Systems and synthetic biology approaches to alter plant cell walls and reduce biomass recalcitrance.

Fine-tuning plant cell wall properties to render plant biomass more amenable to biofuel conversion is a colossal challenge. A deep knowledge of the biosynthesis and regulation of plant cell wall and a high-precision genome engineering toolset are the two essential pillars of efforts to alter plant cell walls and reduce biomass recalcitrance. The past decade has seen a meteoric rise in use of transcriptomics and high-resolution imaging methods resulting in fresh insights into composition, structure, formation and deconstruction of plant cell walls. Subsequent gene manipulation approaches, however, commonly include ubiquitous mis-expression of a single candidate gene in a host that carries an intact copy of the native gene. The challenges posed by pleiotropic and unintended changes resulting from such an approach are moving the field towards synthetic biology approaches. Synthetic biology builds on a systems biology knowledge base and leverages high-precision tools for high-throughput assembly of multigene constructs and pathways, precision genome editing and site-specific gene stacking, silencing and/or removal. Here, we summarize the recent breakthroughs in biosynthesis and remodelling of major secondary cell wall components, assess the impediments in obtaining a systems-level understanding and explore the potential opportunities in leveraging synthetic biology approaches to reduce biomass recalcitrance. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Introduction

Plant cell walls, which include primary walls composed largely of cellulose, pectin and hemicellulose and secondary walls composed mainly of cellulose, lignin and hemicellulose, determine the development and morphology, strength properties, water and solute transport mechanisms, and pest resistance, at both cellular and whole plant levels. For lignocellulosic biofuel production, it is known that chemical as well as structural features of the cell walls and biomass are important determinants of feedstock recalcitrance or its resistance to breakdown to sugars. Several cell wall properties, such as content and quality of lignin and cellulose, discernible chemical (i.e. extent of acetylation and methylation, and heteropolysaccharide composition) and structural features of the wall at the angstrom-level (i.e. inter chain covalent, H-bond and van der Waals interactions) and nanometre level (i.e. tonoplastic space and pore density), have a direct impact on recalcitrance and in turn, on the liquid transportation fuel production per unit area of land (Davison ; Ding ; Himmel ; McCann and Carpita, 2008; Mohnen, 2008; Scheller and Ulvskov, 2010). Fine-tuning these cell wall properties to render plant biomass more amenable to biofuel conversion is therefore, a prime goal of the biofuels research community (Davison ; Kalluri and Keller, 2010).

Within the context of the present report, the term ‘biomass’ is used to refer to lignocellulosic plant material, ‘systems biology’ is used to refer to an integrative approach combining large amounts of biological data to understand and predict emergent complex biological phenomena, and ‘synthetic biology’ is used to refer to the targeted design and redesign of the biological machinery and living systems using a parts-based approach and engineering principles (Schmidt, 2012). The adoption of synthetic biology approaches to precisely engineer metabolic pathways and generate effective designer enzymes holds tremendous potential in improving biomass properties. Promising and/or or well-studied energy crop plants being explored for lignocellulosic biomass-based biofuels production include switchgrass (cultivars of Panicum virgatum), miscanthus (Miscanthus spp.), poplar (Populus trichocarpa, other Populus spp. and interspecific hybrids of Populus spp.), willow (Salix spp.), pine (Pinus spp.), eucalyptus (Eucalyptus spp.), sorghum (Sorghum spp.) and energycane (Saccharum complex). Additionally, plant models such as Arabidopsis (Arabidopsis thaliana), rice (Ozyza sativa), brachypodium (Brachypodium distachyon), foxtail millet (Setaria italica), tobacco (Nicotiana tabacum) and medicago (Medicago truncatula) that have shorter life-cycles and greater amenability to genomics and biotechnology methods continue to be adopted as references for attaining translational breakthroughs in fundamental science.

