Plant Synthetic Metabolic Engineering for Enhancing Crop Nutritional Quality

Journal Pre-proof Plant synthetic metabolic engineering for enhancing crop nutritional quality Qinlong Zhu, Bin Wang, Jiantao Tan, Taoli Liu, Li Li, Yao-Guang Liu PII:

S2590-3462(19)30017-3

DOI:

https://doi.org/10.1016/j.xplc.2019.100017

Reference:

XPLC 100017

To appear in:

PLANT COMMUNICATIONS

Please cite this article as: Zhu, Q., Wang, B., Tan, J., Liu, T., Li, L., Liu, Y.-G., Plant synthetic metabolic engineering for enhancing crop nutritional quality, PLANT COMMUNICATIONS (2020), doi: https:// doi.org/10.1016/j.xplc.2019.100017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019

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Plant synthetic metabolic engineering for enhancing crop nutritional

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quality

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Qinlong Zhu1, 2, Bin Wang1, Jiantao Tan1, Taoli Liu1, Li Li3, 4, Yao-Guang Liu1, 2, *

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1

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Agro-Bioresources; College of Life Sciences, South China Agricultural University,

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Guangzhou 510642, China

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2

Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China

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3

Robert W. Holley Center for Agriculture and Health, USDA-ARS, Cornell University,

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Ithaca 14850, NY, USA

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4

13

University, Ithaca 14850, NY, USA

State

Key

Laboratory

for

Conservation

and

Utilization

of

Subtropical

Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell

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Short summary

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Nutrient deficiencies of crops are a threat to human health. The use of plant synthetic

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metabolic engineering method can effectively fortify crops nutrition and enhance

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crops nutritional quality. In this review, we summarize the latest progress of plant

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synthetic metabolic engineering methods and their application in biofortified crops,

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which provides a useful reference for the development of crops biofortification.

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1

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Abstract

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Nutrient deficiencies in crops are a serious threat to human health, especially for

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populations in poor areas. To overcome this problem, development of crops with

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nutrient-enhanced traits is imperative. Biofortification of crops with improved

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nutritional quality helps combat nutrient deficiencies by increasing the levels of

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specific nutrient components. Compared with agronomic practices and conventional

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plant breeding, plant metabolic engineering and synthetic biology strategies can be

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more

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phytonutrients and/or bioactive components in crops. In this review, we describe

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recent progress in the field of plant synthetic metabolic engineering, specifically in

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research strategies of multigene stacking tools and engineering complex metabolic

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pathways, with a focus on improving traits related to micronutrients, phytonutrients

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and bioactive components. Ultimately, advances and innovations in these areas may

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promote the development of nutrient-enriched crops to meet human's nutritional

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needs.

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Key words: plant metabolic engineering, synthetic biology, transgene stacking, crop

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biofortification

effectively

and

accurately

in

synthesizing

specific

micronutrients,

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INTRODUCTION

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From the beginning of crop domestication, humans have learned to improve crops to

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meet their changing needs. Since the Green Revolution, traditional breeding and the

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use of chemicals have dramatically increased crop yield, enabling modern society to

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achieve food security. However, the reduction of agricultural biodiversity results in

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unbalance of nutrients, thereby increasing the risk of micronutrient and phytonutrient

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malnutrition among consumers. The Food and Agriculture Organization of the United

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Nations estimates that approximately 792 million people worldwide are malnourished,

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out of which 780 million people live in developing countries (McGuire, 2015).

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Besides, about 2 billion people around the world suffer from “hidden hunger”,

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resulted from insufficient intake of micronutrients (vitamins and minerals) in the daily 2

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diet (Muthayya et al., 2013; Hodge J, 2016). Since beginning of the 21st century,

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biofortification has been utilized as an additional and complementary strategy to

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produce “healthier foods”. Biofortification is the process by which nutritionally

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enhanced food crops are developed and grown using modern biotechnology,

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traditional plant breeding and agronomic practices (Saltzman et al., 2017).

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Biofortification differs from conventional fortifications, which increases the nutrient

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levels in crops by breeding and cultivation, rather than by artificial means during food

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processing.

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Long breeding cycles and limited nutrient content variability limit the application

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of traditional breeding for crop nutritional quality improvement. Modern

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biotechnological methods (metabolic engineering and synthetic biology) provide

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alternative and effective approaches for the development of biofortified crops (Garg et

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al., 2018). Metabolic engineering is the process to improve or implement the

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production of target compounds in vivo through modulating one or more genes or

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gene networks (Farre et al., 2014; Fu et al., 2018). Synthetic biology is defined as

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design and creation of new biological pathways to biosynthesize new compounds in

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organisms (Liu and Stewart, 2015; Hanson and Jez, 2018). There is overlap between the

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two fields. At present, most of plant-based approaches for developing and producing

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new bioactive components require a combination of synthetic biology and metabolic

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engineering (Figure 1). Such a method can be called “Synthetic Metabolic

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Engineering” (Nielsen et al., 2011; Ye et al., 2012; Pouvreau et al., 2018). Synthetic

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metabolic engineering involves three major steps (Figure 2): to learn about the

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metabolic pathways or genetic pathways that exist in natural organisms; to reconstruct

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(design, assemble and transform) the artificial metabolic pathways in the target host

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organisms; and to test the practical effects of the reconstructed pathways in the

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transformed target organisms (Pouvreau et al., 2018; Mortimer, 2019). After the first

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cycle, the “learn” step analyses the results of the cycle to enhance system-level

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knowledge and drive subsequent learn/reconstruction/test (L-R-T) cycles. The

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iterative L-R-T cycles promote the development of better synthetic metabolic

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engineering approaches. A good example of the cycles is the development from 3

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Golden Rice to Astaxanthin Rice. The first cycle involves learning the β-carotene

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biosynthesis in plants, yeast and bacteria; reconstructing the pathway using a plant

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PSY gene encoding phytoene synthase and a bacterial CrtI gene encoding carotene

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desaturase in rice; and then evaluating the β-carotene product in the transgenic rice

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(Ye et al., 2000; Paine et al., 2005). The first cycle enhances our understanding of the

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carotenoid synthetic pathway. On the basis of this β-carotene engineering, we

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continued to learn astaxanthin biosynthesis in algae and yeast, reconstruct and

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transform the astaxanthin pathway using PSY, CrtI, BHY (encoding β-carotene

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hydroxylase) and BKT (encoding β-carotene ketolase) genes, and finally successfully

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produced astaxanthin in the rice endosperm (Zhu et al., 2018) (Figure 2).

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With the evolution of molecular biotechnology, a large number of new research

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tools and strategies have been exploited to analyze and reconstruct metabolic

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pathways or genetic circuits in target organisms, which accelerate the development of

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synthetic metabolic biology (García-Granados et al., 2019). In this review, we

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highlight the latest advances in crop biofortification using new strategies and

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technologies for synthetic metabolic engineering.

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STRATEGIES FOR METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY IN PLANTS

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Plant metabolic engineering and synthetic biology involve manipulation of one or

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more key genes or rate-limiting enzyme genes in metabolic or synthetic pathways,

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even all the genes that make the entire pathways. Therefore, the selections of different

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key genes and the methods to deliver and express multiple genes in host plants will

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affect the development of plant synthetic metabolic engineering, which will be

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discussed in the following two aspects.

