scholarly journals Multiplex Genome Editing in Yeast by CRISPR/Cas9 – A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways

2021 ◽  
Vol 12 ◽  
Author(s):  
Joseph Christian Utomo ◽  
Connor Lorne Hodgins ◽  
Dae-Kyun Ro
Keyword(s):  
Genome Editing ◽  
Metabolic Pathways ◽  
Functional Characterization ◽  
Primary Metabolism ◽  
Biosynthetic Pathways ◽  
Post Translational Modification ◽  
Precise Integration ◽  
Specialized Metabolism ◽  
Specialized Metabolites ◽  
Plant Specialized Metabolism

Numerous important pharmaceuticals and nutraceuticals originate from plant specialized metabolites, most of which are synthesized via complex biosynthetic pathways. The elucidation of these pathways is critical for the applicable uses of these compounds. Although the rapid progress of the omics technology has revolutionized the identification of candidate genes involved in these pathways, the functional characterization of these genes remains a major bottleneck. Baker’s yeast (Saccharomyces cerevisiae) has been used as a microbial platform for characterizing newly discovered metabolic genes in plant specialized metabolism. Using yeast for the investigation of numerous plant enzymes is a streamlined process because of yeast’s efficient transformation, limited endogenous specialized metabolism, partially sharing its primary metabolism with plants, and its capability of post-translational modification. Despite these advantages, reconstructing complex plant biosynthetic pathways in yeast can be time intensive. Since its discovery, CRISPR/Cas9 has greatly stimulated metabolic engineering in yeast. Yeast is a popular system for genome editing due to its efficient homology-directed repair mechanism, which allows precise integration of heterologous genes into its genome. One practical use of CRISPR/Cas9 in yeast is multiplex genome editing aimed at reconstructing complex metabolic pathways. This system has the capability of integrating multiple genes of interest in a single transformation, simplifying the reconstruction of complex pathways. As plant specialized metabolites usually have complex multigene biosynthetic pathways, the multiplex CRISPR/Cas9 system in yeast is suited well for functional genomics research in plant specialized metabolism. Here, we review the most advanced methods to achieve efficient multiplex CRISPR/Cas9 editing in yeast. We will also discuss how this powerful tool has been applied to benefit the study of plant specialized metabolism.

The Rise of Chemodiversity in Plants

Science ◽  
2012 ◽  
Vol 336 (6089) ◽  
pp. 1667-1670 ◽  
Author(s):  
Jing-Ke Weng ◽  
Ryan N. Philippe ◽  
Joseph P. Noel
Keyword(s):  
Protein Structure ◽  
Metabolic Pathways ◽  
Metabolic Enzymes ◽  
Structure And Function ◽  
Primary Metabolism ◽  
Catalytic Promiscuity ◽  
Terrestrial Plants ◽  
Specialized Metabolism ◽  
Geographically Dispersed ◽  
And Function

Plants possess multifunctional and rapidly evolving specialized metabolic enzymes. Many metabolites do not appear to be immediately required for survival; nonetheless, many may contribute to maintaining population fitness in fluctuating and geographically dispersed environments. Others may serve no contemporary function but are produced inevitably as minor products by single enzymes with varying levels of catalytic promiscuity. The dominance of the terrestrial realm by plants likely mirrored expansion of specialized metabolism originating from primary metabolic pathways. Compared with their evolutionarily constrained counterparts in primary metabolism, specialized metabolic enzymes may be more tolerant to mutations normally considered destabilizing to protein structure and function. If this is true, permissiveness may partially explain the pronounced chemodiversity of terrestrial plants.

scholarly journals Robust predictions of specialized metabolism genes through machine learning

2018 ◽  
Author(s):  
Bethany M. Moore ◽  
Peipei Wang ◽  
Pengxiang Fan ◽  
Bryan Leong ◽  
Craig A. Schenck ◽  
...  
Keyword(s):  
Machine Learning ◽  
Prediction Model ◽  
Gene Networks ◽  
Experimental Studies ◽  
Plant Genome ◽  
Primary Metabolism ◽  
Specialized Metabolism ◽  
Specialized Metabolites ◽  
General Metabolism ◽  
Pattern Sequence