The rapid progress in our fundamental understanding and improvement of bioenergy crops in the past decade has included gene-centric as well as pathway- and genome-wide studies. The general approach of undertaking a transcriptomics characterization followed by manipulation of a single gene at a time, has recognized limitations and is being addressed by the community, which is now embracing a systems-wide view. In this review, we summarize the recent progress made in our understanding of plant cell wall biosynthesis and remodelling, assess the major impediments in obtaining a systems-level understanding of cell wall formation and explore the potential opportunities in leveraging systems and synthetic biology approaches to reducing biomass recalcitrance.

Cellulose biosynthesis pathway and recent breakthroughs

Cellulose biosynthesis occurs at the plasma membrane, and various soluble and membrane proteins have been reported to play a role in this pathway (Endler and Persson, 2011). Cellulose synthases, designated as CesAs or CESAs, belonging to the Family 2 glycosyltransferases, are the catalytic enzymes that build beta-glucan chains from UDP-glucose substrate (Delmer, 1999). The CesA-containing functional complexes, CSC, or terminal complexes (localized at nonreducing ends of a growing microfibril) from which a cellulose microfibril extends, have been observed to occur as rosette structures (∼30 nm in diameter) in freeze-fracture electron microscopy of plasma membranes obtained from higher plants (Kimura ). Several research reports and reviews have that suggested that factors such as (i) composition, number and arrangement of rosettes, (ii) membrane dynamics, due to action of wall enzymes, changes in lipids in response to developmental cues or environmental stresses, (iii) associated wall proteins KORRIGAN, KOBITO, COBRA (whose molecular functions are still unknown) and (iv) pH [H+ acid growth]/ion [Ca++ levels] changes are determinants of cellulose properties in plant cell walls (Endler and Persson, 2011; Mizrachi ). The regulatory and signalling pathways critical to rapid turnover of CesAs and CSCs and cellulose production are not well understood.

The recent discovery of small microtubule-tethered CesA containing compartments (SmaCCs/MASCs) that move by depolymerization of microtubule ends (Crowell ; Gutierrez ) has provided a working hypothesis on distribution of the Golgi-formed CSC in the plasma membrane (Crowell ). A role for other cytoskeleton-associated proteins in cellulose synthesis is also beginning to emerge. For example, disruption of a dynamin-related protein (Collings ; Xiong ) and a kinesin (Zhong ) has been shown to result in altered cellulose synthesis and microfibril orientation. It has also been proposed that cysteine proteases interact with Zn finger domain of CesA and play a role in the turnover and degradation of rosette complexes (Jacob-Wilk ).

Based on mutant phenotypic analysis, mutant complementation and domain-swapping experiments, it is proposed that distinct sets of at least three primary or secondary wall-specific CesAs isoforms are essential CSC components (Somerville, 2006; Taylor ), where the CesA composition may be nonredundant and the tail region of CesA is important in multisubunit interactions within CSC (Burn ; Wang ). On the basis of high-resolution live-cell fluorescence microscopy, it was shown that CesA moves along microtubules, thereby possibly affecting the direction of cellulose deposition (Paredez ). In a protein interaction screen to identify CSC components, Gu identified an Armadillo repeat containing protein. Further examination confirmed colocalization with CesAs and direct interaction with microtubule and CesA. The precise constitution of functional CSCs is unclarified. The challenge of extending single subunit visualization techniques (e.g. EM-based immunolocalization or live tracking of protein movement) to all CesA proteins, as well as to enable easy visualization of rosettes multisubunits, is a limiting concern in the field (Itoh ).

One of the salient recent developments in the cellulose research field has been the discovery of the structure of the first CesA (Morgan ). This pioneering work included crystallographic analysis of a bacterial cellulose synthesis and transport (channelling) protein subunit pair, BcsA-BcsB, with or without substrate addition. The structure of the more complex plant CesA remains elusive although there are several efforts to experimentally or computationally clarify the structure, such as the 3D atomistic simulation of the catalytic domain of cotton CesA (Sethaphong ).