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Strategies for Synthetic Metabolic Engineering

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A metabolic pathway is a chemical chain reaction in which a series of enzymes

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catalyzes the conversion of substrates to target products and possible by-products in a

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certain order. Therefore, to increase the synthesis of target compounds, several 4

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strategies are usually used: 1) by increasing the expression of upstream genes

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encoding rate-limiting enzymes or multiple key enzymes in the target pathway to

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ensure adequate supply of precursors and increase metabolic flux through the target

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pathway; 2) by inhibiting the expression, or knocking out, of the key enzyme genes

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involved in the competitive pathway of the branch point or the catabolic pathway of

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the target product, to avoid intermediates being diverted and prevent the

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decomposition of target metabolites; 3) by overexpressing transcription factors (or

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together with certain key enzyme genes) to simultaneously activate multiple

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pathway-related endogenous key genes to enhance the synthesis of the metabolites;

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and 4) by combining the above methods to maximize the synthesis of the target

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compounds. In addition, CRISPR/dCas9-based activation/repression systems can also

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be used in metabolic engineering manipulation (Zalatan et al., 2015; Li et al., 2017).

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These strategies are shown in Figure 3.

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Strategies for Multigene Transformation

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Genetic engineering of complex metabolic pathways in plants often requires

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simultaneous expression of multiple target genes, or even entire pathway genes to

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ensure unrestricted metabolic flux. For this purpose, different strategies for multigene

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stacking have been developed (Figure 4), including the early time-consuming and

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labor-intensive iterative strategies (sexual crossing and re-transformation) and

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co-transformation (particle bombardment and mixed Agrobacterium-transformation),

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and the current multi-gene vector transformation (multiple gene expression cassettes

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being linked in a single T-DNA region), polycistronic transgenes (using self-cleavage

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peptide 2A to link different protein sequences) and plastid transformation (Bock et al.,

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2013; Farré et al., 2014). Among these strategies, the multigene vector transformation

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has a profound advantage over the other methods (Dafny-Yelin et al., 2007), which

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makes the multiple transgenes being integrated and inherited as a single unit. The 5

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multigene vector approach requires the assembly of multiple target genes in single

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T-DNA regions of binary vectors for Agrobacterium-mediated transformation, or in

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single plasmid vectors for other transformation methods (see below section).

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Furthermore, plastid transformation is another effective method for certain purposes,

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but it requires transferring multiple genes into the plastid genomes. The differences,

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advantages and disadvantages of the multigene vectors and plastid transformation

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have been described elsewhere (Christian and Ralph, 2019). Due to the obvious

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advantages of the multigene vector transformation, this approach is more widely used

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in many current crop improvement projects.

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PLANT MULTIGENE TRANSFORMATION VECTOR SYSTEMS

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Agrobacterium binary vectors are the crucial tool for plant genetic transformation.

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Delivering multiple genes using a single binary vector has a significant advantage

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over the application of the other multigene transformation strategies as discussed

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above. By only a single transformation, all genes assembled into the T-DNA region of

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a multigene vector can be simultaneously integrated into a single chromosomal site in

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the host plant genome, and these genes are inherited together. Therefore, only a small

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number of transgenic plants are needed to select ideal transgenic events. Although the

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assembly and transformation of a large transformation construct carrying multiple

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genes have been a challenge, various multigene vector systems using a variety of

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approaches have been developed to facilitate the stacking and delivery of different

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numbers of genes into plants (as exemplified below, Figure 5). Currently, these

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approaches have employed the use of rare-cutting homing endonucleases and zinc

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finger nucleases (Zeevi et al., 2012), type IIS restriction enzymes (Golden Gate

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cloning) (Engler et al., 2008), Cre/loxP recombination (Lin et al.2003; Zhu et al.,

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2017), Gateway recombination (Wakasa et al., 2006; Chen et al., 2006), Gibson

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Assembly (Gibson et al., 2009), and homologous recombination in yeast (Shih et al.,

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2016). The multigene vector systems with easy manipulation and large DNA-carrying

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capacity (e.g. using artificial chromosomes as vector backbone) have more advantages. 6

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An example is the recently developed TransGene Stacking II (TGS II) system (Zhu et

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al., 2017; Zhu and Qian, 2017; Shen et al. 2019).

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Using Restriction Enzyme-Based Assembly

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The approach can clone various gene expression cassettes into a multigene binary

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plasmid by utilizing rare-cutting restriction enzymes. Examples include the

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pAUX/pRCS and pSAT vector systems that use rare-cutting homing endonucleases

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(Goderis et al., 2002; Tzfira et al., 2005; Dafny-Yelin et al., 2007), and their upgrade

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pRCS11.1 vector system (Figure 5) using homing endonucleases and zinc finger

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nuclease (Zeevi et al., 2012). Although these vectors can assemble a small number of

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genes, the overall efficiency is low and it is time-consuming due to the low specificity

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and low cutting efficiency of the rare-cutting endonucleases. In addition, the BioBrick

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method using isocaudarner (e.g. Xba I and Spe I) (Knight et al., 2003), Golden

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Gate-related systems using the type IIS restriction enzymes (e.g. Bsa I, Esp3 I and

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Bbs I, which recognize 6-bp sites and can generate 4-nt non-palindromic sticky ends)

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(Engler et al., 2008 ; Emami et al., 2013; Sarrion-Perdigones et al., 2013) also can

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assemble small size of DNA fragments or several genes, which is more commonly

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used in microbial metabolic engineering. However, the high frequent presence of

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these restriction cutting sites in plant genomic DNA sequences limits the use of these

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methods (Liu et al., 2013; Ghareeb et al., 2016).

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Using Cre Site-Specific Recombination for Stacking Multigene

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The strategy using a site-specific recombinase for multigene stacking, mainly

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including Cre/loxP system and Gateway recombination, can overcome the limitations

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of the restriction-ligation methods. In a previous report, Lin et al. (2003) developed

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the first transgene-stacking system that uses a combination of the Cre/loxP-mediated

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recombination system and two homing endonucleases (I-Sce I and PI-Sce I). This

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system contains a transformation-competent artificial chromosome (TAC)-based

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acceptor vector and two donor delivery vectors; the donor vectors with target genes

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are used alternately to recombine with the acceptor vector to sequentially stack

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multiple genes into the acceptor vector. Due to the operational difficulty in using 7

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homing endonuclease digestion and linker ligation for the plasmid circularization, the

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utilization of this early vector system to assemble multigene (more than five)

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remained a challenge.

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In order to solve these problems, on the basis of this early system, Zhu et al.

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(2017) recently developed a simpler and high-efficient multigene assembly and

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transformation vector system, TransGene Stacking II (TGS II). TGS II used the

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Cre-mediated recombination system with the wild-type loxP sites and mutant loxP

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sites (for irreversible recombination), which can automatically remove the donor

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vector backbones during the multigene assembly cycles in a special Escherichia coli

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strian (that expresses the Cre enzyme) (Figure 5). In addition, a selection marker

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gene-deletion construct can be introduced into the acceptor vector (with assembled

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target genes) by a Gateway-reaction, in order to obtain marker-free transgenic plants.

