Light and sucrose up-regulate the expression level of Arabidopsis cystathionine γ-synthase, the key enzyme of methionine biosynthesis pathway

(+)-Borneol is a desirable monoterpenoid with effective anti-inflammatory and analgesic effects that is known as soft gold. (+)-bornyl diphosphate synthase is the key enzyme in the (+)-borneol biosynthesis pathway. Despite several reported (+)-bornyl diphosphate synthase genes, relatively low (+)-borneol production hinders the attempts to synthesize it using microbial fermentation. Here, we identified the highly specific (+)-bornyl diphosphate synthase CbTPS1 from Cinnamomum burmanni. An in vitro assay showed that (+)-borneol was the main product of CbTPS1 (88.70% of the total products), and the Km value was 5.11 ± 1.70 μM with a kcat value of 0.01 s–1. Further, we reconstituted the (+)-borneol biosynthetic pathway in Saccharomyces cerevisiae. After tailored truncation and adding Kozak sequences, the (+)-borneol yield was improved by 96.33-fold to 2.89 mg⋅L–1 compared with the initial strain in shake flasks. This work is the first reported attempt to produce (+)-borneol by microbial fermentation. It lays a foundation for further pathway reconstruction and metabolic engineering production of this valuable natural monoterpenoid.

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Cryptochrome 2 is involved in betacyanin decomposition induced by blue light in Suaeda salsa

Functional Plant Biology ◽

10.1071/fp06073 ◽

2006 ◽

Vol 33 (7) ◽

pp. 697 ◽

Cited By ~ 6

Author(s):

Wang Chang-Quan ◽

Liu Tao

Keyword(s):

Western Blot ◽

Blue Light ◽

Hypocotyl Elongation ◽

Suaeda Salsa ◽

Biosynthesis Pathway ◽

Tyrosine Hydroxylation ◽

Key Enzyme ◽

Cryptochrome 2 ◽

Hydroxylation Activity

Seeds of the halophyte Suaeda salsa (L.) Pall. were cultured in 24 h dark and 14 h blue light / 10 h dark to examine the role of blue light and the blue-light-absorbing photoreceptor cryptochrome 2 (CRY2) in betacyanin accumulation, hypocotyl elongation and cotyledon opening in S. salsa seedlings. Darkness significantly promoted betacyanin accumulation and hypocotyl elongation but inhibited cotyledon opening. Blue light suppressed betacyanin accumulation and hypocotyl elongation but stimulated cotyledon opening. Betacyanin in S. salsa seedlings decomposed with time in blue light. Western blot analysis showed that CRY2 protein accumulated both in hypocotyls and cotyledons of S. salsa seedlings grown in dark, but degraded with time in blue light, which was paralleled by a decrease of tyrosine hydroxylation activity of tyrosinase, a key enzyme involved in the betalain biosynthesis pathway. These results suggest that CRY2 protein mediates betacyanin decomposition via inactivation of tyrosinase in S. salsa seedlings, and the blue-light-dependent degradation of CRY2 protein is crucial to its function.

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A nitrogenase-like enzyme system catalyzes methionine, ethylene, and methane biogenesis

Science ◽

10.1126/science.abb6310 ◽

2020 ◽

Vol 369 (6507) ◽

pp. 1094-1098 ◽

Cited By ~ 3

Author(s):

Justin A. North ◽

Adrienne B. Narrowe ◽

Weili Xiong ◽

Kathryn M. Byerly ◽

Guanqi Zhao ◽

...

Keyword(s):

Dimethyl Sulfide ◽

Enzyme System ◽

Biosynthesis Pathway ◽

Methionine Biosynthesis ◽

Biogenic Methane ◽

Organic Sulfur Compounds ◽

Bond Breakage ◽

Freshwater Bacteria ◽

Volatile Organic ◽

Gaseous Hydrocarbons

Bacterial production of gaseous hydrocarbons such as ethylene and methane affects soil environments and atmospheric climate. We demonstrate that biogenic methane and ethylene from terrestrial and freshwater bacteria are directly produced by a previously unknown methionine biosynthesis pathway. This pathway, present in numerous species, uses a nitrogenase-like reductase that is distinct from known nitrogenases and nitrogenase-like reductases and specifically functions in C–S bond breakage to reduce ubiquitous and appreciable volatile organic sulfur compounds such as dimethyl sulfide and (2-methylthio)ethanol. Liberated methanethiol serves as the immediate precursor to methionine, while ethylene or methane is released into the environment. Anaerobic ethylene production by this pathway apparently explains the long-standing observation of ethylene accumulation in oxygen-depleted soils. Methane production reveals an additional bacterial pathway distinct from archaeal methanogenesis.