The natural evolution of the cellulose research field has steadily yielded valuable insights, yet the pace has been slow relative to the abbreviated timelines, we face in understanding and improvement biomass properties, specifically increasing the content of cellulose. A systems approach offers the potential to identify the molecular and genetic underpinning of the linkages between properties of cellulose synthase and cell wall such as processivity, glucan chain length, degree of polymerization, crystallinity, interactions with other wall components, and sugar release efficiency to facilitate biosystems design of plant cell walls for bioenergy.

Lignin biosynthesis pathway and recent breakthroughs

Lignin, a polyphenolic heteropolymer of monolignols found mainly in secondary walled cells, is to date the plant feature most correlated with recalcitrance of biomass. The abundance, distribution and composition of lignin [i.e. ratio of syringyl (S), guaiacyl (G) and 4-hydroxyphenyl (H) units] is variable across plant species, individuals of a population and even within tissue types of an individual plant. The cross-linkages between lignin and wall polysaccharides confer strength properties to cell wall and plant as a whole, and present a challenge in the form of recalcitrance of biomass to biofuels conversion processes.

Several review articles have provided a detailed view of the lignin biosynthesis pathway in the past (Boerjan ; Bonawitz and Chapple, 2010; Zhao and Dixon, 2011). Additionally, the effect of manipulating lignin pathway genes on sugar release efficiency of biomass was first demonstrated in alfalfa (Chen and Dixon, 2007). The vast amount of knowledge gathered for lignin biosynthesis pathway from multiple plant species over the years has generated a consensus model for the pathway (Vanholme ). The present article elaborates on selected recent works (within past 5 years) pertinent to lignin pathway engineering strategies to reduce biomass recalcitrance.

Among the cellulosic feedstock crops, switchgrass, a fast growing perennial grass, has received the considerable attention in the past 5 years. While a switchgrass genome sequencing effort is underway, the polyploid outcrossing nature of the species is a current impediment to precisely identifying genetic basis of biomass traits such as saccharification or sugar release efficiency. Nonetheless, researchers have made tremendous strides by finding creative solutions in the form of homology driven analyses of RNAseq and EST profiles, design of microarrays, developing a cell culture system and achieving impressive transformation efficacy for switchgrass plants (Shen ). Candidate genes obtained on the basis of the aforementioned approaches are beginning to be validated as genetically controlling lignin content and S : G ratios. A significant breakthrough with switchgrass transgenic studies of cell wall recalcitrance was the discovery that down-regulation of a single caffeic acid O-methyltransferase, COMT, resulted in changes in lignin properties and a 38% increase in ethanol yield (Fu ). There have been several other reports of transgenic switchgrass characterization involving mis-expression of lignin pathway genes such as cinnamoyl CoA reductase, CCR, (Escamilla-Trevino ); cinnamyl alcohol dehydrogenase, CAD (Fu ; Saathoff ) and 4-coumarate CoA ligase, 4CL, (Xu ).

Lignin engineering efforts are primarily focused on modifications of lignin composition, often with introduction of novel lignin forms in plants (Vanholme et al., 2012a), to reduce cross-linking with wall components resulting in easier extractability, digestibility and recalcitrance of usable wall polysaccharides. Vanholme and Weng showed that a combinatorial down-regulation of COMT and overexpression of F5H lead to engineering of cell walls with unusual lignin (>90% benzodiance). Eudes also successfully engineered novel lignin into Arabidopsis stems by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) gene that synthesizes and enhances C6C1 monomers, considered key in reducing lignin polymer length, in cell walls resulting in enhanced saccharification.

Recent studies directed towards a deeper understanding of transcriptome level differences among the collection of Arabidopsis lignin biosynthesis mutants has given rise to an insightful ‘systems biology’ view of impacts of gene perturbation in Arabidopsis (Vanholme ). Such level of systems analysis together with validation of transcriptional nodes and hubs is critical to realizing the potential of synthetic biology in enabling high-precision engineering of biomass properties.

