Next-Generation Plant Metabolic Engineering, Inspired by an Ancient Chinese Irrigation System  Rao Fu, Cathie Martin, Yang Zhang  Molecular Plant  Volume 11, Issue 1, Pages 47-57 (January 2018) DOI: 10.1016/j.molp.2017.09.002 Copyright © 2017 The Author Terms and Conditions

Figure 1 Schematic Representation of Dujiangyan Irrigation System. The Yuzui Levee (A) divides the Min River into inner river (right) and outer river (left). The Feishayan Levee (B) further divides the inner river into inner and outer streams, and carries away excess water and sediment by natural swirling flow. The Baopingkou Channel (C) was caved in the Yulei Mountain to deliver the water into the channel network (D) on the Chengdu plain. Arrows indicate the direction of water flow. Molecular Plant 2018 11, 47-57DOI: (10.1016/j.molp.2017.09.002) Copyright © 2017 The Author Terms and Conditions

Figure 2 Overview of the Different Levels of Metabolic Engineering. Future plant metabolic engineering needs to consider three levels of engineering: the first level introduces new biosynthetic genes; the second level introduces inducing TFs; and the third level enhances the flux of substrates, energy, and reducing power to specialized metabolic pathways. Introduction of new structural genes will allow production of specific metabolites. Further addition of inducing TFs will upregulate the structural genes to synthesize more metabolites. Increasing the supply of substrates, energy, and reducing power will enhance downstream synthetic reactions. Add-ons, such as the use of specific promoters, will induce expression of the structural genes and TFs at directed times and places. Meanwhile, deleting competitive pathway branches using natural mutants, RNAi, or gene editing will further increase the production of specialized metabolites. Combinations of these three engineering levels and add-ons will result in high-level accumulation of specific metabolites in a highly directed manner. Molecular Plant 2018 11, 47-57DOI: (10.1016/j.molp.2017.09.002) Copyright © 2017 The Author Terms and Conditions

Figure 3 Schematic Representation of Multi-level Engineering of Genistin in Tomato Fruit. The first level is labeled in yellow, involving introduction of the new biosynthetic gene, LjIFS, for the production of genistin; the second level is marked in blue, involving using AtMYB12 to active genes of phenylpropanoid metabolism, including PAL, CHS, CHI, F3H, and FLS; the third level is labeled in red, involving increased supply of substrates due to the activation of ENO and DAHPS by AtMYB12, and increased supplies of energy and reducing power via increased flux through glycolysis, the TCA cycle, and the oxidative pentose phosphate pathway. In addition, the fruit-specific E8 promoter was used for expression of LjIFS and AtMYB12 and the natural f3h mutant was introduced to block the competing synthesis of flavonols. All these manipulations resulted in the high-level accumulation of genistin in tomato. PPP, phosphoenolpyruvate; E-4-P, erythrose 4-phosphate; ENO, plastidial enolase; DAHPS, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; PAL, phenylalanine ammonia lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase. Molecular Plant 2018 11, 47-57DOI: (10.1016/j.molp.2017.09.002) Copyright © 2017 The Author Terms and Conditions

Figure 4 New Strategies for Plant Metabolic Engineering Research. Plant metabolic engineering provides a solution to fix the resource scarcity problem. For successful engineering of specialized metabolite production, two aspects need to be investigated. For medicinal plants, the active ingredients need to be identified and then the biosynthetic pathway and key regulators need to be identified. For model plants, the use of specific promoters, specific TFs, and gene-editing options should be investigated. By a combination of these two aspects, new generation of plant metabolic engineering can be carried out. Through construction of new biosynthetic pathways, use of inducing TFs driven by specific promoters, manipulation of flux, and deletion of any competing branch pathways, model plants can be engineered for successful production of high-value metabolites. Molecular Plant 2018 11, 47-57DOI: (10.1016/j.molp.2017.09.002) Copyright © 2017 The Author Terms and Conditions