Identification and Functional Analysis of Escherichia coli Cysteine Desulfhydrases

In Escherichia coli, three additional proteins having L-cysteinedesulfhydrase activity were identified as O-acetylserine sulfhydrylase-A,O-acetylserine sulfhydrylase-B, and MalY protein, in additionto tryptophanase and cystathionine ß-lyase, whichhave been reported previously. The gene disruption for eachprotein was significantly effective for overproduction of L-cysteineand L-cystine. Growth phenotype and transcriptional analysessuggest that tryptophanase contributes primarily to L-cysteinedegradation.

INTRODUCTION

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Introduction

L-cysteine is an important amino acid in terms of its applicationsin the pharmaceutical, food, and cosmetic industries. However,due to feedback inhibition by L-cysteine of serine acetyltransferase(SAT; EC 2.3.1.30), which catalyzes the formation of O-acetyl-L-serinefrom acetyl-coenzyme A (CoA) and L-serine (8, 9, 16), high-levelproduction of L-cysteine from glucose has not been successfullyachieved in microorganisms. In order to obtain L-cysteine producers,we previously constructed Escherichia coli cysE genes that encodealtered SATs. These genes were genetically desensitized to thefeedback inhibition by L-cysteine through site-directed or randommutagenesis (21, 32). We found that, in the recombinant E. colicells expressing the altered cysE gene, there was a marked productionof L-cysteine plus L-cystine.

In the same investigation (21), it was demonstrated that proteinswith L-cysteine desulfhydrase (CD) activity play an importantrole in L-cysteine degradation in E. coli cells. In order tofurther improve L-cysteine production, a host strain havinga lower level of CD activity must be constructed. CD is knownto catalyze the degradation of L-cysteine to pyruvate, ammonia,and sulfide by the following reaction: HSCH₂CH(NH₂)COOH + H₂O $->$ CH₃COCOOH + H₂S + NH₃. This type of enzyme activity has beendemonstrated to be present in several mammalian tissues (15)and in bacteria, such as Salmonella enterica serovar Typhimurium(6, 17) and E. coli (2, 11). In E. coli, cystathionine-ß-lyase(CBL) (17) encoded by metC, which catalyzes mainly the conversionof cystathionine to homocysteine, pyruvate, and ammonia (9),as well as tryptophanase (TNase) encoded by tnaA, which primarilydegrades L-tryptophan to indole, pyruvate, and ammonia (22),has been shown to exhibit CD activity in vitro (9, 23). We havepreviously reported that CBL and TNase catalyzed the CD reactionand acted on L-cysteine degradation in E. coli cells by analyseswith CD activity staining and gene disruption (2). However,the double CD gene-disrupted mutant still had a low level ofCD activity, suggesting that unknown CD proteins remain to beidentified. Thus, we report here further identification andcharacterization of the CDs involved in L-cysteine degradationin E. coli.

Identification of CDs in E. coli.

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Identification of cds in...