In a seminal effort in higher precision engineering of cell wall properties, C4H gene was expressed under a xylem vessel cell-specific VND6 promoter and concomitantly the NST1 was expressed under the IRX8 promoter in Arabidopsis. Yang called this an ‘artificial positive feedback loop system’ as the IRX8 promoter is also induced by NST1. This successful demonstration of simultaneous increase in polysaccharide and decrease in lignin in stems of Arabidopsis plants without gross modifications in plant growth is an example of the potential precision engineering holds. The possibilities of extending this gene modification principle to other combinations of genes are exciting.

A new development in the lignin field is the rapid emergence of interest in maximizing the economic value of lignin as a coproduct (Ragauskas ). For many biomaterial applications, such as carbon fibre production, unbranched lignin with a S/G ratio >2 is a preferred substrate. However, typical lignin polymers are rather random in orientation. System-wide genetic modification of plant biomass to create lignins that are more linear or restricted in their monomer composition, giving them better properties, appears possible. A recent study reported the natural occurrence of a new type of a lignin polymer, catechyl or C-lignin, in high concentrations (>80%) in the seed coat of vanilla orchid plants (Chen ). Furthermore, certain species of cacti exclusively contain C-lignin in seeds (Chen ). The C-lignin is interesting as it is an unbranched, homopolymer of catechyl units with abundant benzodioxane units. Similar benzodioxanes have been reported to occur in ccomt and comt mutant plants. Although the genetic underpinnings of C-lignin production are not specifically worked out, it is believed that well-characterized enzymes in the consensus lignin biosynthesis pathway may be important in production of C-lignin.

Hemicellulose biosynthesis and recent breakthroughs

Noncellulosic polysaccharides such as hemicelluloses (xylan, mannan, glucomannan and xyloglucan) and pectin, composed of mixed sugars with branching and potentially crossed with wall proteins, are variable between primary and secondary walls and between plant groups. The subunit components or in some cases, full-length backbones are synthesized in ER–Golgi and secreted via trans-Golgi network to plasma membrane, the site of integration, and ultimately deposited in the cell walls. There are comprehensive reviews of the biosynthesis of noncellulosic wall polysaccharides recently published (Hao and Mohnen, 2014; Pauly ; Rennie and Scheller, 2014), and hence this topic is dealt in brevity in the present review.

Briefly, several members of cellulose synthase-like gene family (Csl) have been reported to synthesize mixed-linked sugar backbones (Dhugga, 2012). More recently, progress made through characterization of Arabidopsis mutants for stem structural (e.g. irregular xylem) and chemical features (e.g. NMR analyses of signature hemicellulose linkages), and in vitro enzyme assays have revealed a suit of genes coding for enzymes responsible for synthesis of backbone [e.g. (1-4)-beta xylan oligomer], side chain (e.g. 4-O-methyl GlcA) and moieties (e.g. acetyl). These include genes such as IRX8 (GAUT12), IRX9 (GT43), IRX10 (GT47), IRX14 (GT43), IRX15 (DUF579), PARVUS (GATL1), IRX7/FRA8 (GT47), RWA (REDUCED WALL ACETYLATION) and O-acetyl transferases (Carpita, 2012; Hao and Mohnen, 2014; Pauly ). Classic forward and reverse genetics approaches clearly resulted in fundamental insights into the biosynthesis of hemicellulose polymers in the past decade. The diversity of sugars and complexity of biosynthesis processes involved, however, poses a practical challenge in undertaking an immediate systems-level understanding of biogenesis and remodelling of these cell wall components.

Transcription factor-mediated control on biomass recalcitrance-related properties

As secondary cell walls of xylem fibres and vessels are the prime components of biomass, alterations in xylem development and secondary cell wall properties will have a significant effect on biomass quality. Transcript profiling experiments that target secondary cell wall biosynthesis across various species have indicated the involvement of specific transcriptional networks. Mutant and transgenic analysis of many transcription factors from these network studies have confirmed their functional role in secondary wall formation (Figure1).

