoglutathione nearly doubled in OASS overexpressing nodules when compared to control nodules. Metabolite profiling by LC-MS and GC-MS demonstrated that several metabolites related to serine, aspartate, glutamate, and branched-chain amino acid pathways were significantly elevated in OASS overexpressing nodules. Striking differences were also observed in the flavonoid levels between the OASS overexpressing and control soybean nodules. Our results suggest that OASS overexpressing plants compensate for the increase in carbon requirement for sulfur assimilation by reducing the biosynthesis of some amino acids, and by replenishing the TCA cycle through fatty acid hydrolysis. These data may indicate that in OASS overexpressing soybean nodules there is a moderate decease in the supply of energy metabolites to the nodule, which is then compensated by the degradation of cellular components to meet the needs of the nodule energy metabolism.

O-acetylserine(thio)lyase (OAS-TL) molecular expression in Pancratium maritimum L. (Amaryllidaceae) under salt stress.

MAIN CONCLUSION: Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OAS-TL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio)lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root).

Genetic, metabolomic and transcriptomic analyses of the de novo L-cysteine biosynthetic pathway in the enteric protozoan parasite Entamoeba histolytica.

The de novo L-cysteine biosynthetic pathway is critical for the growth, antioxidative stress defenses, and pathogenesis of bacterial and protozoan pathogens, such as Salmonella typhimurium and Entamoeba histolytica. This pathway involves two key enzymes, serine acetyltransferase (SAT) and cysteine synthase (CS), which are absent in mammals and therefore represent rational drug targets. The human parasite E. histolytica possesses three SAT and CS isozymes; however, the specific roles of individual isoforms and significance of such apparent redundancy remains unclear. In the present study, we generated E. histolytica cell lines in which CS and SAT expression was knocked down by transcriptional gene silencing. The strain in which CS1, 2 and 3 were simultaneously silenced and the SAT3 gene-silenced strain showed impaired growth when cultured in a cysteine lacking BI-S-33 medium, whereas silencing of SAT1 and SAT2 had no effects on growth. Combined transcriptomic and metabolomic analyses revealed that, CS and SAT3 are involved in S-methylcysteine/cysteine synthesis. Furthermore, silencing of the CS1-3 or SAT3 caused upregulation of various iron-sulfur flavoprotein genes. Taken together, these results provide the first direct evidence of the biological importance of SAT3 and CS isoforms in E. histolytica and justify the exploitation of these enzymes as potential drug targets.

Two-stage cultivation of Pseudomonas sp. F12 for the production of enzymes converting DL-2-amino-Δ²-thiazoline-4-carboxylic acid to L-cysteine.

Pseudomonas sp. F12 isolated from soil could transform DL-2-amino-Δ(2)-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine. It could grow in minimal medium containing DL-ATC as the sole carbon and nitrogen source, and the apparent activity of L-cysteine synthesis (CS) achieved 122 U/mL in a 5-L bioreactor. Pseudomonas sp. F12 could utilize glucose as carbon source and ammonia as nitrogen source for growth, but no CS activity was formed. To reduce the cost of DL-ATC, the cultivation process was divided into a growth stage on glucose and ammonia and a production stage induced by DL-ATC. The excessive glucose led to the production of byproduct(s) which seriously inhibited cell growth and CS production. Ammonium was accumulated when DL-ATC was consumed, and ammonium did not inhibit CS activity formation until 60 mM. Based on the above features, fed-batch cultivation of the growth stage was developed by supplying glucose restrictively. The volumetric CS activity was enhanced more than two times that obtained under the initial conditions.

Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman-Birk protease inhibitor in seeds.

Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur-containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins have met with limited success. Here, we report a successful strategy to increase the cysteine content of soybean seed through the overexpression of a key sulfur assimilatory enzyme. We have generated several transgenic soybean plants that overexpress a cytosolic isoform of O-acetylserine sulfhydrylase (OASS). These transgenic soybean plants exhibit a four- to tenfold increase in OASS activity when compared with non-transformed wild-type. The OASS activity in the transgenic soybeans was significantly higher at all the stages of seed development. Unlike the non-transformed soybean plants, there was no marked decrease in the OASS activity even at later stages of seed development. Overexpression of cytosolic OASS resulted in a 58-74% increase in protein-bound cysteine levels compared with non-transformed wild-type soybean seeds. A 22-32% increase in the free cysteine levels was also observed in transgenic soybeans overexpressing OASS. Furthermore, these transgenic soybean plants showed a marked increase in the accumulation of Bowman-Birk protease inhibitor, a cysteine-rich protein. The overall increase in soybean total cysteine content (both free and protein-bound) satisfies the recommended levels required for the optimal growth of monogastric animals.

Induction of the Escherichia coli cysK gene by genetic and environmental factors.

Cysteine synthase A encoded by cysK catalyzes the synthesis of cysteine from O-acetylserine. Expression of cysK in Escherichia coli is under the control of CysB, a LysR family transcription factor. Herein we showed that the expression of cysK is regulated by several genetic and environmental factors in addition to CysB: two genetic factors, OmpR and CysE, and lithium. Based on the findings, we constructed the high-level expression system of cysK.

Overexpression of a soybean O-acetylserine (thiol) lyase-encoding gene GmOASTL4 in tobacco increases cysteine levels and enhances tolerance to cadmium stress.

O-Acetylserine (thiol) lyase (OASTL, EC 2.5.1.47) catalyzes the final step in cysteine biosynthesis. The soybean GmOASTL4 gene was overexpressed in tobacco. Transgenic plants showed markedly increased accumulation of transcripts and higher cysteine content compared with the wild-type. Upon exposure to cadmium stress, OASTL activity and cysteine levels increased significantly in transgenic plants. Cadmium accumulation and the activity of both superoxide dismutase and catalase enzymes were enhanced in transformants. These results demonstrate that overexpression of GmOASTL4 in tobacco can enhance cysteine levels and increase tolerance to cadmium stress.

Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress.

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

Cysteine metabolism-related genes and bacterial resistance to potassium tellurite.

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K2TeO3 to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.

Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli.

O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.

Overproduction of SAT and/or OASTL in transgenic plants: a survey of effects.

The last steps of cysteine biosynthesis are catalysed by a bi-enzyme complex composed of serine acetyltransferase (SAT) and cysteine synthase, also called O-acetyl-serine (thiol) lyase (OASTL). SAT is responsible for the production of O-acetyl-serine (OAS) from serine and acetyl-coenzyme A, while OASTL catalyses the formation of cysteine from OAS and hydrogen sulphide. Several distinct nuclear genes for SAT and OASTL enzymes exist in plants. Products of these genes are targeted into at least three cellular compartments: cytosol, chloroplasts, and mitochondria. The SAT and OASTL enzymes are strongly evolutionary conserved, both structurally and functionally. Therefore, isoenzymes from various cellular compartments can be substituted, not only by their plant counterparts from the other cellular compartments but also by their bacterial homologues. During the last decade transgenic plants overproducing SAT, OASTL or both enzymes simultaneously were obtained independently by several research groups. These manipulations led not only to the elevated levels of the respective products, namely OAS and cysteine, but also to increased amounts of glutathione and changes in the levels of other metabolites and enzymatic activities. In several cases, the transgenic plants were also shown to be less susceptible to applied abiotic stresses. In this review, all published and some unpublished results from this laboratory related to heterologous overproduction of SAT and OASTL in transgenic plants are discussed and summarized.

Cysteine synthase overexpression in tobacco confers tolerance to sulfur-containing environmental pollutants.

Cysteine (Cys) synthase [O-acetyl-L-Ser(thiol)-lyase, EC 4.2.99.8; CSase] is responsible for the final step in biosynthesis of Cys. Transgenic tobacco (Nicotiana tabacum; F(1)) plants with enhanced CSase activities in the cytosol and in the chloroplasts were generated by cross-fertilization of two transformants expressing cytosolic CSase or chloroplastic CSase. The F(1) transgenic plants were highly tolerant to toxic sulfur dioxide and sulfite. Upon fumigation with 0.1 microL L(-1) sulfur dioxide, the Cys and glutathione contents in leaves of F(1) plants were increased significantly, but not in leaves of non-transformed control plants. Furthermore, the leaves of F(1) plants exhibited the increased resistance to paraquat, a herbicide generating active oxygen species.

Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex.

Cysteine synthetase from Escherichia coli is a bienzyme complex composed of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS). The effects of the complex formation on the stability of SAT against cold inactivation and proteolysis were investigated. SAT was reversibly inactivated on cooling to 0 degrees C. Ultracentrifugal analysis showed that SAT (a hexamer) was dissociated mostly into two trimers on cooling to 0 degrees C in the absence of OASS, while in the presence of OASS one trimer of the SAT subunits formed a complex with one dimer of OASS subunits. In the presence of OASS, not only the cold inactivation rate was reduced but also the reactivation rate was increased. Furthermore, SAT became stable against proteolytic attack by alpha-chymotrypsin and V8 protease by forming the complex with OASS. On the other hand, SAT was degraded by trypsin in the same manner both in the presence and in the absence of OASS. The different tendency in the stability against proteolysis with the different proteases was discussed with respect to the substrate specificity of the proteases and amino acid sequence of the C-terminal region of SAT that interacts with OASS.

Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8.

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium. L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited O-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T. thermophilus HB8.

Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur.

Subcellular localization and regulation of the spinach (Spinacia oleracea) cysteine synthase (O-acetyl-L-serine[thiol]-lyase, EC 4.2.99.8) isoforms (CysA, CysB, and CysC) were determined in transgenic tobacco (Nicotiana tabacum) and in spinach cell cultures. The 5' regions of CysB and CysC encoding the chloroplastic (CysB-TP) and the putative mitochondrial (CysC-TP) transit peptide (TP) sequences were fused to a bacterial beta-glucuronidase gene (gus) and expressed in tobacco under the control of the cauliflower mosaic virus 35S promoter. Subcellular fractionation of transgenic tobacco showed transportation of beta-glucuronidase proteins to chloroplasts by CysB-TP and to mitochondria by CysC-TP, respectively, indicating that both presequences were sufficient to act specifically as chloroplastic and mitochondrial TPs in vivo. The mRNA expression patterns of CysA (cytoplasmic form), CysB, and CysC genes under nitrogen- and sulfur-starved conditions were characterized in spinach cell cultures. In sulfur-starved cells, only slight differences (approximately 1.2- to 1.5-fold) in the mRNA levels of CysA and CysB were observed during the short-term (0-24 h) cultivation periods compared with cells grown in Murashige-Skoog medium. However, under nitrogen and nitrogen/sulfur double-deficient stress conditions, mRNA levels of CysC increased up to 500% of the original level within 72 h.

The mechanism of the auxotrophy for sulfur-containing amino acids imposed upon Escherichia coli by superoxide.

Defects in both of the genes coding for the cytosolic superoxide dismutases (SODs) of Escherichia coli impose an oxygen-dependent nutritional requirement for cysteine. This is now seen to be a bradytrophy, rather than an absolute auxotrophy, since lack of Cys merely imposed a growth lag and escape from this growth lag did not involve genetic reversion. This Cys bradytrophy was not seen in the SOD-competent parental strain, and it was relieved by a cell-permeant mimic of SOD activity; hence, it was due to O2-.. It was also relieved by an osmolyte, such as sucrose; hence, it appears due to leakage from the cell of some component needed for Cys biosynthesis. Medium conditioned by the aerobic growth of the SOD-defective strain relieved the growth lag. Bioassays with Cys mutants suggested that the conditioned medium contained SO3-3 or its equivalent, and sulfite per se was able to eliminate the growth lag. However, some component of the conditioned medium reacted with added sulfite and interfered with attempts to assay for it colorimetrically. These results suggest that the cell envelope of the SOD-defective strain was weakened, directly or indirectly, by O2 and then leaked sulfite. This prevents cysteine biosynthesis until sulfite accumulates in the medium.