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There are some other Cre/loxP-mediated multigene systems, for examples, the

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MISSA system based on conjugational transfer using the Cre/loxP recombination for

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multigene assembly and Gateway recombination for deletion of the donor vector

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backbone in vivo (Chen et al., 2010), and the recombinase-mediated in planta

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transgene-stacking strategy that requires multi-rounds of plant transformation (Ow,

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2011). Among these systems, TGS II is the mostly simple one in operation and high

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efficient for multiple genes assembly, thus is suitable to engineer complex metabolic

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pathways (Zhu and Qian, 2017; Fang et al., 2018). This system has been successfully

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used to synthesize anthocyanins, canthaxanthin and astaxanthin in rice endosperm by

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stacking and transferring 10 foreign genes (~ 31 kb) involved in anthocyanin

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biosynthesis, and four and six ketocarotenoid biosynthetic foreign genes (~ 15-19 kb),

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respectively, into rice (Zhu et al., 2017; Zhu et al., 2018). By using this system to

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stack multiple genes of photorespiration, Shen et al. (2019) developed a new rice

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germplasm with high light efficiency and productivity. Furthermore, the TGSII

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system currently is the only reported multigene vector system that is marker

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gene-free-competent.

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Using Gateway Recombination Methods 8

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Gateway recombination system is another commonly used method to prepare

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multigene vectors using commercial recombinase LR clonase for attL and attR site

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reactions and BP clonase for attB and attP site reactions (Walhout et al., 2000). The

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multisite Gateway system consists of a set of destination binary vectors with several

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different modified attR sites and a set of three Entry plasmids (each carrying a

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different attL sites); this system uses LR clonase reaction to assemble several genes in

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a single recombination step (Wakasa et al., 2006). However, this system can not stack

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more genes due to the limited number of available att sites (Dafny-Yelin et al., 2007).

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Another Gateway-based system is MultiRound Gateway strategy that alternates use of

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two different Gateway entry vectors to sequentially assemble multiple genes into one

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Gateway destination vector by multiple rounds recombination reactions (Chen et al.,

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2006). Recently, Collier et al (2018) reported an Agrobacterium-based GAANTRY

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system (Gene Assembly in Agrobacterium by Nucleic acid Transfer using

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Recombinase technologY) for assembling multigene into the T-DNA of an

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Agrobacterium rhizogenes pRi virulence plasmid by in vivo multi-round Gateway BP

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recombination. The assembly strategy of MultiRound Gateway and GAANTRY

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system is similar to Cre/loxP-based multigene vector system (Lin et al., 2003), except

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for the use of different recombinases.

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Using Homologous Recombination in Yeast

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Yeast homologous recombination in vivo is a robust technique to assemble large

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linearized DNA fragments (Gibson et al., 2008; Shao et al., 2008). For example,

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Gibson et al. (2008) reported the complete synthesis of the Mycoplasma genitalium

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genome (580 kb) by one-step assembly of 25 overlapping large DNA fragments in

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yeast. For utilizing this method, a jStack vector system was developed for plant

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multigene assembly, which uses yeast-compatible plant binary pYB vectors modified

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from the pCAMBIA2301 binary vector by adding yeast replication and selection

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systems (Shih et al., 2016). By co-transferring linearized multiple DNA fragments and

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pYB vector backbone with overlapping homologous sequences, multiple DNA

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molecules can be assembled into pYB vector in yeast in a single step (Figure 5). This 9

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approach allows efficient assembly of multiple genes or DNA fragments, but the size

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of the assembled DNA molecules hardly exceed 20 kb, limited by the carrying

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capability of the pBR322 backbone of pYB. Therefore, an improvement of this

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system for carrying more genes is possible by adopting the TAC-based vector

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backbone.

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Using DNA Overlapping Sequences Assembly

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The DNA overlapping assemblies use the ‘chew-back-and-anneal’ strategy, which

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requires digesting one strand of overlapping DNA ends to produce short

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complementary end sequences for annealing without ligation. These methods include

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uracil-specific excision reagent (USER) (Nour-Eldin et al., 2010), InFusion (Sleight et

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al., 2010), sequence- and ligase-independent cloning (SLIC) (Li and Elledge, 2007),

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and Gibson Assembly (Gibson et al., 2009). They are suitable for the assembly of

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small DNA fragments or gene expression cassettes. Among these methods, Gibson

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Assembly is a powerful technique for joining multiple overlapping DNA fragments

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simultaneously in a single reaction to synthesize large DNA molecules in vitro.

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Gibson Assembly uses a mixed reagent with a 5′ exonuclease for generating single

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strands of overlapping sequences, a DNA polymerase for filling gaps of annealed

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products, and a DNA ligase for sealing the nicks (Figure 5). Nevertheless, application

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of these approaches is limited for the assembly of DNA fragments with repetitive

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sequences. Also, as the number of DNA fragments to be assembled in one reaction

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increases, the efficiency and accuracy rate decrease (Liu et al., 2013; Ghareeb et al.,

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2016).

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ENGINEERING PHYTONUTRIENTS AND MICRONUTRIENTS IN CROPS

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Phytonutrients (nutraceuticals) and micronutrients play important roles in human

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nutrition and health, but they are often deficiency in various degrees in major staple

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crops (Mattoo et al., 2010). Moreover, some health-promoting nutraceuticals (e.g.

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astaxanthin) are lacking in food crops. Therefore, biofortification is of great

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significance for enhancing nutritional and health-promoting qualities of food crops. 10

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The use of synthetic metabolic engineering is a suitable strategy for such endeavor

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(De Steur et al., 2017). To date, a variety of biofortified crops have been developed

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through these approaches, such as the most well known β-carotene-enriched ‘‘Golden

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Rice’’ (Ye et al., 2000; Paine et al., 2005), anthocyanin-enriched ‘‘Purple Tomatoes’’

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(Butelli et al., 2008), anthocyanin-enriched ‘‘Purple endosperm rice’’ (Zhu et al.,

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2017), and astaxanthin-enriched ‘‘aSTARice’’ (Zhu et al., 2018).

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Flavonoid and Anthocyanin Biofortification in Crops

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Anthocyanins are a class of flavonoid compounds widely distributed in fruits and

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vegetables, which have strong antioxidant properties for promoting human health

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(Deng et al., 2013). In cereal grains, anthocyanins are only present in the pericarp of

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particular species or varieties (such as black rice, black corn and purple wheat), and

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are completely absent in cereal endosperms. In addition, the health-promoting

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properties of the grains are further reduced due to the habit of eating refined grains

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without the pericarp in the East Asian people. Anthocyanin biosynthesis is one of the

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well-understand metabolic pathways (Figure 6). It involves multiple structural genes

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(encoding enzymes for forming a series of anthocyanin metabolites), decorating genes

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and transporters, as well as the crucial transcription factors that form a

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MYB-bHLH-WD40 (MBW) complex to control anthocyanin structural gene

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expression (Hichri et al., 2011; Dixon et al., 2013; Zhang et al., 2014; Yuan and

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Grotewold, 2015). By using the key structural genes (Muir et al., 2001; Ogo et al.,

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2013), the transcription factor genes (Bovy et al., 2002; Butelli et al., 2008; Zhang et

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al., 2015; Jian et al., 2019), or the key structural genes plus the transcription factor

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genes (Zhu et al. 2017; Liu et al., 2018a; Liu et al., 2018b ), biofortification with

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flavonoids and anthocyanins have been achieved in some crops (e.g. rice, maize, and

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tomato) (Table 1).