Figure 1

A conceptual model of transcription factor-mediated control on biomass recalcitrance-related properties such as xylem development, secondary cell wall formation, lignin and other cell wall components. The model is based on current knowledge from bioenergy crop types such as Populus, Pinus, Eucalyptus and Panicum spp. as well as the plant model Arabidopsis thaliana presented in several recent publications (Hussey ; Ko ; Shen ; Wang ; Zhong , 2011). Transcription factors (activators or repressors) reported to be control lignin biosynthesis pathway are indicted in red circles. Additional unidentified transcription factors relating to each of these properties are indicated by a ‘?’ symbol embedded within circles of respective colours. The bottom panel of the figure reflects the phenotypic properties of lignocellulosic biomass such as stem development, secondary wall formation under the influence of reported transcription factors.

It has been shown in Arabidopsis that SND1 and its close homologs, NST1 (fibres), NST2, VND6, and VND7 (fibres) (Kubo ; Mitsuda , 2007; Yamaguchi ; Zhong , 2007), act as ‘master switches’ of secondary wall formation, which in turn regulate genes encoding other transcription factors such as MYB46, MYB83, SND3, MYB103 and KNAT7 (McCarthy ; Zhong , 2007) (Figure1). PtrMYB3 and PtrMYB20 have been shown to be involved in secondary cell wall formation in poplar (McCarthy ) and MYB85, MYB63, and MYB58 are reported to regulate lignin biosynthesis (Zhong ; Zhou ). Ko followed the transcriptome dynamics across a time series of secondary wall formation by generating a dexamethasone-inducible MYB46 secondary wall thickening system in Arabidopsis. Arabidopsis GeneChip microarray and Illumina digital gene expression profiles at 0, 1, 3 and 6 h postinduction and confirmatory in vivo assays showed that AtC3CH14, MYB52 and MYB63 are downstream to MYB46. Furthermore, their work suggests that the C3H-type zinc-finger protein AtC3H14 is possibly another master regulator of genes involved in secondary wall biosynthesis (such as CesA4, CesA7, CesA8, PAL4, CCoAOMT, LAC10). In Arabidopsis, one AP2/EREBP transcriptional factor (SHINE2) has been identified as a key regulator responsible for the increasing of cellulose and decreasing in lignin biosynthesis (Aharoni ; Ambavaram ). A common set of transcription factors, along with pathway-specific transcription factors, may regulate secondary wall biosynthetic pathways (Ko ; Mellerowicz ; Mellerowicz and Sundberg 2008).

Mis-expression of these transcription factors has been reported to often result in weak walls, short stature, disease susceptibility, or imbalance in other wall chemicals, suggesting that a robust systems biology model of cell wall changes and a strategy for achieving the desired wall property precisely without compromising other critical attributes is still not available. Detailed knowledge about how cellulose and lignin biosynthetic genes are regulated may provide an opportunity to design the reorganization of plant cell wall compositions.

Bonawitz recently showed that the dwarf and lignin-deficient phenotype of Arabidopsis ref8 mutant is rescued by disrupting MED5a/REF4 and MED5b/RFR1, which are subunits of a transcriptional co-regulatory Mediator complex. med5a/5b ref8 plants have a wildtype phenotype except that the synthesis of guaiacyl and syringyl is not rescued and a novel p-hydroxyphenyl-enriched lignin is formed, and the saccharification efficiency is enhanced. This work suggests a possible role of Mediator underlying the other dwarf lignin mutant phenotype reports. It also underscores the fact that molecular components of transcriptional machinery and signalling pathways are important to evaluate as potential higher precision targets in biomass improvement efforts.

MyB4, a secondary cell wall transcription factor, has previously been shown to repress lignin biosynthesis in Pinus spp. (Patzlaff ) (Figure1). Overexpression of the transcription factor gene, PvMYB4, in switchgrass dramatically improved the yield of cellulosic ethanol yield from switchgrass when compared to nontreated controls or COMT transgenic plants (Shen ). These results extend the proven utility of transcriptional regulators into perennials and potential biofeedstocks. PvMYB4-OX switchgrass is a complementary model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production.