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Overexpression of an optimized Aspergillus sulphureus β-mannanase gene in Pichia pastoris

Biologia ◽

10.2478/s11756-009-0043-5 ◽

2009 ◽

Vol 64 (2) ◽

Cited By ~ 8

Author(s):

Xiaoling Chen ◽

Jiaoyun Qiao ◽

Haifeng Yu ◽

Yunhe Cao

Keyword(s):

Pichia Pastoris ◽

Expression Level ◽

Wild Type ◽

Feed Supplement ◽

Bias Effect ◽

Key Enzyme ◽

Aspergillus Sulphureus ◽

Code Bias ◽

High Level ◽

High Expression Level

Abstractβ-Mannanase (EC 3.2.1.78) is a key enzyme to hydrolyze the β-mannosidic linkages in mannan and heteromannan. The expression of a wild type β-mannanase (manWT) of Aspergillus sulphureus in Pichia pastoris is not high enough for its application in feed supplement. To earn a high expression level, the manWT gene was firstly optimized to manM according to the code bias of P. pastoris, which was then inserted into pPICzαA and transformed into P. pastoris strain X-33. In the induction by methanol, β-mannanase was expressed in high level with 32% increase in comparison with the manWT gene expressed in P. pastoris in shaken flask. In a 10-L fermenter, the manM was expressed in 9-fold higher level than that in shaken flask, which yielded the enzyme activity of 1100 U/mL. This is the first study on codon bias effect on the β-mannanase gene expression level, which helps to achieve high β-mannanase yield and enzymatic activity in P. pastoris.

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Transcriptome-wide Identification and Quantification of Caffeoylquinic Acid Biosynthesis Pathway and Prediction of their Putative BAHDs Gene Complex in A. spathulifolius

10.20944/preprints202105.0527.v1 ◽

2021 ◽

Author(s):

SeonJoo Park ◽

Jean Claude Sivagami ◽

Sunmi Park

Keyword(s):

Phenylpropanoid Pathway ◽

Biosynthesis Pathway ◽

Gene Complex ◽

Biotic And Abiotic Stress ◽

Acid Biosynthesis ◽

Gene Encoding ◽

Caffeoylquinic Acid ◽

Key Enzyme ◽

By Products

The phenylpropanoid pathway is a major secondary metabolite pathway that helps plants overcome biotic and abiotic stress and produces various by-products that promote human health. Its byproduct, chloroquinic acid (CQA), is a soluble phenolic compound present in many angiosperms. Hy-droxycinnamate-CoA shikimate/quinate transferase(BAHDs superfamily enzyme) is a significant en-zyme that plays a role in accumulating CQA biosynthesis. This study analyzed transcriptome-wide identification of the phenylpropanoid to chloroquinic acid biosynthesis candidate genes in A. spathulifolius flowers and leaves. Transcriptomic analyses of the flowers and leaves showed a differential expression of the PPP and CQA biosynthesis regulated unigenes. An analysis of PPP captive unigenes revealed the following: the major duplication of the key enzyme, PAL, 120 unigenes in leaves and 76 in flowers; the gene encoding C3’H, 169 unigenes in leaves and 140 unigenes in flowers; duplicated unigenes of 4CL, 41 in leaves and 27 in flowers. In addition, C4H unigenes had 12 unigenes in the leaves of A. spathulifolius and four in the flowers. The characterization of the BAHDs superfamily members identified 82 in leaves and 72 in flowers. Among them, phylogenetic analysis showed that five unigenes encoded HQT and three en-coded HCT in A. spathulifolius. The three HQT are common to both leaves and flowers, whereas the two HQT were specialized for leaves. The pattern of HQT synthesis was upregulated in flowers, whereas HCT was expressed strongly in the leaves of A. spathulifolius. Overall, 4CL, C4H, and HQT are expressed strongly in flowers, and caffeic acid and HCT show more expression in leaves. Therefore, CQA biosynthesis occurs in the flowers of A. spathulifolius rather than leaves.

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Structural analysis of mycobacterial homoserine transacetylases central to methionine biosynthesis reveals druggable active site

Scientific Reports ◽

10.1038/s41598-019-56722-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Catherine T. Chaton ◽

Emily S. Rodriguez ◽

Robert W. Reed ◽

Jian Li ◽

Cameron W. Kenner ◽

...

