Discussion on the Biochemical Similarity in Metabolic Principles Based on Leucine Biosynthesis Pathway

Artemisinin-based medicines are the most effective treatment for malaria. To date, the wormwood plants (Artemisia annua L.) are the main source of artemisinin. Due to the limited nature of this source, considerable efforts are directed towards the development of methods for artemisinin production via heterologous expression systems. We used in this study agrobacterial transformation to transfer the genetic module of the artemisinin biosynthesis pathway into plants and then analyzed its transcription in a heterologous host. Tobacco plants were transformed with the artemisinin biosynthesis genes encoding amorpha-4,11-diene synthase, artemisin-aldehyde All(13) reductase, amorpha-4,11-diene monooxygenase, cytochrome P450 reductase from A. annua and yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase cloned in the pArtemC vector; farnesyl diphosphate synthase and aldehyde dehydrogenase were used to transform the plants as parts of vector p2356. As a result of transformation with the pArtemC and p2356 vectors, in twos transgenic lines with all target genes were obtained. Five genes of artemisinin biosynthesis and two genes of biosynthesis of its precursors were successfully transferred into the genome of transgenic tobacco lines as a result of the co-transformation with abovementioned vectors. Thus, the entire artemisinin biosynthesis pathway was first reconstructed in heterologous plants: the transcription of the artemisinin biosynthesis genes in the tobacco plants was shown via RT-PCR. The obtained results will be used in further research on expression systems for the production of artemisinin and other non-protein substances in heterologous host plants. artemisinin, malaria, metabolic engineering, tobacco, transgenic plants This work was supported by a Grant from the Russian Science Foundation no. 19-14-00190.

Download Full-text

Monosaccharide Biosynthesis Pathways Database

Glycobiology ◽

10.1093/glycob/cwab030 ◽

2021 ◽

Author(s):

Jaya Srivastava ◽

P Sunthar ◽

Petety V Balaji

Keyword(s):

Expression Profiling ◽

Experimental Studies ◽

Structural Complexity ◽

Evolutionary Relationships ◽

Enzyme Assays ◽

Biosynthesis Pathway ◽

Knock Out ◽

Set Up ◽

Function Relationship ◽

Natural Glycans

Abstract A distinctive feature of glycans vis-à-vis proteins and nucleic acids is its structural complexity which arises from the huge repertoire of monosaccharides, isomeric linkages and branching. A very large number of monosaccharides have so far been discovered in natural glycans. Experimentally, pathways for the biosynthesis have been characterized completely for 55 monosaccharides and partially for a few more. However, there is no single platform which provides information about monosaccharide biosynthesis pathways and associated enzymes We have gathered 572 experimentally characterized enzymes of 66 biosynthesis pathways from literature and set up a first of its kind database called the Monosaccharide Biosynthesis Pathways Database http://www.bio.iitb.ac.in/mbpd/). Annotations such as the reaction catalysed, substrate specificity, biosynthesis pathway and PubMed IDs are provided for all the enzymes in the database. Sequence homologs of the experimentally characterized enzymes found in nearly 13,000 completely sequenced genomes from Bacteria and Archaea have also been included in the database. This platform will help in the deduction of evolutionary relationships among enzymes such as aminotransferases, nucleotidyltransferases, acetyltransferases and SDR family enzymes. It can also facilitate experimental studies such as direct enzyme assays to validate putative annotations, establish structure–function relationship, expression profiling to determine the function, determine the phenotypic consequences of gene knock-out/knock-in and complementation studies.

Download Full-text

Protein complex formation in methionine chain-elongation and leucine biosynthesis

Scientific Reports ◽

10.1038/s41598-021-82790-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Li-Qun Chen ◽

Shweta Chhajed ◽

Tong Zhang ◽

Joseph M. Collins ◽

Qiuying Pang ◽

...

Keyword(s):

Protein Complexes ◽

Complete Characterization ◽

Bimolecular Fluorescence Complementation ◽

Chain Elongation ◽

Substrate Channeling ◽

Leucine Biosynthesis ◽

Protein Complex Formation ◽

Genes Encoding ◽

Isopropylmalate Dehydrogenase

AbstractDuring the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

Download Full-text

Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene

Bioengineering ◽

10.3390/bioengineering8010014 ◽

2021 ◽

Vol 8 (1) ◽

pp. 14

Author(s):

Yu Zhang ◽

Tsan-Yu Chiu ◽

Jin-Tao Zhang ◽

Shu-Jie Wang ◽

Shu-Wen Wang ◽

...

Keyword(s):

Copy Number ◽

Chromosome Rearrangement ◽

Fold Increase ◽

Host Strain ◽

Target Product ◽

Biosynthesis Pathway ◽

Suitable Host ◽

Enhanced Production ◽

Condition Optimization ◽

Heterologous Pathway

Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product.

Download Full-text

Spectroscopic methods to quantify molecules of the heme‐biosynthesis pathway – a review of laboratory work and point‐of‐care approaches

Translational Biophotonics ◽

10.1002/tbio.202000026 ◽

2021 ◽

Author(s):

Christian Heckl ◽

Maximilian Eisel ◽

Alexander Lang ◽

Christian Homann ◽

Michael Paal ◽

...

Keyword(s):

Point Of Care ◽

Heme Biosynthesis ◽

Laboratory Work ◽

Spectroscopic Methods ◽

Biosynthesis Pathway

Download Full-text

The role of pH in the melanin biosynthesis pathway.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)34190-5 ◽

1982 ◽

Vol 257 (15) ◽

pp. 8738-8744

Author(s):

F G Cánovas ◽

F García-Carmona ◽

J V Sánchez ◽

J L Pastor ◽

J A Teruel

Keyword(s):

Biosynthesis Pathway ◽

Melanin Biosynthesis

Download Full-text

Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor

Scientific Reports ◽

10.1038/s41598-021-85486-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yu-Cheng Chang ◽

Yi-Ching Chiu ◽

Nai-Wen Tsao ◽

Yuan-Lin Chou ◽

Choon-Meng Tan ◽

...

Keyword(s):

Transcriptome Analysis ◽

Natural Antioxidants ◽

Biosynthesis Pathway ◽

Comparative Transcriptome ◽

Amaranthus Tricolor ◽

Enzymatic Assays ◽

Comparative Transcriptome Analysis ◽

Elevated Expression ◽

Green Coloration

AbstractAmaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1, AmDODAα1, AmDODAα2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAα1 and AmDODAα2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae.

Download Full-text