An E. coli cell has 4,388 kinds of open reading frame (ORF),including the function-unknown and -deduced genes. The librarywas constructed by placing each ORF under the lacZ promoterin vector pCA24N (20). E. coli wild-type strain JM39 was independentlytransformed with nine libraries, each consisting of 480 kindsof plasmids, and then approximately 1,200 colonies appearedfrom each library on Luria-Bertani (LB) medium (27) containingchloramphenicol (100 µg/ml). As a whole, more than 10,000independent E. coli clones were obtained. The transformed cellsfrom each library were mixed and grown at 37°C in 5 ml ofLB medium containing chloramphenicol (100 µg/ml). Whenthe absorbance at 600 nm reached 0.5, isopropyl-ß-D-thiogalactopyranosidewas added to the culture medium to a final concentration of0.01 mM to induce gene expression. After cultivation for 4 hat 37°C, cell extracts were separated by native polyacrylamidegel electrophoresis (PAGE) and the gel was stained by gentlyshaking in the solution containing L-cysteine, pyridoxal 5'-phosphate(PLP), and BiCl₃ as described previously (2, 35). When the gelwas kept at 4°C for 1.5 h, which was a shorter time thanin the previous condition (3 h), three CD proteins were newlydetected in JM39 in addition to CBL and TNase (Fig. 1): O-acetylserinesulfhydrylase-A(OASS-A), encoded by cysK (lane 2), O-acetylserinesulfhydrylase-B(OASS-B), encoded by cysM (lane 3), and MalY, encoded by malY(lane 4). OASS-A primarily catalyzes the synthesis of L-cysteinefrom O-acetylserine and sulfide along the L-cysteine biosyntheticpathway (3, 7, 19). OASS-B is considered to be an isomer ofOASS-A (30, 31), but its function(s) is not yet clearly understood.Because both OASS-A and -B require PLP for CD activity in asimilar manner as CBL, we think that these enzymes can catalyzethe analogous reaction. MalY protein is also a PLP-dependentenzyme with the activity of the carbon-sulfur bond cleavage(ßC-S lyase) of cystathionine (35), as well as a transcriptionalregulator in mal gene expression (5, 26, 28). The secondaryand tertiary structures of MalY are highly homologous to thoseof CBL (4, 5, 9, 18). Consequently, the five CD proteins inE. coli were identified as TNase, CBL, OASS-A, OASS-B, and MalY.

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FIG. 1. Detection of OASS-A, MalY, and OASS-B by CD activity staining. Preparation of native PAGE gel, cell extracts, and CD activity staining were carried out according to the method described previously (2). Lane 1, wild-type JM39 harboring pBluescript II SK+ (vector control; Toyobo Biochemicals, Osaka, Japan); lane 2, JM39 harboring pcysK (cysK plasmid; OASS-A is overexpressed); lane 3, JM39 harboring pcysM (cysM plasmid; OASS-B is overexpressed); lane 4, JM39 harboring pmalY (malY plasmid; MalY is overexpressed).

Effect of gene disruption on total CD activities and L-cysteine production in E. coli.

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Effect of gene disruption...

For the construction of cysK-, cysM-, and malY-disrupted strains,internal fragments of the cysK, cysM, and malY genes of strainJM39 were amplified by PCR and cloned in pEL3, which has a thermosensitivereplicon (1). The primers used were 5'-CGC CGC GGA TCC CAA TCTACC GGT TAT TTT GAT AAC C-3' and 5'-CGC CGC GGA TCC CAA GCTGGC ATT ACT GTT GCA ATT C-3' for cysK, 5'-GCG GCG GGA TCC TAGGTT GAG TGA ATG TTA AAC GCC C-3' and 5'-GCG GCG GGA TCC ATACTG CAT TTG TCG GCA GCA ACA-3' for cysM, and 5'-ATC CAG TCGATG ATC GAT ACC GGG ATC C-3' and 5'-CGC GGG ATC CTT AAC GAACAG CGC GGA TGG CGT TA-3' for malY. Gene disruption was performedas described previously (2). The ampicillin-sensitive strainsobtained were characterized and designated as JM39 ${Delta}$ cysK, JM39 ${Delta}$ cysM,and JM39 ${Delta}$ malY. JM39 ${Delta}$ tnaA ${Delta}$ metC ${Delta}$ cysM ${Delta}$ malY and JM39 ${Delta}$ tnaA ${Delta}$ metC ${Delta}$ cysK ${Delta}$ cysM ${Delta}$ malY were constructed from JM39 ${Delta}$ tnaA ${Delta}$ metC (2) in the samemanner as described above. CD gene disruption was confirmedby PCR and by CD activity staining. In each CD gene-disruptedmutant, the corresponding CD protein bands disappeared on theactivity staining gel. No CD bands were detected in the quintetmutant, JM39 ${Delta}$ tnaA ${Delta}$ metC ${Delta}$ cysK ${Delta}$ cysM ${Delta}$ malY (data not shown). TotalCD activities of the cell extracts prepared from wild-type JM39and CD gene-disrupted strains cultured in LB medium were measuredas described previously (2) (Table 1). CD activity was measuredby colorimetric assay of the sulfide formed from L-cysteineby the enzyme sources (2, 29). The total CD activities of allmutants were lower than that of wild-type JM39. Interestingly,even the quintet mutant still had a low level of CD activity.The reason for the residual activity remains unclear; however,the presence of another CD protein(s), which fails to be separatedby native PAGE or is inactivated by oxygen during native PAGE,is possible.