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Anthocyanins are naturally existent in many fruits and vegetables, but not in fruit of

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most tomato cultivars. Overexpression of a single key enzyme gene or a transcription

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factor gene (such as the petunia chalcone isomerase or the regulator AtMYB12) (Muir

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et al., 2001; Zhang et al., 2015), or transcription factor genes of the regulatory 11

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complex (such as maize anthocyanin transcription factors Lc and C1) (Bovy et al.,

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2002), all could increase the content of flavonols in tomato, but did not produce

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anthocyanins. However, co-expression of the snapdragon anthocyanin regulator

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complex genes AmDel and AmRos1 (Butelli et al., 2008), or expression of the tomato

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regulatory gene SlMYB75 (Jian et al., 2019), could achieve a large accumulation of

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anthocyanins in tomato fruit (Figure 6B). These results indicate that the ability of the

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transcription factors from different sources is different in activating the structural

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genes. Although the genetic manipulation is simple, the strategy of only using the

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transcription factor genes or their combination may not be successful.

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Compared with anthocyanin-engineering of tomato, biofortification of cereal

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crops with anthocyanins is more complicated and difficult. For example, in rice

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endosperm, some of the structural and transcription factor genes involved in the

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anthocyanin biosynthetic pathway are functionally defective. As a result, expression

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of structural genes or co-expression of the bHLH- and MYB-type regulatory genes

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(such as maize anthocyanin regulators ZmR-S and ZmC1) could not complete the

341

target anthocyanin biosynthesis, but only produced the intermediate flavonoid

342

products (the anthocyanin upstream precursors) in the rice endosperm (Shin et al.,

343

2006; Ogo et al., 2013).

344

Recently, we explored a new strategy involving transformation of the maize

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regulatory genes (ZmLc and ZmPl) and a complete set of the six structural genes from

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the coleus anthocyanin biosynthesis pathway, all driven by endosperm-specific

347

promoters (Zhu et al., 2017). By using the multigene stacking system TGSII, the eight

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genes were assembled and transformed into rice to generate a novel biofortified

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germplasm ‘‘Purple Endosperm Rice’’ with high anthocyanin content and antioxidant

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activity in the endosperm (Figure 6C and 6D). Using a similar strategy, the embryo

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and endosperm anthocyanin-enriched “Purple Maize” (Figure 6E) was developed by

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using seed-specific bidirectional promoters to drive the target gene coding sequences

353

linked by the self-cleavage peptide 2A linker (Liu et al., 2018a; Liu et al., 2018b).

354

These results indicate that the strategy of using transcription factor genes plus

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multiple structural genes has a wider adaptability in anthocyanin metabolic 12

356

engineering. With this strategy, it is also possible to obtain other purple endosperm

357

cereals.

358 359

Carotenoid Biofortification in Crops

360

Carotenoids are a large class of important lipid-soluble phytonutrients that play

361

important roles in promoting human nutrition and health. For example, β-carotene is

362

the precursor for vitamin A synthesis and astaxanthin is the most powerful antioxidant.

363

Carotenoids are rich in vegetables and fruits but low in cereal grains (Saltzman et al.,

364

2017). Therefore, biofortification of carotenoids in crop grains is crucial and urgent.

365

Carotenoid biosynthesis is another well-studied pathway (Figure 7A). The use of

366

metabolic engineering strategies for carotenoid enrichment is an effective method,

367

such as the famous β-carotene-enriched “Golden Rice” (Ye et al., 2000; Paine et al.,

368

2005).

369

Although the key enzyme genes in the core carotenoid biosynthesis pathway have

370

been cloned, little is known about the regulator genes (Stanley et al., 2019), except for

371

the ORANGE (OR) gene that encodes a key regulator for carotenoid accumulation by

372

interacting with the PSY protein and expanding the plastid metabolic sink strength

373

(Lu et al., 2006; Zhou et al., 2015; Chayut et al., 2017; Yazidani et al., 2019; Sun et

374

al., 2018). Thus, the main engineering strategy for carotenoid biofortification is to use

375

a combination of multiple key enzyme genes, from plants, algae and microbe, to

376

directly synthesize target products in crops, by Agrobacterium-mediated multigene

377

vector transformation (Huang et al., 2013; Pierce et al., 2015; Zhu et al., 2018; Ha et

378

al., 2019; Tian et al., 2019) or particle bombardment (e.g. maize and wheat) (Zhu et

379

al., 2008; Wang et al., 2014). One notable example is the de novo synthesis of

380

astaxanthin in rice endosperm (Zhu et al., 2018; Ha et al., 2019).

381

Astaxanthin is a red-colored ketocarotenoid synthesized from β-carotene in

382

astaxanthin-producing organisms, and has very high antioxidant activity. However,

383

astaxanthin is not synthesized in most higher plants due to lacking the BKT gene

384

(encoding β-carotene ketolase) (Zhu et al., 2009). Recently, Zhu et al. (2018) reported

385

the first real sense of synthesis of astaxanthin in rice endosperm de novo, named 13

386

“aSTARice”, by using the TGSII vector system to assemble and transfer four

387

synthetic gene expression cassettes (PSY1, CrtI, BHY, and BKT), driven by the rice

388

endosperm-specific promoters, into transgenic rice. In addition, Ha et al. (2019) used

389

the polycistronic transgene strategy, which used the fused genes linking with

390

self-cleavage peptide 2A, to produce lower content of astaxanthin in rice endosperm.

391

Since the generation of “Golden Rice”, a number of biofortified crops have been

392

developed in the past 20 years for production of various nutritive carotenoids (Table

393

2). However, the contents of the synthesized carotenoids are still not high enough in

394

the biofortified crops, especially in rice, for better human nutrition and health.

395

Therefore, introduction of more genes for increasing contents of the precursors (e.g.

396

the upstream genes DXS, HGRM) (Bai et al., 2016; Tian et al., 2019), for improving

397

the carotenoid stability (e.g. the HGGT gene) Che et al., 2016), and for effective

398

regulators (e.g. OR) (Kim et al., 2019; Yazidani et al., 2019), may produce higher

399

levels and more stable carotenoids in biofortified crops. Further research and

400

understanding of the carotenoid metabolic regulation mechanism, combined with the

401

use of metabolic engineering strategies and new research tools (e.g. genome editing),

402

will dramatically improve our capacity to manipulate plant carotenoid biosynthesis for

403

crops biofortification. Some carotenoid-enriched biofortified crops (e.g. rice, maize,

404

wheat, tomato and soybean) are shown in Figure 7B-F and summarized in Table 2.

405 406

Vitamin Biofortification in Crops

407

Vitamins (e.g. vitamin B9, vitamin B6, vitamin C and vitamin E) are a group of

408

essential micronutrients for the growth and development of animals. Human cannot

409

synthesize these compounds, thus must obtain them from the diet, especially from

410

plant foods (Garcia-Casal et al., 2017). However, as the main dietary components for

411

human population, the most consumed stable crops and many edible plants deliver

412

inadequate amounts of these micronutrients (Strobbe et al., 2018). Biofortification via

413

genetic engineering provides a crucial tool to enrich crop vitamins in the fight against

414

micronutrient malnutrition.

415

At present, the contents of some vitamins in certain crops have been improved by 14

416

increasing the biosynthetic efficiency and promoting the storage stability though

417

metabolic engineering methods (Strobbe and Van Der Straeten, 2018). Expressing one

418

or multiple key enzyme genes involved in folate (vitamin B9) biosynthesis and

419

stability, i.e. GTPCHI (GTP cyclohydrolase I), ADCS (aminodeoxychorismate

420

synthase), FPGS (folylpolyglutamate synthetase) or FBP (folate binding protein),

421

significantly increases the amount of folates in rice, maize, potato, and tomato (de la

422

Garza et al., 2004; Storozhenko et al., 2007; Naqvi et al., 2009; Blancquaert et al.,

423

2015; De Lepeleire et al., 2017). For example in rice, expression of the transgene

424

GTPCHI resulted only about 10-fold increase of the folate content, but co-expression

425

of GTPCHI and ADCS led to 100-fold folate enhancement (Storozhenko et al., 2007).