Status of collective goal to reduce biomass recalcitrance

As noted in the previous sections, the number of research efforts attempting to improve biomass properties have increased dramatically. In general, the targeting points include the following: key transcriptional factors that control expression of several enzymes, enzymes or transporters involved in nucleotide sugar conversion, and enzymes that generate or modify polysaccharides (Doblin ) (Table1).

Table 1

Recent (within past 5 years) examples of engineering plant cell walls to improve biofuel production

Species	Engineering approach	Outcome	References
Panicum virgatum	RNAi of Caffeic acid O-methyltransferase	Increase ethanol yield by up to 38%	Fu et al. (2011a)
P. virgatum	Down-regulation of Cinnamyl Alcohol Dehydrogenase (CAD)	Improved saccharification efficiency	Fu et al. (2011b), Saathoff et al. (2011)
P. virgatum	Overexpression of Myb transcriptional factor PvMYB4	Increase ethanol yield by 2.6-fold	Shen et al. (2013)
Saccharum spp. hybrids	RNAi of Caffeic acid O-methyltransferase	Reduction in total lignin by 6% improved saccharification efficiency by 19%–23%	Jung et al. (2012a)
Arabidopsis thaliana	pVND6::C4H+ pIRX8::NST	Increased polysaccharide deposition in the fibre cell	Yang et al. (2013)
A. thaliana	Overexpression of bacterial hydroxycinnamoyl-CoA hydratase-lyase	Improve saccharification and reduce lignin polymerization degree	Eudes et al. (2012)
P. virgatum	Silencing of 4-coumarate:coenzyme A ligase	Reduce lignin content and increase the efficiency of fermentable sugar	Xu et al. (2011)

Many emerging technologies in genomics, cell biology and cell wall chemistry have been critical to advances in our understanding of plant cell wall formation. Such techniques include (i) molecular techniques relevant to robust, reliable and rapid single cell genomics, single-molecule detection, protein profiling from very small samples, genetic engineering, and transient and stable transformation, (ii) phenotyping techniques for in situ cell wall measurements include label-free methods for chemical imaging methods such as coherent Raman scattering microscopy (Zeng ), 3D time-of-flight secondary-ion mass spectrometry (Jung ), fluorescence tag-based single-molecule tracking of protein and carbohydrate-binding modules for in situ cellulose biosynthesis and cellulose degradation studies (Gu ; Liu ), and imaging of fluorescent lignin monomers in cell walls (Ding ; Tobimatsu ). Additional key resources include (iii) appreciably annotated genome sequences from dozens of plant species and hundreds of genotypes; and (iv) standards-adhered data analysis, and reconstructions of transcriptional networks and nodes.

As a general observation, these genomics-based plant improvement efforts typically involve an initial phase of genome-wide ‘omics’ studies (such as transcriptomics, proteomics and metabolomics) and pathway analyses followed by formulation and testing of hypotheses for genetic basis of observed functional feature that simply involves a single candidate gene. The adoption of such an approach of manipulating expression of a single gene under a ubiquitous promoter, while fundamentally informative, has often resulted in pleiotropic and undesirable effects and therefore considered an impediment in the path to rapid and precise improvement of biomass properties.

Despite the extensive efforts, there are few examples shown to be effective in engineering the cell wall components in a predictable manner. This may partly be explained by the (i) failure to identify and target the most effective gene mix or master regulator, (ii) existence of functional endogenous copies of the same or related gene and (iii) lack of knowledge of the best allele- and stoichiometry-related to paralogous or distinct protein family members required in the pathway.

Opportunities, potential and promise of emerging systems and synthetic biology approaches

Precision genome engineering requires advanced knowledge base, informed design approaches and versatile biotechnological tools. With the availability of extensive genome sequence and expression resources, systems biology has emerged as a multidisciplinary research in understanding from interactions of molecules, signal transduction pathways to functional modules of biological systems (Bruggeman and Westerhoff, 2007; Chuang ; Sheth and Thaker, 2014).

