From qualitative to quantitative: the state of the art and challenges for plant synthetic biology

Abstract

Backgrounds: As an increasing number of synthetic switches and circuits have been created for plant systems and of synthetic products produced in plant chassis, plant synthetic biology is taking a strong foothold in agriculture and medicine. The ever-exploding data has also promoted the expansion of toolkits in this field. Genetic parts libraries and quantitative characterization approaches have been developed. However, plant synthetic biology is still in its infancy. The considerations for selecting biological parts to design and construct genetic circuits with predictable functions remain desired.

Results: In this article, we review the current biotechnological progresses in field of plant synthetic biology. Assembly standardization and quantitative approaches of genetic parts and genetic circuits are discussed. We also highlight the main challenges in the iterative cycles of design-build-test-learn for introducing novel traits into plants.

Conclusion: Plant synthetic biology promises to provide important solutions to many issues in agricultural production, human health care, and environmental sustainability. However, tremendous challenges exist in this field. For example, the quantitative characterization of genetic parts is limited; the orthogonality and the transfer functions of circuits are unpredictable; and also, the mathematical modeling-assisted circuits design still needs to improve predictability and reliability. These challenges are expected to be resolved in the near future as interests in this field are intensifying.

Graphical abstract

Keywords

plant synthetic biology / quantitative characterization / genetic parts / genetic circuits

1 PLANT SYNTHETIC BIOLOGY: THE CURRENT STATUS

As a bottom-up approach, synthetic biology is a multi-disciplinary field aiming to rewire a biological process/behavior and design/build man-made life systems for the benefit of human beings. Simple biological parts are assembled together into complex genetic circuits to obtain programmed cells or organisms. Tremendous progress have been made in prokaryotic microbial systems [1,2], the next wave of synthetic biology has now advanced from single-celled organisms to complicated multicellular systems.

Plants are attractive platforms for engineered traits because of their advanced ability to adapt to living environment, variety of growth forms, and their natural ability to produce useful metabolites. Plant synthetic biology includes not only the introduction of foreign transgenes or gene editing, but the design and construction of predictable novel circuits, new metabolic pathways, biological sensors, and physiological traits to fulfill the evolving needs [3–5]. Despite the difficulties in engineering the complexity of eukaryotic systems compared to prokaryotes, versatile genetic circuits can be achievable in the synthetic biology of plants. By introducing a set of genes into engineered plants, multiple plant systems have been successfully utilized to produce value-added metabolites and therapeutic proteins, such as taxadiene, L-DOPA, and vaccine of SARS-CoV-2 [6–9]. Engineered rice with a high content of essential nutrients, such as GABA, β-carotene, or anthocyanins, have the potential to solve the nutritional deficiency [10,11]. Optogenetically regulated controllers have been achieved in plants, such as the red light-controlled switch based on phytochrome B (PhyB) and the green-light-responsive system based on CarH [12,13]. A set of synthetic hormone activated Cas9-based repressors (HACRs) was introduced into Arabidopsis thaliana to track the levels of hormones, such as auxin, gibberellins, and jasmonates [14]. The reversible memory switch can invert the activated/deactivated states of target genes through the inversion of promoter sequences dependent on the site-specific recombinases using the phiC31 integrase and its cognate proteins [15,16]. Complete logic gates provide new possibilities for programmable manipulation of previously unrealized traits [17,18].

To some degree, these groundbreaking advancements have enabled the artificial manipulation of synthetic systems in space-time. On the other hand, there is a delay in the control of protein dosages, which is involved with the output trait in regulatory networks. The dosage imbalance between the expression of B- and C-class genes triggered by a cis-regulatory change contributed to the transformation in stamen morphology [19]. The mutation of TIL reduces the amplitude of the root clock, a temporal series of oscillating changes in gene expression, and effect the lateral root branching [20]. A HACR targeting the auxin transporter PIN-FORMED1 (PIN1) can even change the levels of the relationship between PIN1 and auxin and reduce the number of branches [14]. In addition to the expression levels, different spatial patterns were generated by combining the activities of input promoters using logic gates in A. thaliana roots [18].

