In Escherichia coli, three additional proteins having L-cysteine desulfhydrase activity were identified as O-acetylserine sulfhydrylase-A, O-acetylserine sulfhydrylase-B, and MalY protein, in addition to tryptophanase and cystathionine ␤-lyase, which have been reported previously. The gene disruption for each protein was significantly effective for overproduction of L-cysteine and L-cystine. Growth phenotype and transcriptional analyses suggest that tryptophanase contributes primarily to L-cysteine degradation.L-cysteine is an important amino acid in terms of its applications in 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-serine from acetyl-coenzyme A (CoA) and L-serine (8, 9, 16), high-level production of L-cysteine from glucose has not been successfully achieved in microorganisms. In order to obtain L-cysteine producers, we previously constructed Escherichia coli cysE genes that encode altered SATs. These genes were genetically desensitized to the feedback inhibition by L-cysteine through site-directed or random mutagenesis (21, 32). We found that, in the recombinant E. coli cells expressing the altered cysE gene, there was a marked production of L-cysteine plus L-cystine.In the same investigation (21), it was demonstrated that proteins with L-cysteine desulfhydrase (CD) activity play an important role in L-cysteine degradation in E. coli cells. In order to further improve L-cysteine production, a host strain having a lower level of CD activity must be constructed. CD is known to catalyze the degradation of L-cysteine to pyruvate, ammonia, and sulfide by the following reaction: HSCH 2 CH(NH 2 )COOH ϩ H 2 O 3 CH 3 COCOOH ϩ H 2 S ϩ NH 3 . This type of enzyme activity has been demonstrated 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-␤-lyase (CBL) (17) encoded by metC, which catalyzes mainly the conversion of cystathionine to homocysteine, pyruvate, and ammonia (9), as well as tryptophanase (TNase) encoded by tnaA, which primarily degrades L-tryptophan to indole, pyruvate, and ammonia (22), has been shown to exhibit CD activity in vitro (9, 23). We have previously reported that CBL and TNase catalyzed the CD reaction and acted on L-cysteine degradation in E. coli cells by analyses with CD activity staining and gene disruption (2). However, the double CD gene-disrupted mutant still had a low level of CD activity, suggesting that unknown CD proteins remain to be identified. Thus, we report here further identification and characterization of the CDs involved in L-cysteine degradation in E. coli.

A toxic L-proline analogue, L-azetidine-2-carboxylic acid (AZC), causes misfolding of the proteins into which it is incorporated competitively with L-proline, thereby inhibiting the growth of the cells. AZC enters budding yeast Saccharomyces cerevisiae cells primarily through the general amino acid permease Gap1, not through the proline-specific permease Put4. We isolated an AZChypersensitive mutant that cannot grow even at low concentrations of AZC because of the accumulation of intracellular AZC. By screening through a yeast genomic library, the mutant was found to carry an allele of RSP5 encoding an E3 ubiquitin ligase. A single amino acid change replacing Ala (GCA) at position 401 with Glu (GAA) showed that Ala-401 in the third WW domain (a protein interaction module) is not conserved in the domain. The addition of NH 4 ؉ to yeast cells growing on L-proline induced rapid ubiquitination, endocytosis, and vacuolar degradation of the plasma membrane protein Gap1. However, immunoblot and permease assays indicated that Gap1 in the rsp5 mutant remained stable and active on the plasma membrane probably with no ubiquitination, leading to AZC accumulation and hypersensitivity. The rsp5 mutants also showed hypersensitivity to various stresses (toxic amino acid analogues, high temperature in a rich medium, and oxidative treatments) and defects in spore growth. These results suggest that Rsp5 is involved in selective degradation of abnormal proteins and specific proteins for spore growth, in addition to nitrogenregulated degradation of Gap1. Furthermore, Ala-401 of Rsp5 was considered to have an important role in the ubiquitination of targeted proteins.A ddition of some amino acid analogues can induce a transient physiological stress response in cells comparable to that of heat shock stress (1-3). Most analogues are transported into cells via amino acid permeases and cause misfolding of the proteins as they compete with naturally occurring amino acids. The accumulation of abnormal proteins, in turn, inhibits cell growth. Recently, Trotter and colleagues (4, 5) found that L-azetidine-2-carboxylic acid (AZC), a toxic four-membered ring analogue of L-proline, arrests proliferation in the G 1 phase of the cell cycle by the same mechanism as temperature up-shift. AZC is an unusual imino acid found only in several plants belonging to the Lilaceae family (6, 7), but can replace L-proline in proteins of bacteria and animal cells (8, 9), presumably in those of yeast cells (5). When AZC was added to cells of yeast Saccharomyces cerevisiae growing in minimal medium, cell viability gradually decreased, causing cell death (M. Nomura and H.T., unpublished work).The accumulation of abnormal or misfolded proteins in cells under stress is a serious problem. To overcome it, the following two strategies can be considered: (i) to degrade the proteins through a ubiquitin-proteasome system or (ii) to refold the proteins by molecular chaperones. We isolated AZC-resistant mutants and strains and elucidated the mechanisms of AZC resistance. Lar...