Systems-level strategies and methodologies will be essential for interpreting and leveraging data/knowledge from different omics platforms (Bruggeman and Westerhoff, 2007; Sheth and Thaker, 2014). In general, top-down and bottom-up methods are two fundamental approaches to coordinate various levels of information in systems biology (Bruggeman and Westerhoff, 2007; Edwards and Thiele, 2013; Klipp ; Schneider, 2013). Top-down methods characterize multivariate data as a whole, and perform global analyses, such as principal components analysis (PCA), machine learning and pattern recognition, to fit a priori and post priori models. Bottom-up methods, on the other hand, are designed to build models or test hypotheses by utilizing existing knowledge of network. For example, due to the redundancy of a large laccase gene family in P. trichocarpa, the molecular functions of laccases are difficult to elucidate. Transcriptomics and metabolomics analyses of transgenic P. trichocarpa plants overexpressing Ptr-MIR397a have revealed the post-transcriptional regulation of multiple laccases genes and their roles in the regulation of lignin biosynthesis (Lu ). Practically, systems biology, more than simply collecting huge amount of omics data, relies heavily on mathematical modelling and computational simulations to aid the understanding of complex biological systems (Bruggeman and Westerhoff, 2007; Edwards and Thiele, 2013).

Synthetic biology aims to create living systems with designed functions, a distinguishing feature from traditional genetic selection or manipulation. The potential applications of synthetic biology have been extended to many aspects of life sciences from production of new drugs to biofuels (Church ). Despite the ambitious scope, recent progress in plants has not reached previous expectations mainly due to a lack of efficient genetic tools (Khalil and Collins, 2010; Lienert ). Thus in order to precisely reprogram biological parts to construct new bio-systems with novel functions, building solid foundations of well-defined tools (e.g. DNA synthesis, genome assembly, activation circuits) is the key to the success of synthetic biology (Brophy and Voigt 2014; Khalil and Collins, 2010; Lienert ). Many tools have recently been developed for creating multigene constructs, precision genome editing and site-specific gene stacking and removal (Liu ).

High-throughput assembly of multigene constructs

To design and optimize the metabolic pathways leading to a desirable production of low recalcitrant biomass feedstock requires transformation of multiple genes. A vector harbouring large DNA molecules with changeable elements provides an opportunity to efficiently deliver multiple genes. For example, the development of multigene vector systems, such as GoldenGate, ePathBricks and BioBrick, allows assembly of several genes in a predictable manner (Appleton ; DePaoli ; Liu ; Sarrion-Perdigones , 2014; Xu and Koffas, 2013). By combining de novo DNA synthesis with seamless assembly methods, it is now feasible to construct multigene vectors (Kosuri and Church, 2014).

Precision genome editing

As an emerging discipline, precise genome editing creates DNA deletion, mutation or integration at a specific site in the host genome, and represents a profound potential for plant biotechnology. The engineering of zinc-finger nucleases or TALE nucleases could also target any genomic sequence specifically, and generate DNA breaks (Sanjana ). This technology has been successfully used in many plant species to generate targeted mutants (Osakabe ; Shan ; Shukla ). Similarly, a recent method called clustered regularly interspaced short palindromic repeats (CRISPRs) generate DNA mutation using precursor RNA guided mechanism to edit genomic sequences (Haurwitz ; Liu ). The CRISPR-Cas9 system is best characterized between the different types of CRISPRs, and successful examples have been reported in tobacco, Arabidopsis and rice (Chen and Gao, 2014; Li ; Shan ).

Site-specific gene stacking and removal

As opposed to generating mutation in targeted regions of a genome, site-specific gene stacking and removal offers the ability of controlling the transgene insertion and removal. Reuse of selectable markers for multiple rounds of transformation and integration of transgene into specific target sites can reduce the risks of unintended consequences (Yau and Stewart, 2013). The application of recombinases systems in plants has been reported. For example, combining the expression of CRE recombinase and ϕC31 integrases in Arabidopsis, De Paepe have shown about 9% of transformants generated site-specific integration of T-DNA and removal of the resident selectable marker.