In this manuscript, we consider the current progress in genetic parts and genetic circuits in terms of quantitative characterization and standardization, and we also highlight key challenges facing the advance of quantitative prediction, design, and application in plants.

2 QUANTITATIVE PLANT SYNTHETIC BIOLOGY IS EMERGING

2.1 Surging genetic parts from big data in plant science

Genetic parts are footstones for synthetic biology design with programmed purposes. The functional identification and standardized assembly are prerequisites for plant synthetic biology. Genetic parts can be mined from both prokaryotic and eukaryotic cells. Sequenced plant genome information can be fully utilized to predict available DNA parts, and the number of available DNA parts is increasing. At present, several existing genetic part libraries have been created (Tab.1).

The genetic parts in these databases are mainly derived from model plants. For example, PlantProm DB contains 7723 predicted promoters by mapping full-length cDNAs on the genome of Arabidopsis and rice. Promoters collected in EPDNew are derived from Arabidopsis and maize [22]. Recently, a published web server, TDTHub, contains pre-processed data for 40 plant species, enabling more quick and intuitive research for transcriptional regulatory networks [23].

Genetic information on model plants has been widely used in the in-depth mining of bioparts. Many toolkits have also been established aiming to mine the novel genetic parts. TSSP-TCM is the first predictive tool that has been trained and adapted for promoters [35]. The transductive confidence machine (TCM) provides confidence in the predictions for each individual example in the test set. 35 promoters can be correctly predicted in a set of 40 TATA promoters; besides, 21 promoters can be predicted in a set of 25 TATA-less promoters. These regulatory element databases in turn facilitate the development of predictive tools in plants (Tab.1). Based on large promoter collections from PPDB and PlantProm, TSSPlant has been developed and achieves significantly high accuracy in prediction [36].

CREs in plant pathogens promoters were revealed through the identification of A. thaliana transcription factor binding sites. Adjustments of the identity, density and position of these CREs can regulate the relative expression of output genes [37]. In recent years, a mass of genomic information about various plants has been revealed [21,38–40]. It means that there are extremely large amounts of potential bioparts need to be further tapped. And also, toolkits have been established aiming to mine the novel genetic parts. Three water-deficit and one salt-stress candidate genetic elements from hybrid poplar transcriptomes were used to design synthetic promoters, showing an outperformed endogenous promoter in transgenic plants [41]. 49,802,023 transposable elements (TE) records from 67 plant genomes were retrieved, and there are 62.85% retrotransposons and 37.15% DNA transposons for all known TEs [42].

The potential number of bioparts available for plant circuits is virtually unlimited. After standardization and characterization, more practical and powerful circuits can be designed with these potential bioparts.

2.2 Standardized assembly

Well-characterized and standardized building parts enable efficient engineering of versatile synthetic bio-circuits [43]. Several individual parts (promoters, localization peptides, terminators, UTRs, CDS) can be selected and assembled to produce circuits designed with predictable functions [44,45]. Such circuits in plants include toggle switches, positive feedback circuits, and logic gates to form complex functions. The DNA fragments allow different sequences with the same basic utility to be exactly exchanged with larger designs, enabling any difference in function conferred by sequence variations between parts to be functionally quantified.

With the aim of facilitating the interoperable exchange, a standardized producer for DNA fragments assembly was established for defining DNA parts. The first widely adopted standard is BioBrick [46,47]. BioBrick simplified the assembly of DNA using iterative, pairwise assembly of standardized parts (Fig.1). New methods for simultaneous assembly of multiple DNA fragments are more popular in plants. The Multisite Gateway method utilizes site-specific recombination based on attL and attR sites (Fig.1) [48,49]. A plant molecular biology toolbox with basic versatile genetic elements in the MultiSite Gateway framework was created by the Department of Plant Systems Biology (Ghent, Belgium) [50]. A cellular resolution can be achieved with cell type-specific promoters which have been cloned into Multisite Gateway-compatible entry vectors in different root cell types [51]. According to the features of Type IIS restriction enzymes the Golden Gate system allows seamless cloning through careful design of the restriction sites (Fig.1) [52]. The UK Biotechnological and Biological Sciences Research Council (BBSRC) defined the standard syntax based on the Golden Gate cloning method and established a common grammar for plant synthetic biology (Fig.1) [53]. The convenience of plug-and-play comes from the standard libraries [54–56]. Nevertheless, it is still a heavy workload to define two ends and remove conflicting intrinsic recognition sites of elements in the library, largely depending on the laboratory accumulation [56–60]. Although some toolkits are available, they are far from adequate in plant synthetic biology [54]. Additionally, the sticky ends, which have been predefined, will introduce unnecessary 1 bp or 4 bp traces into the connection of adjacent elements in the constructions.