Since some amino acids, polyols and sugars in cells are thought to be osmoprotectants, we expected that several amino acids might also contribute to enhancing freeze tolerance in yeast cells. In fact, proline and charged amino acids such as glutamate, arginine and lysine showed a marked cryoprotective activity nearly equivalent to that of glycerol or trehalose, both known as major cryoprotectants for Saccharomyces cerevisiae. To investigate the cryoprotective effect of proline on the freezing stress of yeast, we isolated proline-analogue-resistant mutants derived from a proline-non-utilizing strain of S. cerevisiae. When cultured in liquid minimal medium, many mutants showed a prominent increase, two- to approximately tenfold, in cell viability compared to the parent after freezing in the medium at -20 degrees C for 1 week. Some of the freeze-tolerant mutants were found to accumulate a higher amount of proline, as well as of glutamate and arginine which are involved in proline metabolism. It was also observed that proline-non-utilizer and the freeze-tolerant mutants were able to grow against osmotic stress. These results suggest that the increased flux in the metabolic pathway of specific amino acids such as proline is effective for breeding novel freeze-tolerant yeasts.

We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular L-proline derived from L-proline analogue-resistant mutants. The mutation responsible for the analogue resistance and L-proline accumulation was a single nuclear dominant mutation. By introducing the mutant-derived genomic library into a non-L-proline-utilizing strain, the mutant was found to carry an allele of the wild-type PRO1 gene encoding ␥-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC). Interestingly, the allele of PRO1 was shown to enhance the activities of ␥-glutamyl kinase and ␥-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis from L-glutamate and which together may form a complex in vivo. When cultured in liquid minimal medium, yeast cells expressing the mutated ␥-glutamyl kinase were found to accumulate intracellular L-proline and showed a prominent increase in cell viability after freezing at ؊20°C compared to the viability of cells harboring the wild-type PRO1 gene. These results suggest that the altered ␥-glutamyl kinase results in stabilization of the complex or has an indirect effect on ␥-glutamyl phosphate reductase activity, which leads to an increase in L-proline production in Saccharomyces cerevisiae. The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains.Frozen-dough technology has recently been used in the baking industry to supply oven-fresh bakery products to consumers. Many freeze-tolerant yeasts have been isolated from natural sources and under natural culture conditions, and many have also been constructed by conventional mutation techniques (11,13,21,23,25). However, the mechanism of freeze tolerance is not well understood, and a baker's yeast that provides good leavening qualities for both sweet-and lean-thawed doughs after frozen storage has not yet been developed.We previously investigated the cryoprotective effects of amino acids on freezing stress in the yeast Saccharomyces cerevisiae and found that L-proline, which is known to be an osmoprotectant (5, 9), has cryoprotective activity that is nearly equal that of glycerol or trehalose (38). In bacteria, L-proline biosynthesis from L-glutamate has been shown to be regulated by end product inhibition of ␥-glutamyl kinase (␥-GK) activity (26, 35). L-Proline-overproducing mutants of Escherichia coli (7), Salmonella enterica serovar Typhimurium (4), and Serratia marcescens (27) have mutations which result in desensitization of L-proline feedback inhibition of ␥-GK. S. cerevisiae synthesizes L-proline from L-glutamate via the intermediates ␥-glutamyl phosphate (␥-GP), glutamate-␥-semialdehyde (GSA), and ⌬ 1 -pyrroline-5-carboxylate (P5C) by almost the same pathway found in bacteria (Fig. 1). Three enzymes, ␥-GK (the PRO1 gene product), ␥-GP reductase (␥-GPR) (the PRO2 gene product), and P5C reductase (the PRO3 gene product), are involved...

Organisms that overproduced l-cysteine andl-cystine from glucose were constructed by usingEscherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg ofl-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production ofl-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production ofl-cysteine plus l-cystine was markedly increased compared to production in JM39.