CAD1 Gene Research Articles

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate

The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440mUmg−1 and 30mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60mM (7.8gl−1), a molar yield of 0.4molmol−1, and a volumetric productivity of 2.1mmoll−1h−1.

Functional analysis of cis-aconitate decarboxylase and trans-aconitate metabolism in riboflavin-producing filamentous Ashbya gossypii

In Ashbya gossypii, isocitrate lyase (ICL1) is a very crucial enzyme for riboflavin production. Itaconate, the inhibitor of ICL1, has been used as an antimetabolite for mutagenic studies in A. gossypii. It has been reported that itaconate is produced from cis-aconitate by cis-aconitate decarboxylase (CAD1) in Aspergillus terreus. In this study, identification of CAD1 gene and determination of the presence of itaconate in the riboflavin biosynthetic pathway in A. gossypii were carried out to confirm itaconate metabolism. Although no CAD1 candidate gene was found and no itaconate production was observed, cis- and trans-aconitate were detected in the riboflavin production phase. It is known that trans-aconitate inhibits aconitase (ACO1) in the tricarboxylic acid cycle. In A. gossypii, the transcription level of AGR110Wp, the homolog of trans-aconitate 3-methyltransferase (TMT1), was enhanced by almost threefold during riboflavin production than that during its growth phase. TMT1 catalyzes the methylation reaction of trans-aconitate in Saccharomyces cerevisiae. Thus, these results suggest that the enhancement of the transcription level of this TMT1 homolog decreases the trans-aconitate level, which may mitigate the inhibition of ACO1 by oxidative stress in the riboflavin biosynthetic pathway in A. gossypii. This is a novel finding in A. gossypii, which may open new metabolic engineering ideas for improving riboflavin productivity.

Salicylic Acid and a Chitin Elicitor Both Control Expression of theCAD1Gene Involved in the Plant Immunity ofArabidopsis

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.

Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A.

The cotton (+)-delta-cadinene synthase (CAD1), a sesquiterpene cyclase, catalyzes a branch-point step leading to biosynthesis of sesquiterpene phytoalexins, including gossypol. CAD1-A is a member of CAD1 gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors. We isolated several WRKY cDNAs from Gossypium arboreum. One of them, GaWRKY1, encodes a protein containing a single WRKY domain and a putative N-terminal Leu zipper. Similar to genes encoding enzymes of cotton sesquiterpene pathway, GaWRKY1 was down-regulated in a glandless cotton cultivar that contained much less gossypol. GaWRKY1 showed a temporal and spatial pattern of expression comparable to that of CAD1-A in various aerial organs examined, including sepal, stigma, anther, and developing seeds. In suspension cells, expression of both GaWRKY1 and CAD1-A genes and biosynthesis of sesquiterpene aldehydes were strongly induced by a fungal elicitor preparation and methyl jasmonate. GaWRKY1 interacted with the 3x W-box derived from CAD1-A promoter in yeast (Saccharomyces cerevisiae) one-hybrid system and in vitro. Furthermore, in transgenic Arabidopsis plants, overexpression of GaWRKY1 highly activated the CAD1-A promoter, and transient assay in tobacco (Nicotiana tabacum) leaves demonstrated that W-box was required for this activation. These results suggest that GaWRKY1 participates in regulation of sesquiterpene biosynthesis in cotton, and CAD1-A is a target gene of this transcription factor.

The Arabidopsis CAD1 gene that controls cell death and defense activation encodes a novel protein containing MACPF domain

The Arabidopsis CAD1 gene that controls cell death and defense activation encodes a novel protein containing MACPF domain

Isolation of a (+)- δ-cadinene synthase gene CAD1-A and analysis of its expression pattern in seedlings ofGossypium arboreum L.

The cotton sesquiterpene cyclase, (+)-delta-cadinene synthase, is encoded by a gene family, which can be divided into two subfamilies:CAD1-A and CAD1-C. The geneCAD1-A was isolated fromG. arboreum. In situ hybridization performed on seven-day-old cotton seedlings localized transcripts of both the CAD1 -A and CAD1 -C mainly in lateral root primordium and apical ground meristem, vascular tissues of emerging lateral roots, and also in procambium and some subepidermal cells of the hypocotyl. The CAD1 -A promoter showed a similar tissue-specificity in transgenic tobacco plants. Histochemistry showed occurrence of sesquiterpene aldehydes in outer cells of the lateral root tips, as well as in pigment glands. The CAD1 gene expression in G.arboreum seedlings and the spatial pattern of sesquiterpene biosynthesis constitute a chemical defense machinery in cotton seedlings.