RNA-editing

A promising new technology is based on pentatricopeptide repeat (PPR) proteins known for their affinity to bind RNA rather DNA molecules (Yagi ). The nucleotide sequence specificity of PPR proteins for RNA binding and editing leads itself amenable to design of customizable enzymes suited to precision RNA engineering.

Here, we present a conceptual scheme of integration of systems biology and synthetic biology for understanding the molecular basis of cell wall biosynthesis and its regulation (Figure2). Firstly, from a top-down view, the various omics data are processed and analysed by common platform, and further integrated to form mathematical models; from a bottom-up view, to test hypotheses or models, individual perturbations are designed to generate new data sets for analysis. Secondly, through the systems biology iterations, meaningful models or genetic circuits would guide the design of synthetic biology to perform high-precision engineering tasks (Figure2). Specifically, systems biology studies could provide biological ‘parts’ (e.g. genes, promoters) and framework (e.g. gene network, spatial and temporal expression, metabolic flux and balance) for synthetic biology approaches such as precision genome editing and multiple gene transfer to reduce biomass recalcitrance.

Figure 2

A conceptual scheme describing the integration of systems and synthetic biology. In the upper panel, a ‘cloud’ of systems biology integrates top-down and bottom-up approaches to exploit the omics data. The knowledge from systems biology can guide the design of precision engineering for synthetic biology. The lower panel highlights that the key element of synthetic biology is to develop versatile biotechnological tools for its various applications.

In addition to the emerging tools of genetic engineering mentioned above, methods also exist for ‘rational’ engineering of enzyme structure. There are two main approaches to improving or creating new protein functions, rational design of proteins and directed evolution. Put simply, rational design is the is the generation of new designer proteins based on prior knowledge, and directed evolution is the selection of new functions on the basis of screening of randomly generated variant proteins.

Enzymes of cellulase or glucohydrolase family have been employed in ‘rational design’ strategies to improve the catalytic efficiency during biomass conversion, (Buckeridge and Gustavo, 2011). In general, site-directed mutagenesis and random mutagenesis are two common strategies by which modifications of key residues of proteins of interest. Through three-dimensional structure and molecular dynamics simulation analyses of normal and mutated protein, the knowledge of biochemical importance of key residues would guide the rational design to alter protein structure (McLaughlin ). For example, based on the structures of class II cellobiohydrolases, Heinzelman screened over 6000 of chimeras and identified three thermostable fungal chimeric cellulases.

Conclusion and future perspective

It is an exciting time for plant biologists addressing grand challenge questions. The vast amount of knowledge being garnered on the basis of widely available sequencing, omics profiling, genome engineering and higher resolution phenotyping technologies is moving the field towards the targeted goal of designer plants with improved biomass properties.

It is clear that the recalcitrance of lignocellulosic biomass is determined by plant properties of growth, xylem development and patterning, secondary cell wall formation and cell wall composition. However, the complex sets of biosynthetic and remodelling pathways, and their interactions with transcription factors and signalling effectors underlying recalcitrance properties are not fully understood. Therefore, there is an immediate need to fill the critical knowledge gaps using advanced systems biology studies. While single gene manipulations may have measureable effects on target phenotype, it is also clear that synthetic biology approaches provide an unprecedented opportunity to engineer the cell wall composition, minimize off-target effects and reduce biomass recalcitrance for bioenergy production. The example of ref8 mutant study in A. thaliana has demonstrated a two-step strategy of genetic engineering wherein lignin composition is altered, saccharification efficiency is improved and negative growth effects are restored. A timely convergence of systems and synthetic biology is necessary to address the challenging goal of plant cell wall modification for efficient conversion of biomass to biofuels.

Beyond improved cell wall characteristics, an ideotypic bioenergy crop will also require improved plant performance resulting in high productivity under conditions of suboptimal soil structure, nutrient composition, temperature, moisture and resiliency to various biotic stresses. The systems biology and synthetic biology tools, barriers and opportunities discussed in the context of feedstock improvement for cell wall properties, are expected to be largely universal to research in other areas of plant improvement as well as studies of other plant-derived biofuels and biomaterials.