AbstractPlant specialized metabolism (SM) enzymes produce lineage-specific metabolites with important ecological, evolutionary, and biotechnological implications. Using Arabidopsis thaliana as a model, we identified distinguishing characteristics of SM and GM (general metabolism, traditionally referred to as primary metabolism) genes through a detailed study of features including duplication pattern, sequence conservation, transcription, protein domain content, and gene network properties. Analysis of multiple sets of benchmark genes revealed that SM genes tend to be tandemly duplicated, co-expressed with their paralogs, narrowly expressed at lower levels, less conserved, and less well connected in gene networks relative to GM genes. Although the values of each of these features significantly differed between SM and GM genes, any single feature was ineffective at predicting SM from GM genes. Using machine learning methods to integrate all features, a well performing prediction model was established with a true positive rate of 0.87 and a true negative rate of 0.71. In addition, 86% of known SM genes not used to create the machine learning model were predicted as SM genes, further demonstrating its accuracy. We also demonstrated that the model could be further improved when we distinguished between SM, GM, and junction genes responsible for reactions shared by SM and GM pathways. Application of the prediction model led to the identification of 1,217 A. thaliana genes with previously unknown functions, providing a global, high-confidence estimate of SM gene content in a plant genome.SignificanceSpecialized metabolites are critical for plant-environment interactions, e.g., attracting pollinators or defending against herbivores, and are important sources of plant-based pharmaceuticals. However, it is unclear what proportion of enzyme-encoding genes play roles in specialized metabolism (SM) as opposed to general metabolism (GM) in any plant species. This is because of the diversity of specialized metabolites and the considerable number of incompletely characterized pathways responsible for their production. In addition, SM gene ancestors frequently played roles in GM. We evaluate features distinguishing SM and GM genes and build a computational model that accurately predicts SM genes. Our predictions provide candidates for experimental studies, and our modeling approach can be applied to other species that produce medicinally or industrially useful compounds.

scholarly journals Using Gene Expression to Study Specialized Metabolism—A Practical Guide

2021 ◽  
Vol 11 ◽  
Author(s):  
Riccardo Delli-Ponti ◽  
Devendra Shivhare ◽  
Marek Mutwil
Keyword(s):  
Gene Expression ◽  
Chemical Compounds ◽  
Cell Types ◽  
Biosynthetic Pathways ◽  
Expression Data ◽  
Specialized Metabolism ◽  
Specialized Metabolites ◽  
Expression Of Genes ◽  
Organ Specific ◽  
User Friendly

Plants produce a vast array of chemical compounds that we use as medicines and flavors, but these compounds’ biosynthetic pathways are still poorly understood. This paucity precludes us from modifying, improving, and mass-producing these specialized metabolites in suitable bioreactors. Many of the specialized metabolites are expressed in a narrow range of organs, tissues, and cell types, suggesting a tight regulation of the responsible biosynthetic pathways. Fortunately, with unprecedented ease of generating gene expression data and with >200,000 publicly available RNA sequencing samples, we are now able to study the expression of genes from hundreds of plant species. This review demonstrates how gene expression can elucidate the biosynthetic pathways by mining organ-specific genes, gene expression clusters, and applying various types of co-expression analyses. To empower biologists to perform these analyses, we showcase these analyses using recently published, user-friendly tools. Finally, we analyze the performance of co-expression networks and show that they are a valuable addition to elucidating multiple the biosynthetic pathways of specialized metabolism.

Combinatorial Control of Plant Specialized Metabolism: Mechanisms, Functions, and Consequences

2020 ◽  
Vol 36 (1) ◽  
pp. 291-313 ◽  
Author(s):  
Elia Lacchini ◽  
Alain Goossens
Keyword(s):  
Transcription Factors ◽  
Metabolic Pathways ◽  
Biological Research ◽  
Selective Activation ◽  
Specialized Metabolism ◽  
Hierarchical Levels ◽  
External Cues ◽  
Combinatorial Control ◽  
Intuitive Concept ◽  
Plant Specialized Metabolism

Plants constantly perceive internal and external cues, many of which they need to address to safeguard their proper development and survival. They respond to these cues by selective activation of specific metabolic pathways involving a plethora of molecular players that act and interact in complex networks. In this review, we illustrate and discuss the complexity in the combinatorial control of plant specialized metabolism. We hereby go beyond the intuitive concept of combinatorial control as exerted by modular-acting complexes of transcription factors that govern expression of specialized metabolism genes. To extend this discussion, we also consider all known hierarchical levels of regulation of plant specialized metabolism and their interfaces by referring to reported regulatory concepts from the plant field. Finally, we speculate on possible yet-to-be-discovered regulatory principles of plant specialized metabolism that are inspired by knowledge from other kingdoms of life and areas of biological research.