426

When simultaneously expressing three genes (GTPCHI, ADCS and FPGS) or four

427

genes (GTPCHI, ADCS, FPGS and FBP) the folate levels were increased by 100-fold

428

and 150-fold, respectively, and the product was more stable during post-harvest

429

storage (Blancquaert et al., 2015). Thus, the multigene stacking strategy is a

430

high-efficiency method for folate biofortification in crops.

431

Similarly, overexpressing the PDX gene (encoding a pyridoxal phosphate synthase,

432

a key enzyme of vitamin B6 biosynthesis) also could significantly increase the content

433

of vitamin B6 in cassava (Li et al., 2015). In addition, using a similar strategy

434

expressing one or more key enzyme genes involved in vitamin C or E biosynthesis,

435

vitamin C biofortification was achieved in tomato, potato and maize (Naqvi et al.,

436

2009; Qin et al., 2011; Bulley et al., 2012), and vitamin E content was enhanced in

437

rice, maize, and barley (Babura et al., 2017; Chen et al., 2017; Strobbe et al., 2018).

438 439

Omega-3 Fish Oil in Crops

440

Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic

441

acid (EPA, 20: 5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a vital role in

442

human health and development, and the lack of these fatty acids increases the risk or

443

severity of cardiovascular and inflammatory diseases (Ruxton et al., 2007). However,

444

these two unsaturated fatty acids are not present in higher plants (Napier et al., 2015),

445

and their intake depends only on marine fish oil. The research on engineering plants 15

446

with accumulation of LC-PUFAs began in the late 1990s (Broun et al., 1999).

447

Biosynthesis of EPA and DHA involves many enzymes that do not exist in higher

448

plants.

449

Recently, several research groups have successfully synthesized EPA and DHA

450

in Arabidopsis, Brassica juncea, Camelina sativa, and canola (Ruiz-Lopez et al., 2012;

451

Ruiz-Lopez et al., 2013; Amjad Khan et al., 2017). Two different strategies are

452

employed: one is the introduction of seven desaturase and elongase genes from algae

453

or fungal (Wu et al., 2005; Petrie et al., 2012; Ruiz-Lopez et al., 2014; Petrie et al.,

454

2014 ); another is transferring three unsaturated fatty acid synthase subunit genes and

455

one phosphopantetheinyl transferase gene from microalgae (Walsh et al., 2016).

456

Currently, several research groups in the United Kingdom, the United States, and

457

Australia have performed animal feeding experiments and confirmed that the

458

plant-derived sources of EPA and DHA can completely replace marine fish oil

459

(Betancor et al., 2015; Tejera et al., 2016).

460 461

Mineral Iron (Fe) and Zinc (Zn) Biofortification in Crops

462

Iron and zinc are essential elements for many metabolic processes in human

463

(Underwood, 1977; Prasad, 1978). Among the top 10 global risk factors for disease

464

burden, zinc and iron deficiencies rank fifth and sixth, respectively (Kumar et al.,

465

2011). Plants absorb these minerals from the surrounding environment. The transgenic

466

strategy to increase the contents of iron and zinc in crops is mainly to improve the

467

absorption and utilization efficiency of iron and zinc by enhancing the expression of

468

related transporter genes (Kerkeb et al., 2008; Blancquaert et al., 2017), and reducing

469

the contents of anti-nutrient factors (e.g. phytic acid) (Aluru et al., 2011). In addition,

470

the co-expression of Lactoferrin (Fe-chelating glycoprotein) and FERRITIN also

471

increases the iron contents in crops (Goto et al., 1999; Lucca et al., 2001; Drakakaki

472

et al., 2000; Borg et al., 2012). Simultaneous expression of FERRITIN and NAS

473

(nicotianamine synthase) increases not only zinc but also iron content in crops (Wirth

474

et al., 2009; Lee et al., 2009; Zheng et al, 2010). The collaborative expressions of four

475

genes, FERRITIN, NAS, PSY and CrtI, significantly increased iron, zinc and 16

476

β-carotene contents in transgenic rice, producing a multi-nutrient enriched biofortified

477

crop (Singh et al., 2017). By using these methods, a number of biofortified crops

478

(such as rice, maize and wheat) with enriched iron and zinc have been developed

479

(Kumar et al., 2019). The multiple nutrient-biofortified crops have great potential for

480

combating global human mineral deficiencies.

481 482

CURRENT CHALLENGES AND OPPORTUNITIES

483

Although significant progress has been made in using synthetic metabolic engineering

484

to biofortify crops, there are still some challenges (García-Granados et al., 2019). The

485

primary problem facing the development of synthetic metabolic engineering is to

486

understand metabolic pathways and key regulators in an organism. Although more

487

and more plant genomes have been sequenced, the lack of effective gene functional

488

annotations makes it difficult to determine the composition of genes encoding key

489

enzymes in complete metabolic pathways. The combined analysis of genomics,

490

transcriptomics, proteomics, and metabolomics will accelerate well-understanding of

491

metabolic pathways and the key components (Farre et al., 2014). The constitutive

492

synthesis of metabolites is likely to cause abnormal cell development and growth in

493

plants. At the same time, metabolic pathways are susceptible to influence by feedback

494

regulation and other factors. The development and application of tissue-specifically

495

expressing transgenes can accomplish the synthesis and accumulation of metabolites

496

in specific tissues and avoid their negative effects on normal plant growth (Peremarti

497

et al., 2010). Furthermore, the development of synthetic metabolic engineering is also

498

limited by molecular techniques. Most metabolic pathways involve multiple

499

regulatory factors and enzymes. Thus the use of the high-efficiency multigene

500

expression vector systems (e.g. the TGSII system) and the CRISPR gene editing tool

501

(e.g. CRISPR/dCas9-based knockout or transcriptional activation or inhibition) can

502

simultaneously enable the expression and regulation of the upstream and downstream

503

gene networks of entire metabolic pathways in more flexible and precise ways (Zhu et

504

al., 2017; Zalatan et al., 2015; Li et al., 2017).

505

With a well understanding of biosynthesis pathways and advances in metabolic 17

506

engineering technology, the synthetic metabolic engineering will more accurately

507

achieve the reconstruction and regulation of multi-step complex metabolic networks,

508

and produce more novel varieties of biofortified crops with multiple nutrients (such as

509

phytonutrients, vitamins, minerals and functional nutraceuticals), which will meet the

510

needs for better human nutrition and health.

511 512

ACKNOWLEDGMENTS

513

This work was supported by grants from the National Natural Science Foundation of

514

China (31971915) and the Major Program of Guangdong Basic and Applied Research

515

(2019B030302006).

516 517

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872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 30

897

Figure legends

898

Figure 1. Schematic diagram of synthetic metabolic engineering.

899

The synthetic product (D) in natural organisms is accompanied by intermediary

900

products (A, B, C) and by-products (E, F, G). Metabolic engineering minimizes the

901

synthesis of by-products (indicated by smaller letters) in the natural anabolic pathway

902

and increases the flux of the main synthetic pathway (represented by larger letters and

903

thicker blue arrows). Synthetic biology introduces a complete exogenous pathway for

904

metabolic synthesis. At present, biofortification projects in many crops include these

905

two methods, so they are termed as synthetic metabolic engineering.