Gene stacking using a polycistronic strategy in a single construct for the introduction of multiple genes into plants is required for manipulating complex traits and pathways. Many efficient technologies have been developed in plants based on the superposition of modular cloning systems with predefined standardization. Multiple cloning sites (MCS) were used in combination with MultiSite Gateway technology to assemble multiple gene cassettes [61]. Building on the standard Type IIS syntax, jStack leverages in vivo yeast homologous recombination to assemble large DNA fragments, such as multiple gene expression cassettes, into plant transformation vectors by one-pot [62]. TransGene Stacking II (TGSII) using improved Cre/loxP-mediated recombination and a transformation-competent artificial chromosome (TAC)-based acceptor vector, which is capable of large-size (>100 kb) DNA fragments [63-65]. Up to 10 gene cassettes in a single T-DNA were successfully transferred into rice [64]. Unique nucleotide sequence-guided nicking endonuclease (UNiE)-mediated DNA assembly (UNiEDA) was introduced into TGSII system and developed TGSII-UNiE to assemble more genes in parallel into acceptor/donor vectors [66]. 11 genes with three different synthetic pathways were successfully assembled into a T-DNA region with the TGSII-UNiE approach [66].

Overall, standardization is considered to be a facilitating factor in the field of plant synthetic biology. It extensively promotes genetic design and construction with engineering principles, enabling high-throughput, quantitative characterization, and large-scale constructions and comparisons of synthetic bioparts and circuits. A more traceless and standardized synthetic platform is desired.

2.3 Quantitative characterization of genetic parts

Types of genetic parts include promoters, 5′ and 3′ UTRs, protein coding sequences, terminators, activators, transcription factors, and simple regulator elements etc. The genetic parts are assembled with a fluorescent protein encoding gene to form a simple circuit, which is further transferred into the plant cells for qualitative and quantitative characterization. The biochemical parameters, that can be measured for each part, include binding affinities, transcriptional rate, quantity of repressor or activator, promoter strength, rate of protein synthesis, and RNA or protein degradation rate.

Several DNA delivery approaches, including particle bombardment, Agrobacterium infiltration, and protoplast transformation, allow rapid evaluation of the behaviors of introduced circuits in plant [67]. The unavoidable noise with using fluorescent proteins in plant cells is that chlorophyll may overwhelm the output signal. Therefore, ratiometric dual reporter systems, for example, firefly luciferase/Renilla luciferase and E-Luc/Red-F, have been developed and widely applicated in quantitative evaluation of genetic parts [55,68,69]. Self-transcribing active regulatory region sequencing (STARR-seq) has also been used for the in-depth characterization of enhancers and core promoters (Fig.2) [70–73]. By constructing STARR libraries with unique barcodes, up to 30,000 or even more transcriptional strength of core promoters can be obtained through a single sequencing, avoiding errors caused by multiple transformations and detections [72].

Whole plants take a long time for stable transformation, while transient expression in plant cells or protoplasts serves as a proxy. There have been several remarkable studies aiming at large-scale characterization of plant bioparts in transiently transformed Nicotiana benthamiana leaves [56], BY2 suspension cells [74], Arabidopsis protoplasts, sorghum protoplasts [68,75], and lima bean cotyledons [76]. However, the characterizations mainly focus on specific cells or tissues of these widely used model plants with an effective convenient genetic transformation system. It is known that genetic parts do not behave consistently in different plants, even in different cells of a particular species due to cell/tissue-specific or environmentally regulated expression of transacting factors and epigenetic regulators [74]. The outputs of synthetic circuits need to be carefully evaluated, because the performance of the building blocks may be varied in testing systems.