scholarly journals Nephromyces represents a diverse and novel lineage of the Apicomplexa that has retained apicoplasts

2019 ◽  
Author(s):  
Sergio A Muñoz-Gómez ◽  
Keira Durnin ◽  
Laura Eme ◽  
Christopher Paight ◽  
Christopher E Lane ◽  
...  
Keyword(s):  
Life Style ◽  
Metabolic Pathways ◽  
Evolutionary History ◽  
Phylogenetic Analyses ◽  
Phylogenetic Position ◽  
Biosynthetic Pathways ◽  
History Of ◽  
Sea Squirts ◽  
Shed Light ◽  
Apicoplast Genome

Abstract A most interesting exception within the parasitic Apicomplexa is Nephromyces, an extracellular, probably mutualistic, endosymbiont found living inside molgulid ascidian tunicates (i.e., sea squirts). Even though Nephromyces is now known to be an apicomplexan, many other questions about its nature remain unanswered. To gain further insights into the biology and evolutionary history of this unusual apicomplexan, we aimed to (1) find the precise phylogenetic position of Nephromyces within the Apicomplexa, (2) search for the apicoplast genome of Nephromyces, and (3) infer the major metabolic pathways in the apicoplast of Nephromyces. To do this, we sequenced a metagenome and a metatranscriptome from the molgulid renal sac, the specialized habitat where Nephromyces thrives. Our phylogenetic analyses of conserved nucleus-encoded genes robustly suggest that Nephromyces is a novel lineage sister to the Hematozoa, which comprises both the Haemosporidia (e.g., Plasmodium) and the Piroplasmida (e.g., Babesia and Theileria). Furthermore, a survey of the renal sac metagenome revealed 13 small contigs that closely resemble the genomes of the non-photosynthetic reduced plastids, or apicoplasts, of other apicomplexans. We show that these apicoplast genomes correspond to a diverse set of most closely related but genetically divergent Nephromyces lineages that co-inhabit a single tunicate host. In addition, the apicoplast of Nephromyces appears to have retained all biosynthetic pathways inferred to have been ancestral to parasitic apicomplexans. Our results shed light on the evolutionary history of the only probably mutualistic apicomplexan known, Nephromyces, and provide context for a better understanding of its life style and intricate symbiosis.

scholarly journals To Stretch the Boundary of Secondary Metabolite Production in Plant Cell-Based Bioprocessing: Anthocyanin as a Case Study

2004 ◽  
Vol 2004 (5) ◽  
pp. 264-271 ◽  
Author(s):  
Wei Zhang ◽  
Chris Franco ◽  
Chris Curtin ◽  
Simon Conn
Keyword(s):  
Secondary Metabolites ◽  
Plant Cell ◽  
Metabolic Pathways ◽  
Anthocyanin Biosynthesis ◽  
Rational Approach ◽  
Great Promise ◽  
Primary Metabolism ◽  
Global Correlation ◽  
Empirical Level

Plant cells and tissue cultures hold great promise for controlled production of a myriad of useful secondary metabolites on demand. The current yield and productivity cannot fulfill the commercial goal of a plant cell-based bioprocess for the production of most secondary metabolites. In order to stretch the boundary, recent advances, new directions and opportunities in plant cell-based bioprocessing, have been critically examined for the 10 years from 1992 to 2002. A review of the literature indicated that most of the R&D work was devoted predominantly to studies at an empirical level. A rational approach to molecular plant cell bioprocessing based on the fundamental understanding of metabolic pathways and their regulations is urgently required to stimulate further advances; however, the strategies and technical framework are still being developed. It is the aim of this review to take a step forward in framing workable strategies and technologies for molecular plant cell-based bioprocessing. Using anthocyanin biosynthesis as a case study, an integrated postgenomic approach has been proposed. This combines the functional analysis of metabolic pathways for biosynthesis of a particular metabolite from profiling of gene expression and protein expression to metabolic profiling. A global correlation not only can thus be established at the three molecular levels, but also places emphasis on the interactions between primary metabolism and secondary metabolism; between competing and/or complimentary pathways; and between biosynthetic and post-biosynthetic events.

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