906 907

Figure 2. Schematic illustration of the basic procedure for Learn/Reconstruct/

908

Test cycle.

909

The Learn/Reconstruct/ Test cycle includes first to “learn” the metabolic synthesis

910

pathways to understand the key nodes in the natural organisms, then to “reconstruct”

911

the metabolic pathways using the molecular biology techniques to design, assemble

912

and transfer the artificial pathways into the model organisms, and finally to “test” the

913

products of the reconstructed pathways in target organisms following stable

914

transformation. After one cycle, the original hypothesis is refined with all data

915

processed in the “learn” phase, and the next cycle can begin to test the new

916

hypothesis.

917 918 919 920

Figure 3. Strategies for synthesizing target products in plants. A

,

B

and

C

are the precursors of a target product

D

.

E

is a branch or by-

921

product. The key enzymes of the pathway are indicated as E1~E6. (A) Enhancing the

922

activity of the rate-limiting enzyme (E5) by overexpression (OX) or improving

923

upstream precursor content by overexpression of one or more key enzymes (such as

924

E4-OX). (B) Increasing upstream precursors by overexpressing one or more

925

rate-limiting enzymes (E1) , and simultaneously blocking the branch point (E6) by

926

RNA interference or CRISPR/Cas-mediated knockout (KO), to enhance the metabolic 31

927

flux in the main pathway. (C) Improving levels of the precursors by overexpressing

928

transcription factors (TFs) to activate multiple key enzyme genes of the pathway, and

929

simultaneously knock-down/out of the branch point (E6). (D) Comprehensively

930

increasing contents of the precursors by simultaneously overexpressing all key

931

enzyme genes and the transcription factor genes, along with blocking the branch point.

932

(E) Enhanced accumulation of the target metabolite by overexpressing a possible

933

multi-functional or fused enzyme (Ex) that can catalyze

934

original reaction steps, so as the consumption of the intermediates is reduced, together

935

with improving rate-limiting enzymes activity by overexpression and blocking the

936

by-product production.

A

into

C

, and blocking the

937 938

Figure 4. Schematic representation of multigene transformation strategies.

939

(A) In the sexual crossing method, plants carrying transgenes A and B, respectively,

940

are crossed to bring both genes into the same line. (B) In the retransformation method,

941

transgene B is retransformed into transgenic plant already carrying transgene A to

942

stack both genes into the same line. In methods (A) and (B), the integration of

943

transgenes is independent. (C) In the co-transformation approach, transgenes A and B

944

in separate plasmids are introduced into plants by particle bombardment or mixed

945

Agrobacteria transformation. (D) In the multigene vector transformation, multiple

946

genes A, B and C are assembled in a single binary vector. By a single

947

Agrobacterium-mediated transformation, all the linked transgenes are integrated into a

948

chromosomal locus. (E) In the polycistronic transgene approach, two or more target

949

genes are expressed as a fusion protein linked by the self-cleavage 2A peptide. The

950

expressed polyprotein is self-cleaved into individual proteins A, B and C, each

951

carrying part of the 2A peptide. (F) In the plastid transformation, one or more genes

952

can be expressed as an operon in plastid genome transformed by particle

953

bombardment and plastid homologous recombination. This method is suitable for

954

genes that can be expressed in plastids and for species that is competent for stable

955

plasmid transformation (such as tobacco). At present this method is difficult in cereal

956

crops (such as rice, maize and wheat). 32

957 958

Figure 5.Typical methods for constructing multigene assembly vectors for plant

959

transformation.

960

(A) The TransGene Stacking II system for multigene assembly based on

961

Cre/loxP-mediated reversible and irreversible recombination in an E. coli strain

962

(NS3529) expressing Cre (Zhu et al., 2017). The first round assembly (Round I)

963

includes the processes: (i) Cre/loxP recombination of the pYL322d1-A/B plasmid

964

(containing target genes A and B) into the pYLTAC380GW acceptor binary plasmid,

965

and (ii) automatic removing of the pYL322d1 backbone by irreversible recombination

966

between the mutant loxP1L (1L) and loxP1R (1R) sites. These processes form the first

967

recombinant plasmid pYLTAC380GW-A/B. Round II: Cre/loxP recombination of the

968

pYL322d2-C/D plasmid into pYLTAC380GW-A/B and deletion of the pYL322d2

969

backbone by irreversible recombination between the mutant loxP1L (2L) and loxP1R

970

(2R) to form plasmid pYLTAC380GW-A/B/C/D. Round III is similar with Round I.

971

After all target genes are assembled by repeated assembly cycles, the selectable

972

marker/marker-excision cassette on the marker-free donor plasmid is recombined into

973

the acceptor vector by Gateway BP reaction to generate the final binary construct. In

974

transgenic plants, the Cre expression driven by the pollen-specific promoter Pv4 (or

975

another inducible promoter) deletes the marker/Cre cassettes. (B) The pRCS11.1

976

vector system using homing endonucleases and zinc finger nucleases (Zeevi et al.,

977

2012). Assembly of a multigene binary plasmid is achieved by successive cloning of

978

various gene expression cassettes into pRCS11.1 vector using cutting and ligation. (C)

979

Multigene assembly using homologous recombination in yeast (Patrick et al., 2016).

980

Various gene expression cassettes with homologous end sequences and the linearized

981

pYB vector are co-transferred into yeast cells, and the target genes can be recombined

982

into the pYB vector in vivo. (D) Gibson cloning for multiple genes (Gibson et al.,

983

2009). Multiple genes or fragments with overlapping end sequences (20-30 bp) are

984

mixed with a linearized vector, then the collaborative reactions of a 5′ exonuclease, a

985

DNA polymerase and a DNA ligase produce single strand ends of the overlapping 33

986

sequences, annealing between the ends, filling of the gaps, and finally ligating the

987

nicks, to complete the multigene assembly.

988 989

Figure 6. Anthocyanins biofortification in crops.

990

(A) The anthocyanin biosynthesis pathway in plants. The key enzymes of general

991

upstream phenylpropanoid pathway include phenylalanine ammonia lyase (PAL),

992

cinnamate 4-hydroxylase (C4H), and 4-coumaryol CoA ligase (4CL). Structural genes

993

encoding enzymes involved in anthocyanin biosynthesis are chalcone synthase (CHS),

994

chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavanone 3’, 5’-

995

hydroxylase (F3’5’H), and flavanone 3’-hydroxylase (F3’H). Anthocyanins are

996

synthesized by dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS).

997

Decorating genes encoding enzymes such as GT: glucosyltransferase; MT:

998

methyltransferase are involved in the formation of stable anthocyanins from

999

anthocyanidins. GST, glutathione S-transferase. The flavonoid intermediate products,

1000

flavonols, are synthesized by flavonol synthase (FLS) from dihydroflavonol (marked

1001

by gray background) and flavones are synthesized by flavone synthase (FNS). The

1002

transcription regulators form a MBW complex (MYB-bHLH-WD40) to control the

1003

expression of the anthocyanin biosynthesis pathway genes. (B) The anthocyanin

1004

biofortified purple tomato by fruit-specific co-expression of the anthocyanin

1005

regulators AmRos1 and AmDel (Butelli et al., 2008). (C, D) Purple anthocyanins

1006

accumulated in the transgenic developing rice seeds (C) , and polished and

1007

cross-sections of the anthocyanin biofortified purple rice endosperm (D), by

1008

introducing two maize regulator genes (ZmPl and ZmLc), and six anthocyanin

1009

structural genes (SsCHS, SsCHI, SsF3H, SsF3’H, SsDFR and SsANS from coleus), all

1010

driven by endosperm-specific promoters (Zhu et al., 2017). (E) Grains and

1011

cross-sections of purple embryo and endosperm of maize by biodirectional promoter

1012

driving co-expression of four maize anthocyanin biosynthetic genes (ZmR2, ZmC1,

1013

ZmBz1 and ZmBz2) linked by 2A self-cleavage peptide (Liu et al., 2018a and 2018b).