Therefore, in addition to the characterization of these elements, redesign new elements that can meet specific requirements is also an indispensable approach. Recent studies have also shown the potential for fine-tuning transgene expression levels by regulatory elements while spatiotemporal specificity is preserved [37,72,77–79]. In order to achieve quantitative expression at various levels, a rational design by combining multiple regulatory elements from various species, such as the core promoters, enhancers, 5′UTR, 3′UTR, and introns, was used to construct expression cassettes with quantitative intensities in multiple species and tissues [54,57,68,80,81]. The theoretical transcriptional activity (TTA) of cassettes with different promoter/terminator combinations is coincident with the experimental activity values in transiently transformed Nicotiana leaves [56].

Computer tools accelerate the processing and modeling of plant bioparts, including their structures and quantitative activities. They enable the rational design of new bioparts with a quantitative prediction (Fig.3). Machine learning (ML) algorithms, such as support vector machine (SVM) or neural networks, greatly facilitate the qualitative/quantitative prediction and de novo design of bioparts. Sequence motifs identified by POsition-sensitive WoRd Set (POWERS) algorithm are used to generate novel elements, whose property of expression is comparable to plant native elements across multiple tissues and developmental stages [81,82]. Another way to obtain customizable elements is de novo design. Generation of random sequences with key features of native short plant core promoters that exhibit similar function and transcriptional intensity in Arabidopsis and maize [72]. A convolutional neural network (CNN) model, which can predict the strength of core promoters, was further used for in silico evolution and a large increase in strength was observed after three rounds [72]. Based on the detailed and sufficient characterization of the core promoter both in sequences and intensity by transcriptome sequencing approach, these synthetic elements were successfully constructed. The comprehensive understanding of plant regulatory elements will lead to the flexibility and controllability in genetic circuit design [83].

2.4 Quantitative artificial circuits

By introducing multi-genes, many value-added metabolites have been successfully produced in different plants (Tab.2). Multi-gene transformation is the inevitable trend of modern synthetic biology. Diverse regulatory elements and regulatory systems have been used in plants [13,16,98]. However, when introducing multiple genes, the elements in practical applications are actually limited and the number has begun to flatten out. Hence, developing new elements and new modes of expression control with specific quantities is of great significance. Quantitative expression of genes is decisive and how to design complex synthetic systems with quantitative predictions with different purposes in plants will be a long-term research topic in the future. Transposons-mediated and signal-response synthetic switches have been developed and applied to manipulate the physiological processes and metabolic pathways in plants [99–101]. Many valuable synthetic tools have also been developed in plants, such as reversible memory switches, optogenetic switches, and logic gates [16,17,102].

The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been successfully used to manipulate gene knockout/knock-in/knockdown in plants. CRISPR-Cas9 system has been used to engineer quantitative variation for yield-related traits in maize by editing CLE promoter sequences [103]. Cis-regulatory variation and promoter engineering have also been used to improve tomato and rice properties [104,105].

Synthetic transcriptional repressors or activators based on nuclease-inactivated CRISPR/dCas are increasingly popular for their high accuracy and multiplexing capacity, providing new strategies for designing synthetic gene circuits [106,107]. A simple CRISPR activator, which contains a single VP64 domain fusion to dCas9, shows low activation levels [108]. Effective strategies from the genetic engineering of microbes and mammalian cells are developed to improve the capacities in plant synthetic biology [109–112]. Some studies focus on the engineering of dCas by fusing a Suntag, a tripartite complex VPR, EDLL, TV, and so on [113,114]. A few studies focus on the modification of gRNA or crRNA, such as SAM and scaffold RNA [111,115]. A comprehensive, quantitative, and systematic comparison of these designs was reported in N. benthamiana [115]. A variant of the scRNA-gRNA2.0 scaffold (used in dCas EV2.1) with a spontaneous mutation shows the highest transcriptional activation [115]. It has been applied to re-route metabolic fluxes towards the accumulation of flavonoids with simultaneous regulation of up to six enzymes [116].