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TABLE 1. Effect of CD gene disruption on total CD activities and L-cysteine plus L-cystine production in E. coli

In order to confirm whether CDs were involved in L-cysteinedegradation in E. coli cells, we analyzed the L-cysteine productivitiesof wild-type JM39 and each CD gene-disrupted mutant harboringpEAS-m (24, 33), which carries the cDNA encoding feedback-insensitiveSAT from Arabidopsis thaliana. It should be noted that two mutants,JM39 ${Delta}$ cysK and JM39 ${Delta}$ tnaA ${Delta}$ metC ${Delta}$ cysK ${Delta}$ cysM ${Delta}$ malY, were not tested,because OASS-A encoded by cysK is cysteine synthetase, whichis essential for L-cysteine biosynthesis. In contrast, it wasfound that OASS-B encoded by cysM was not an isomer of OASS-Abased on the fact that JM39 ${Delta}$ cysM grew on a medium lacking L-cysteine(data not shown). For the production of L-cysteine and L-cystine,a loopful of cells cultured for 24 h on LB solid medium containingampicillin (50 µg/ml) at 30°C was inoculated into20 ml of C1 plus TS medium in a 500-ml flask and cultured at30°C on a reciprocal shaker maintained at 120 strokes permin (33). Growth was measured by optical density at 562 nm (OD₅₆₂)of culture broth after appropriate dilution with 0.1 N HCl.The amounts of L-cysteine and L-cystine were determined by microbioassaywith Pediococcus acidilactici IFO3076, as described by Tsunodaet al. (34). As L-cysteine in the culture fluid was easily oxidizedto L-cystine, which was slightly soluble in water, the culturefluids were assayed after L-cystine was dissolved with 0.5 NHCl. As shown in Table 1, the production of L-cysteine and L-cystineby these mutants was higher than that observed in the case ofJM39. The amounts of L-cysteine and L-cystine produced after72 h of cultivation increased by a factor of 1.8 to 2.3. Theseresults clearly indicate that CD proteins played important rolesin L-cysteine degradation in E. coli cells and that the correspondinggene disruption was effective in the production of L-cysteineand L-cystine by E. coli cells. However, the amounts of L-cysteineand L-cystine decreased significantly after 96 h of cultivationin all the strains, probably because of the remaining CD enzyme(s)(data not shown).

TNase contributes primarily to L-cysteine degradation in E. coli.

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Tnase contributes primarily to...

Although the mechanisms are not yet understood, it has previouslybeen observed that the growth of E. coli cells is inhibitedby excess L-cysteine (12-14, 25). Therefore, the effect of CDgene disruption on the growth of E. coli cells in the presenceof L-cysteine was examined. All of the strains were grown inLB plus 30 mM L-cysteine at 37°C, and cell growth was measuredby optical density at 610 nm. As shown in Table 1, the growthof the tnaA disruptant JM39 ${Delta}$ tnaA was significantly inhibited,while all of the strains showed the same level of growth whencultured in LB medium (data not shown). This result suggeststhat TNase is a key enzyme in L-cysteine degradation in E. colicells.