1014

These panels (B-E) are reproduced with permission from Ref Butelli et al., 2008, Zhu

1015

et al., 2017 and Liu et al., 2018a and 2018b, respectively. 34

1016 1017

Figure 7. Carotenoids biofortification in crops.

1018

(A) Schematic carotenoid biosynthesis pathway in plants and the expanded

1019

ketocarotenoids/astaxanthin-producing pathway in plants. The precursors of

1020

carotenoid biosynthesis are from the upstream plastidial 2-C-methyl-D-erythritol

1021

4-phosphate (MEP) pathway. The foreign transgene-encoding enzymes (in blue) are

1022

phytoene synthase (PSY), bacterial carotene desaturase (CrtI), β-carotene hydroxylase

1023

(BHY) and β-carotene ketolase (BKT), which catalyze precursors of carotenoids to

1024

generate the target end-product astaxanthin in transgenic plants. The other enzymes

1025

(in black) are encoded by endogenous carotenogenic genes in plants. DXS,

1026

1-deoxy-D-xylulose 5-phosphate (DXP) synthase; DXR, DXP reductoisomerase;

1027

GGPPS, geranylgeranyl diphosphate synthase; PDS, phytoene desaturase; ZISO,

1028

ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerase;

1029

LCYE, lycopene ε-cyclase; LCYB, lycopene β-cyclase; EHY, ε-carotene hydroxylase;

1030

VDE, violaxanthin de-epoxidase; ZEP, zeaxanthin epoxidase; NXS, neoxanthin

1031

synthase. (B) The diverse phenotypes (Ph) of carotenoids biofortified corns by

1032

co-transformation of different combinatorial carotenoid biosynthesis-related genes

1033

using particle bombardment (Zhu et al., 2008). Ph-1, expression of Psy1; Ph-2,

1034

expression of CrtI; Ph-3, co-expression of Psy1 and CrtI; Ph-4: co-expression of Psy1,

1035

CrtI and Lycb; Ph-5: co-expression of Psy1, CrtI and Lych; Ph-6: co-expression of

1036

Psy1, CrtI, Lycb and CrtW (bacterial β-carotene ketolase gene); Ph-7: co-expression

1037

of Psy1, CrtI, Lycb, Lcyh and CrtW. (C) β-carotene enriched wheat by co-expression

1038

of CrtB (bacterial phytoene synthase) and CrtI using particle bombardment (Wang et

1039

al., 2014). (D) Astaxanthin enriched tomato by co-expression of BHY and BKT using

1040

Agrobacterium-mediated transformation (Huang et al., 2013). (E) Astaxanthin

1041

enriched

1042

Agrobacterium-mediated transformation (Zhu et al., 2018). (F) Ketocarotenoid

1043

production of soybean by combinatorial expression of CrtB, CrtB and BKT, CrtB and

1044

CrtW (Pierce et al., 2015). These panels (B-F) are reproduced with permission from

rice

by

co-expression

of

Psy1,

35

CrtI,

BHY

and

BKT

using

1045

Ref Zhu et al., 2008; Wang et al., 2014; Huang et al., 2013; Zhu et al., 2018 and

1046

Pierce et al., 2015, respectively.

1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 36

Table 1 Flavonoid and anthocyanin biofortification in major transgenic crops Species

Transgene

promoters

ZmR-S, ZmC1

Endosperm-s pecific promoter npr33

OsPAL, OsCHS

Rice

OsPAL, OsCHS, AtF3H, AtFLS

Target tissue

Major products

Total content (µg/g DW)

Reference

Binary vector and Agrobacterium-mediate d transformation

Pericarp (dark brown) and the outer layer endosperm

Flavonoids (dihydroquercetin, 3’-O-methyl dihydroquercetin, and 3’-O-methyl quercetin)

12800*

Shin et al., 2006

Endosperm-s pecific promoter GluB-1

Endosperm

Seed-specific promoter Ole

Embryo

1~70

Endosperm

10~60

Endosperm-s pecific promoter GluB-1 Seed-specific promoter Ole

OsPAL, OsCHS, GmIFS

Transgene assembly and transformation

1~12 Flavonoids (Naringenin,)

Multigene vectors (Multisite Gateway) and Agrobacterium-mediate d transformation

Flavonol (Kaempferol) Embryo

Endosperm-s pecific promoter GluB-1

Endosperm

Seed-specific

Embryo

37

10~700

Isoflavone (genistein)

10~40

10~350

Ogo et al., 2013

promoter Ole

OsPAL, OsCHS, PcFNSI, GmFNSII

OsPAL, OsCHS, PcFNSI, GmFNSII, OsOMT, ViolaF3’5’H

Maize

Endosperm-s pecific promoter GluB-1

Endosperm

Seed-specific promoter Ole

Embryo

Flavone (Apigenin)

Endosperm-s pecific promoter GluB-1

ZmLc,ZmPl, SsCHS,SsCHI, SsF3H, SsF3’H, SsDFR, SsANS

Endosperm-s pecific promoters GluC, GluB-1, GluB-4, Glb1, GluB-5, Npr33, 10KDa, 16KDa

ZmC1, ZmR2, ZmANS, ZmGST

Embryo-specif ic bidirectional promoter PZmBD1 Seed-specific

40~120

Endosperm

Multigene vectors (Cre/loxP-based TGSII system) and Agrobacterium-mediate d transformation

Multigene vectors (linked by 2A) and Agrobacterium-mediate d transformation

Endosperm

5~60

Flavone (Tricin)

Anthocyanin (cyanidin 3-O-glucoside and peonidin 3-O-glucoside)

110

~1000

Zhu et al., 2017

Embryo

Anthocyanin (cyanidin and pelargonidin)

200~1035

Liu et al., 2018a

Embryo and

Anthocyanin (cyanidin,

896~1534

Liu et al.,

38

bidirectional promoter P2R5SGPA

endosperm

pelargonidin and peonidin)

PhCHI

CaMV 35S

Fruit peel

Flavonols

1900

Muir et al., 2001

ZmLc, ZmC1

Fruit-specific promoter E8

Flavonol (Kaempferol)

40~80

Bovy et al., 2002

AmDel, AmRos1

Fruit-specific promoter E8

2830

Butelli et al, 2008

1860

Jian et al., 2019

1545

Park et al., 2015

Tomatoa

Sweet potato

SlMYB75

CaMV 35S

IbMYB1

storage roots-specific promoter SPO1

Binary vector and Agrobacterium-mediate d transformation

Fruit

2018b

Anthocyanins

Tuber

Anthocyanins

The asterisk represents the measured value of brown rice uses the taxifolin as stand at 280 nm and the value may be much higher. a. The content is µg/g FW (fresh weight). b. All gene names can be found in note of Figure 6, except for the OMT (O-methyltransferase). Abbreviations: DW, dry weight; Zm, Zea mays; Os, Oryza sativa; At, Arabidopsis thaliana; Gm, Glycine max; Pc, Petroselinum crispum; Ss, Solenostemon scutellarioides; Ph, Petunia hybrid; Am, Antirrhinum majus; Ib, Ipomoea batatas.