RNA scaffold is a powerful machinery coordinating the transcriptional regulation of multiple genes due to their ability in activating and repressing sets of genes simultaneously, permitting more sophisticated designs in yeast and human cells [111]. A combination system, CRISPR-Combo, permits simultaneous genome editing and gene activation in plants [117]. This suggests that achieving the complex multilayered regulation in plants is promising.

3 THE CHALLENGES FOR QUANTITATIVE SYNTHETIC BIOLOGY IN PLANTS

Considering the natural characteristics of plant cells of multi-organelle and multi-tissue, it is intractable to construct multilayered synthetic systems in plants. There are several current challenges for quantitative plant synthetic biology.

3.1 Detailed quantitative descriptions on genetic parts are limited

The limited understanding of plant genetic components restricts the application and upgrade of potential bioparts in plants. Relying on pre-existing knowledge of CREs, more efficient and more diverse synthetic biology parts can be obtained through the mutation, rearrangement, and combination of fragments or motifs in promoters. The engineering of synthetic circuits remains heavily dependent on the interaction of specific CREs and transcription factors [118–120]. However, the current understanding of transcription factors is still insufficient, potentially millions of TF-CRE interactions need to be validated even in Arabidopsis genome [121]. Besides, not all functionalities of CREs are clearly performed. It has been shown that some core promoters display enhancer activity, while some enhancers also potentially carry sequence motifs of core promoters such as TATA-box [73,122]. Therefore, although a number of regulatory elements are predicable from genomic information with the assist of genetic tools, their functions still need to be experimentally tested. In addition to the function of elements, the unclear mechanism of distal regulatory elements has severely hindered their applications. Enhancers and silencers affect gene expressions through chromatin interactions, and in some case the distances of these regulatory sequences can be several dozens of megabases [123]. 34% of enhancers potentially regulate multi-genes and most of them target immediately flanking genes, skipping at least one gene [124]. The complexity of distal regulatory elements is an important reason that few of them were applied in the rational design of synthetic promoters. Poor analysis of these element sequences in plants, especially the CREs, hinders their reconstruction. Elucidation of these plant genetic components can provide useful information for functional design.

Genetic parts with biochemical parameters have been well-characterized and used to produce circuits in single-celled microorganisms, including promoter strengths, transcriptional rates, translation rates, quantity of repressors/activators, binding affinities, and protein degradation rates [125–129]. Artificial genetic circuits can be rationally designed with a software tool, Cello, in Escherichia coli and in Saccharomyces cerevisiae [130,131]. Previous synthetic biology research has demonstrated that even if the function of each element is known, it is still probably out of work as expected [132]. Only 4 of 10 synthetic repressors decreased the expression level of green fluorescent protein (GFP) and none achieved complete repression [18]. Except for the functional properties, a quantitative understanding of these CREs and TF-CRE interactions are also critical to select the optimal parts to assemble into functional devices with quantitatively predictable functions. The contradiction between a large number of element resources and a small number of applications stems from inadequate quantitative annotation. The DNA sequences of output promoters and their ON/OFF signal strength in standardized units were necessary in designing artificial synthetic circuits in E. coli (Fig.4). The quality of the mathematical models describing the gate is central of these genetic design automation software [133,134]. Quantitative characterization of plant bioparts lags that of prokaryotes. As we discussed above, the dual-luciferases systems, a mathematical model, and barcode tagging-based sequencing were used to accurately characterize individual parts in N. benthamiana leaves and plant protoplasts with transient assays. The quantitative data will not tell us about the performance of these parts in other-type untested cells or in a stably transformed state. Novel high-throughput biopart characterization techniques in multiple-type cells are underexplored.

3.2 Genetic parts should be orthogonal

Many plant bioparts are not orthogonal enough across chassis cells. Orthogonality means the bioparts can function independently of the host and other bioparts, consequently ensuring predictability. The established construction may be crashed because of this consequent variability. The regulatory networks vary widely in individual plant cells. Some plant-derived promoters have inconsistent performances for gene expression in N. benthamiana leaves and BY2 suspension cells [74]. Refactoring is a process in which the genetic part’s native design parameters are simplified, and inefficiencies and endogenous regulation are removed, while still retaining the essential function of the part [83]. The position effect and the interference of the surrounding sequence can also lead to variability. Insulators can mitigate this effect through the modulation of chromatin structure, but these elements work differently in different plant cells [135]. How to build synthetic circuits as desired and achieve complete orthogonality between the system and the host chassis remains to be further developed.