We examined the role of CD enzymes on L-cysteine degradation.Wild-type JM39 and CD gene-disrupted mutants were cultivatedin LB plus 10 mM L-cysteine, and total CD activities were measured(Table 1). All of the single mutants, except for JM39 ${Delta}$ tnaA, showeda prominent increase in CD activity, ranging from 38% to 84%of that observed in the absence of L-cysteine. The CD activityfrom the tnaA-disrupted strain was virtually unchanged in thepresence or absence of L-cysteine. Northern blot analysis fortnaA was carried out by using a Gene Images Random-Prime Labelingand Detection System (Amersham Pharmacia Biotech, Buckinghamshire,England). Strain JM39 was cultivated in LB medium or LB plus10 mM L-cysteine, and total RNA was prepared using an RNeasyProtect Bacteria Mini kit (QIAGEN, Valencia, Calif.). As a DNAprobe, the DNA fragment of tnaA was prepared by PCR with oligonucleotideprimers 5'-CCG TTC CGC ATT CGT GTT AT-3' and 5'-TGC GGT GAAGTG ACG CAA TA-3'. As shown in Fig. 2, the addition of L-cysteineresulted in an elevated level of the specific transcript (ca.1.7 kb), while the basal level of expression in LB medium wasfairly low. Transcription of the E. coli tna operon, consistingof two major structural genes, tnaA encoding tryptophanase andtnaB encoding tryptophan permease, have been studied in detail(10). This operon also contains a 319-bp transcribed leaderregulatory region, tnaC, preceding tnaA and specifying a 24-residueleader peptide, TnaC, and expression of the tna operon is inducedby L-tryptophan. Interestingly, the tnaA DNA probe detecteda tnaC-tnaA transcript of ca. 1.7-kb in the presence of L-cysteine(lane 2), because no transcript was observed when the DNA fragmentof tnaB was used as a probe (data not shown). These resultsindicate that TNase synthesis is induced by L-cysteine, in agreementwith previous data on native PAGE (2). Our results may suggestthat TNase contributes mainly to L-cysteine degradation andthat a novel transcriptional regulation system is involved intnaA expression.

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FIG. 2. TNase induction by the addition of L-cysteine. Total RNA of JM39 was prepared from cells cultivated in LB medium (lane 1) and LB plus 10 mM L-cysteine (lane 2). Each lane was loaded with 20 µg of total RNA. The arrow indicates a tnaC-tnaA transcript of ca. 1.7 kb. The 23S and 16S rRNAs are total RNA-loading controls detected by UV spectrometer.

In conclusion, five CD enzymes were identified in E. coli cells,and the gene disruption for each protein was significantly effectivefor overproduction of L-cysteine and L-cystine. However, itis noteworthy that the quintet mutant JM39 ${Delta}$ tnaA ${Delta}$ metC ${Delta}$ cysK ${Delta}$ cysM ${Delta}$ malY in the presence of L-cysteine showed higher CD activitythan that observed in the absence of L-cysteine (Table 1). Itappears that other CDs, in addition to the five proteins identified,could be induced by L-cysteine in E. coli. We must further analyzethe quintet mutant in order to investigate the mechanism. Throughthe CD activity staining described here, some faint bands werestill seen in the gel and some proteins appeared not to be migratedinto the gel. We therefore think that unidentified proteinswith CD activity, which may be induced by L-cysteine, are stillpresent. Development of alternative methods to detect the remainingCDs, including enzyme purification and CD activity staining,is necessary and is currently in progress. We will also analyzethe genome-wide expression profile in each CD gene-disruptedmutant using DNA microarray technology. It is possible thatone gene disruption would affect the expression of other CDproteins and the whole metabolic profile in E. coli.

ACKNOWLEDGMENTS

The technical assistance of S. Yamada of our laboratory is greatlyappreciated.

This work was supported in part by a grant-in-aid from JapanSociety for the Promotion of Science for Young Scientists (no.01978 to N.A.) and by a grant from Ajinomoto Co., Inc., to H.T.

FOOTNOTES

* Corresponding author. Mailing address: Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan. Phone: 81-776-61-6000. Fax: 81-776-61-6015. E-mail: hiro{at}fpu.ac.jp.

Present address: Division of Applied Bioscience, Graduate Schoolof Agriculture, Hokkaido University, Sapporo 060-8589, Japan.

REFERENCES

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