39

Table 2 Carotenoid biofortification in major transgenic crops

Species

Rice

a

Total carotenoid, µg/g DW (fold change relative WT)

Reference

Paine et al., 2005

Transgene

Transgene assembly and transformation

ZmPSY1, PaCrtI,

Multigene vectors (restriction-ligation) and Agrobacterium-mediated transformation

β-carotene, 30.9

36.7

sCaPSY, sPaCrtI,

Multigene vectors (linked by 2A) and Agrobacteriummediated transformation

β-carotene, 2.35

4.18

ZmPSY1, PaCrtI, tHMGR1

Multigene vectors (restriction-ligation) and Agrobacterium-mediated transformation

β-carotene, 10.5

14.5

ZmPSY1, PaCrtI, sCrBKT

Co-transformation and particle bombardment

Canthaxanthin, 4.0

8.8

β-carotene, 24.7

27.6

Canthaxanthin, 25.8

31.4

Astaxanthin, 16.2

21.9

ZmPSY1, PaCrtI sZmPSY1, sPaCrtI, sCrBKT sZmPSY1, sPaCrtI,, sCrBKT,

Target tissue

Target products, µg/g DW (fold change relative WT)

Endosperm

Multigene vectors (Cre/loxP-based TGSII system), and Agrobacterium-mediated transformation

40

Jeong et al., 2017

Tian et al., 2019

Bai et al., 2017

Zhu et al., 2018

sHpBHY CaPSY, PaCrtI, stCaBch, CaPSY, PaCrtI, stCaBch, stHpBKT

Maize

Wheat

Multigene vectors (polycistronic transgene with 2A) and Agrobacteriummediated transformation

Zeaxanthin, 0.8

1.9

Astaxanthin, 1.1

1.8 Ha et al., 2019

CaPSY, PaCrtI, CaBch,,CaCcs

Intercrossing a CaPSY, PaCrtI, stCaBch transgenic rice line with a CaCcs transgenic rice line

Capsanthin, 0.33

2.2

ZmPSY1, PaCrtI,

Co-transformation and particle bombardment

β-carotene, 57.35 (410)

156.1 (142)

ZmPSY1, PaCrtI, GlLycB

Co-transformation and particle bombardment

β-carotene, 48.87 (349)

148.8 (135)

ZmPSY1, PaCrtI , GlLycB, CrtW

Co-transformation and particle bombardment

Astaxanthin, 4.5

146.8 (134)

ZmPSY1,CrtZ, BKT, RNAi-ZmLycE,

Co-transformation and particle bombardment

Astaxanthin, 16.8

35.1 (2.6)

Farré, et al., 2016

ZmPSY1, PaCrtI

Co-transformation and particle bombardment

β-carotene, 59.3 (148)

163.2 (109)

Naqvi et al., 2009

CrtB, CrtI

Co-transformation and particle bombardment

β-carotene, 3.2 (64)

4.1 (7)

Wang et al., 2014

CrtB

particle bombardment

β-carotene, 2.7 (16)

7.4 (6)

RNAi-BHY

particle bombardment

β-carotene, 2.3 (14)

3.7 (3)

Endosperm

Endosperm and kernels Endosperm

Endosperm

41

Zhu et al., 2008

Zeng et al., 2015

CrtB, RNAi-BHY ZmPSY1, PaCrtI, Sorghum

ZmPSY1, PaCrtI, AtDXS ZmPSY1, PaCrtI, AtDXS, HGGT

Endosperm

β-carotene, 9.1 (8)

26.3 (4)

β-carotene, 5.2 (11)

26.41 (3)

Lipkie et al., 2013

Che et al., 2016

Canthaxanthin, 52 , β-carotene, 666

915 (61)

Canthaxanthin, 45, β-carotene, 195

324 (22)

CrtI

β-carotene, 520 (2)

1372 (0.5)

Romer et al, 2000

CrtB

β-carotene, 825 (3)

5912 (2)

Fraser et al., 2002

β-carotene, 205 (47)

215 (2)

D’Ambrosio et al., 2004

Canthaxanthin, 2249.7, Astaxanthin, 926.1

5813.1 (5)

Canthaxanthin, 338.4, Astaxanthin, 16104.7

19054.4 (17)

Agrobacterium-mediated transformation

β-carotene, 22.6

90.8 (1)

β-carotene, 25.6

160.7 (2)

Binary vectors (restrictionligation) and Agrobacterium-mediated transformation

Zeaxanthine, 67.6 (1.6)

88.3 (1.6)

b

SlLycB CrBKT

Co-transformation and particle bombardment

Binary vectors and Agrobacterium-mediated transformation

Seed

Fruit

CrBKT, HpBHY AtORWT AtOR

His

OR Potato

9.3 (8)

31.7 (6)

CrtB, BKT

Tomato

Multigene vectors (multiple Gateway) and Agrobacterium-mediated transformation

β-carotene, 5.6 (35)

β-carotene, 9.3 (19)

CrtB, CrtW Soybean

Co-transformation and particle bombardment

CrtZ, CrtW CrtZ, CrtW, OR

Tuber

Astaxanthin, 28 Astaxanthin, 48.6

42

Pierce et al., 2015

Huang et al., 2013

57.7 (1.1) 93.5 (1.7)

Yazdani et al., 2018 Campbell et al., 2015

a. Rice endosperm does not contain carotenoids. b In wild type soybean (cv.Jack), lutein is only detected. AtORWT, Arabidopsis thaliana wild-type OR; AtORHis, Arabidopsis thaliana OR with mutant at R90H; AtDXS, Arabidopsis thaliana 1-deoxy-D-xylulose 5-phosphate synthase gene; CaPSY, Capsicum annuum phytoene synthase gene; CaBch, Capsicum annuum β-carotene hydroxylase gene; CaCcs, Capsicum annuum capsanthin-capsorubin synthase gene; CrtB, bacterial phytoene synthase gene; CrtI, bacterial phytoene desaturase gene; CrtW, bacterial β-carotene ketolase gene; CrtZ, bacterial β-carotene hydroxylase gene; CrBKT, Chlamydomonas reinhardtiiβ-carotene ketolase gene; HGGT, homogentisate geranylgeranyl transferase gene; HpBHY, Haematococcus pluvialis β-carotene hydroxylase gene; OR, cauliflower ORANGE gene; PaCrtI, Pantoea ananatis phytoene desaturase gene; SlLycB, Solanum lycopersicum lycopene β-cyclase gene; sCrBKT, a rice codon-optimized synthetic Chlamydomonas reinhardtii β-carotene ketolase gene; stCaBch, a rice codon-optimized synthetic Capsicum annuumβ-carotene hydroxylase gene; stHpBKT, a rice codon-optimized synthetic Haematococcus pluvialis β-carotene hydroxylase gene; sHpBHY, a rice codon-optimized synthetic Haematococcus pluvialis β-carotene hydroxylase gene; sPaCrtI, a rice codon-optimized synthetic Pantoea ananatis phytoene desaturase gene; sZmPSY1, a rice codon-optimized synthetic Zea may phytoene synthase gene; tHMGR, truncated 3-Hydroxy-3-methylglutaryl coenzyme A reductase from Saccharomyces cerevisiae; ZmPSY1, synthetic Zea may phytoene synthase gene; ZmLycE, Zea may lycopene ε-cyclase gene.

43