3.3 Building capacity is limited

As discussed above, various standardized large fragments assembly methods have been applied in plants. Different standardizations have been combined at different levels to realize the design and assembly of more diversity and longer sequences. In addition to the genetic constructs, genomic sequence re-editing is also an essential step in achieving synthetic regulation. Rapid progress has been made in the development of base editing, gene targeting, and gene insertion. CRISPR-associated nucleases play a critical role in genomic reprogramming [136,137]. It is feasible to simultaneously edit multiple target genes and modulate gene expressions. Yeast-mediated modification of the chloroplast genome of the green alga, Chlamydomonas reinhardtii, demonstrates a new approach to modify the chloroplast genome [136,137]. Cleavage-free genomic engineering approaches of large sequences still have a lot of development potential. There are still no reports about synthetic chromosomes in plants, while single-chromosome yeast has been successfully created [138].

3.4 Genetic compiler is used to predict the behaviors of genetic circuits

Genetic compiler is a system that allows computers and robotics to select and assemble the optimal parts for a specified behavior of a genetic circuit. Digital quantitative descriptions of parts and circuits will provide credible information to reduce the laborious process of trial-and-error. Biological processes can be described as complex networks of signaling pathways and translated into mathematical models for computer simulation [139]. An accurate mathematical model enables complex computational control by synthetic circuits.

Whole-cell models of Mycoplasma genitalium and E. coli have been published [140–142]. As an important eukaryotic model organism, more than 14 versions of S. cerevisiae genome-scale metabolic models have been created [143]. The Virtual Physiological Rat (VPR) projects have made significant progress in predictable medicine [144].

Compared to these advances, research in plant cells still lags far behind. Flourishing plant omics data collected from plant tissues or cells provide gene expression, metabolite formation, and protein biochemical information. Many robust models have been developed in plants, such as the C3 and C4 photosynthetic process, flowering, gene regulatory network, and root growth [145,146]. There is a vacancy of multi-scale mathematical models for quantitative description of plant regulatory networks, even of the most used model plant, A. thaliana.

4 CONCLUSION

Synthetic biology has made rapid progresses in microbial chassis in recent years. Many biological activities or intercellular signals of microorganisms and human cells have already been described and fine-tuned by mathematical models [147–150]. Quantitative models of input-output relationships have been used to design multicellular circuits in yeast. Computational predictions with high predictability and reliability have been tracked by experiments [151]. Compared to the above, the quantitative research in plants is still in its infant stage, confined to a single group of quiescent cells, with little involvement in cellular dynamics. Therefore, plant synthetic biology researchers can learn from these extraordinary works on microorganisms and human cells.

With the consideration of plant species and different tissues, the differences between plant cells are greater than those of microorganisms. More tissue- and condition-specific element resources are required. The hottest technical, single-cell sequencing (SCS), is a promising approach to explore potential elements with distinct patterns [152–155]. Cell-specific regulatory elements can be identified by comparing the data from different single-cells.

In addition, plants are sensitive to the environment. Light, temperature, and air quality can affect the expected performance of synthetic gene circuits. These various variables should be considered in the relevant mathematical models, which hinder the development of real-time quantitative monitoring methods and tools for plant elements and logic circuits. For complex regulatory systems, especially some metabolic networks and multicellularity interactions, how to accurately describe the functional status and properties of synthetic systems should be taken into consideration.

Plant synthetic biology is an important approach that can provide solutions to tackle many problems in field of agriculture, health care, and energy resources in the future. In the process of its application and commercialization, quantitative engineering principles are required, which can guarantee some more practical and robust systems in plants. In conclusion, the limited knowledge of plant elements, the low compatibility of toolkits, and the backwardness of quantitative functional characterization are the most important challenges in plant synthetic biology. These challenges should be emphasized in further research.

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