Expression pattern of (+)-delta-cadinene synthase genes and biosynthesis of sesquiterpene aldehydes in plants of Gossypium arboreum L.

The cotton (+)-delta-cadinene synthase, a sesquiterpene cyclase, is encoded by a complex gene family which, based on homology, can be divided into two subfamilies: cad1-A and cad1-C. Southern blots revealed several members of the cad1-C subfamily, and a single member of the cad1-A subfamily, in the diploid Gossypium arboreum genome. One of the cad1-C genes, cad1-C3, was isolated from this species. According to reverse transcriptase-polymerase chain reaction, transcripts of both cad1-C and cad1-A genes appeared in roots from the second day post germination and in 1-d-old cotyledons, whereas the transcription levels were too low to be detected in the hypocotyls. Initially, sesquiterpene cyclase activities were found to be high in the seedlings, then dropped in aerial organs but increased in roots during development. Sesquiterpene aldehyde contents followed the same pattern. In fully developed plants, the transcripts of cad1-C were detected in stems, leaves and pericarps, as well as in the sepals and petals 3 d before anthesis, but not at the day of anthesis. In contrast, cad1-A transcripts were not detected in any of these aerial organs. The sesquiterpene aldehyde contents increased in petals but decreased in sepals after anthesis. Treatment of G. arboreum stems with a Verticillium dahliae elicitor-preparation activated cad1-A transcription, but a significant level of cad1-C transcripts was detected both before and after elicitation. In G. hirsutum cv. GL-5, a glandless cultivar, the cad1-C gene was activated by the same fungal elicitor, followed by the synthesis of the sesquiterpene cyclase, and accumulation of sesquiterpene aldehydes. The cad1 gene expression during development and in response to elicitation, as well as the spatial and temporal pattern of sesquiterpene biosynthesis, constitute a chemical defense machinery in cotton plants.

Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe.

Phytochelatins (PCs), a family of heavy metal-inducible peptides important in the detoxification of heavy metals, have been identified in plants and some microorganisms, including Schizosaccharomyces pombe, but not in animals. PCs are synthesized enzymatically from glutathione (GSH) by PC synthase in the presence of heavy metal ions. In Arabidopsis, the CAD1 gene, identified by using Cd-sensitive, PC-deficient cad1 mutants, has been proposed to encode PC synthase. Using a positional cloning strategy, we have isolated the CAD1 gene. Database searches identified a homologous gene in S. pombe, and a mutant with a targeted deletion of this gene was also Cd sensitive and PC deficient. Extracts of Escherichia coli cells expressing a CAD1 cDNA or the S. pombe gene catalyzing GSH-dependent, heavy metal-activated synthesis of PCs in vitro demonstrated that both genes encode PC synthase activity. Both enzymes were activated by a range of metal ions. In contrast, reverse transcription-polymerase chain reaction experiments showed that expression of the CAD1 mRNA is not influenced by the presence of Cd. A comparison of the two predicted amino acid sequences revealed a highly conserved N-terminal region, which is presumed to be the catalytic domain, and a variable C-terminal region containing multiple Cys residues, which is proposed to be involved in activation of the enzyme by metal ions. Interestingly, a similar gene was identified in the nematode, Caenorhabditis elegans, suggesting that PCs may also be expressed in some animal species.

Yeast bZip proteins mediate pleiotropic drug and metal resistance.

Saccharomyces cerevisiae contains a group of transcription factors related to mammalian c-Jun. This yeast Jun-family of proteins consists of GCN4, a regulator of genes involved in amino acid biosynthesis, and yAP-1, a factor conferring pleiotropic drug resistance when overexpressed. In the work described here, we show that a third member of the yeast Jun-family exists. This protein has been designated CAD1 and provides resistance to cadmium when present on a high-copy plasmid. CAD1 and yAP-1 are related in their amino-terminal DNA binding domains and can recognize the same DNA target site in vitro. Overproduction of CAD1 leads to transcriptional activation of an artificial reporter gene in delta yap1 cells. High level production of either CAD1 or yAP-1 causes cells to acquire a pleiotropic drug-resistant phenotype and to be able to tolerate normally toxic levels of iron chelators and zinc. Surprisingly, disruption of the CAD1 gene has no effect on the normal cellular resistance to cadmium but delta yap1 mutants are hypersensitive to this cytotoxic metal. The cadmium hypersensitivity of the delta yap1 mutant described here indicates that one major role of YAP1 in the yeast cell is to mediate resistance to this metal.