Keyword(s):

Infectious Disease ◽

Mycobacterium Tuberculosis ◽

Active Site ◽

Fungal Species ◽

Biosynthesis Pathway ◽

Methionine Biosynthesis ◽

Structure Based Drug Design ◽

High Degree ◽

Homoserine Transacetylase ◽

Host Metabolism

AbstractMycobacterium tuberculosis is the cause of the world’s most deadly infectious disease. Efforts are underway to target the methionine biosynthesis pathway, as it is not part of the host metabolism. The homoserine transacetylase MetX converts l-homoserine to O-acetyl-l-homoserine at the committed step of this pathway. In order to facilitate structure-based drug design, we determined the high-resolution crystal structures of three MetX proteins, including M. tuberculosis (MtMetX), Mycolicibacterium abscessus (MaMetX), and Mycolicibacterium hassiacum (MhMetX). A comparison of homoserine transacetylases from other bacterial and fungal species reveals a high degree of structural conservation amongst the enzymes. Utilizing homologous structures with bound cofactors, we analyzed the potential ligandability of MetX. The deep active-site tunnel surrounding the catalytic serine yielded many consensus clusters during mapping, suggesting that MtMetX is highly druggable.

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Pathways of Assimilative Sulfur Metabolism inPseudomonas putida

Journal of Bacteriology ◽

10.1128/jb.181.18.5833-5837.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5833-5837 ◽

Cited By ~ 47

Author(s):

Paul Vermeij ◽

Michael A. Kertesz

Keyword(s):

Pseudomonas Aeruginosa ◽

Pseudomonas Putida ◽

Sulfur Metabolism ◽

Sulfur Source ◽

Methionine Biosynthesis ◽

Cysteine Synthase ◽

Transsulfuration Pathway ◽

Key Enzyme ◽

Low Levels

ABSTRACT Cysteine and methionine biosynthesis was studied inPseudomonas putida S-313 and Pseudomonas aeruginosa PAO1. Both these organisms used direct sulfhydrylation of O-succinylhomoserine for the synthesis of methionine but also contained substantial levels of O-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine γ-synthase and cystathionine β-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In P. aeruginosa, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows P. aeruginosa to grow rapidly with methionine as the sole sulfur source. P. putida S-313 also grew well with methionine as the sulfur source, but no cystathionine γ-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway, P. putida desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine γ-lyase, which was upregulated under these conditions. A transposon mutant of P. putida that was defective in the alkanesulfonatase locus (ssuD) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in P. putida methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine.

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Potent Anti-Trypanosoma cruzi Activities of Oxidosqualene Cyclase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.4.1210-1215.2001 ◽

2001 ◽

Vol 45 (4) ◽

pp. 1210-1215 ◽

Cited By ~ 50

Author(s):

Frederick S. Buckner ◽

John H. Griffin ◽

Aaron J. Wilson ◽

Wesley C. Van Voorhis

Keyword(s):

Trypanosoma Cruzi ◽

High Energy ◽

Sterol Biosynthesis ◽

Biosynthesis Pathway ◽

Lead Compound ◽

Major Health Problem ◽

Major Health ◽

Oxidosqualene Cyclase ◽

Key Enzyme ◽

Related Compounds

ABSTRACT Trypanosoma cruzi is the protozoan agent that causes Chagas' disease, a major health problem in Latin America. Better drugs are needed to treat infected individuals. The sterol biosynthesis pathway is a potentially excellent target for drug therapy againstT. cruzi. In this study, we investigated the antitrypanosomal activities of a series of compounds designed to inhibit a key enzyme in sterol biosynthesis, oxidosqualene cyclase. This enzyme converts 2,3-oxidosqualene to the tetracyclic product, lanosterol. The lead compound,N-(4E,8E)-5,9, 13-trimethyl-4,8, 12-tetradecatrien-1-ylpyridinium, is an electron-poor aromatic mimic of a monocyclized transition state or high-energy intermediate formed from oxidosqualene. This compound and 27 related compounds were tested against mammalian-stage T. cruzi, and 12 inhibited growth by 50% at concentrations below 25 nM. The lead compound was shown to cause an accumulation of oxidosqualene and decreased production of lanosterol and ergosterol, consistent with specific inhibition of the oxidosqualene cyclase. The data demonstrate potent anti-T. cruzi activity associated with inhibition of oxidosqualene cyclase.

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