Recent advances and current challenges in synthetic biology of the

Plant Physiology Preview. Published on September 4, 2018, as DOI:10.1104/pp.18.00767

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Title

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Recent advances and current challenges in synthetic biology of the plastid genetic system and

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metabolism

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Short title

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Plastid synthetic biology

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Authors’ byline

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Christian R. Boehm1 and Ralph Bock1,*

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Authors’ addresses

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Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm,

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Germany

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Corresponding author

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Ralph Bock

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Max Planck Institute of Molecular Plant Physiology

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Am Mühlenberg 1

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D-14476 Potsdam-Golm

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Germany

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email: [email protected]

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One-Sentence Summary: Recent technological developments narrow the existing complexity

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gap between bacterial and plastid synthetic biology.

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SUMMATIVE PARAGRAPH

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Building on recombinant DNA technology, leaps in synthesis, assembly and analysis of DNA have

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revolutionized genetics and molecular biology over the past two decades (Kosuri and Church,

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2014). These technological advances have accelerated the emergence of synthetic biology as a

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new discipline (Cameron et al., 2014). Synthetic biology is characterized by efforts targeted at

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the modification of existing and the design of novel biological systems based on principles 1 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

Copyright 2018 by the American Society of Plant Biologists

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adopted from information technology and engineering (Andrianantoandro et al., 2006; Khalil

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and Collins, 2010). As in more traditional engineering disciplines such as mechanical, electrical

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and civil engineering, synthetic biologists utilize abstraction, decoupling and standardization to

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make the design of biological systems more efficient and scalable. To facilitate the management

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of complexity, synthetic biology relies on an abstraction hierarchy composed of multiple levels

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(Endy, 2005): DNA as genetic material, “parts” as elements of DNA encoding basic biological

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functions (e.g., promoter, ribosome-binding site, terminator sequence), “devices” as any

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combination of parts implementing a human-defined function, and “systems” as any

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combination of devices fulfilling a predefined purpose. Parts are designated to perform

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predictable and modular functions in the context of higher-level devices or systems, which are

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successively refined through a cycle of designing, building and testing.

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Within the past two decades, the synthetic biology approach has produced several

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notable successes, especially in microbial systems. These include, for example, the design of a

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minimal bacterial genome (Hutchison et al., 2016) and a highly modified yeast genome

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(Richardson et al., 2017) as well as the metabolic engineering of yeast for the biosynthesis of the

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antimalarial drug precursor artemisinic acid (Ro et al., 2006) and the opioid compounds

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thebaine and hydrocodone (Galanie et al., 2015). Compared to synthetic biology in bacteria and

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yeast, synthetic biology in algae and plants is still lagging behind. While the potential of

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photoautotrophic organisms for environmentally sustainable bioproduction has long been

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recognized (Ryan Georgianna and Mayfield, 2012; Fesenko and Edwards, 2014; Liu and Stewart,

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2015; Boehm et al., 2017), their relatively slow growth, scarcely available tools for genetic

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manipulation and the physiological as well as genomic complexity of plant systems have delayed

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their widespread adoption as synthetic biology chassis. However, especially the small genome of

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the plastid (chloroplast) represents a highly promising platform for engineering the

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sophisticated metabolism and physiology of the eukaryotic cell it is embedded in (Fig. 1).

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The chloroplast originated through the endosymbiotic uptake of a cyanobacterium by a

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heterotrophic eukaryote more than a billion years ago (Palmer, 2003). Following this event, the

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endosymbiont evolved mechanisms for facilitated exchange of metabolites with the host cell,

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underwent radical streamlining of its genome (by gene loss and large-scale transfer of genes to

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the host nuclear genome) and established an import machinery for the uptake of nucleus-

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encoded proteins. The resulting organelle serves as the major biosynthetic compartment in

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photoautotrophic organisms, and has been exploited as a platform for metabolic engineering

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and molecular farming since the successful development of transformation technologies in the

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late 1980s (Boynton et al., 1988; Svab et al., 1990). Compared to nuclear genetic engineering,

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plastid transformation offers several notable advantages relevant to plant biotechnology. These

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include (i) the high precision of genetic engineering enabled by efficient homologous

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recombination, (ii) the possibility of transgene stacking in synthetic operons, (iii) the potential

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for high-level expression of gene products, (iv) the absence of epigenetic transgene silencing,

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and (v) the reduced risk of unwanted transgene transmission due to maternal inheritance of

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plastid DNA (Bock, 2015). In this article, we provide an update on tools and technologies available for extending the

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synthetic biology approach to plastids, and highlight key challenges to be addressed through

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future research. Guided by an abstraction hierarchy of biological design, we identify a scarcity of

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well-characterized genetic parts, tightly controlled expression devices and quantitative

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knowledge of plastid gene expression as current key limitations to plastid synthetic biology. We

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highlight recent technological developments narrowing the existing complexity gap between

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bacterial and plastid synthetic biology, and provide an outlook to the implementation of complex

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systems such as synthetic metabolic feedback loops, designer subcompartments and tailor-made

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genomes in chloroplasts.

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MAIN SECTION

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Parts 3 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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The Registry of Standard Biological Parts (http://parts.igem.org) currently contains over 20,000

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genetic elements which can be requested by researchers for use in synthetic biology

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applications. From this collection, approximately 100 parts each have been designed for use in

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the unicellular green alga Chlamydomonas reinhardtii and in multicellular plants (e.g., the seed

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plants Nicotiana tabacum and Arabidopsis thaliana, the moss Physcomitrella patens and the

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liverwort Marchantia polymorpha). The majority of these parts are designated for nuclear

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engineering, with only about two dozen suitable for gene expression from the chloroplast

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genome. One explanation for the relative paucity of plastid genetic elements in the Registry of

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Standard Biological Parts lies in the half-year timeframe of projects pursued as part of the

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international Genetically Engineered Machine (iGEM) competition (Smolke, 2009), which is

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barely compatible with the generation and characterization of stable plastid-engineered

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(transplastomic) organisms. Beyond iGEM, the repertoire of regulatory sequences routinely

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used for transgene expression in plastids has remained similarly small: it is comprised of a few

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preferred promoters (e.g., from the plastid ribosomal RNA operon, Prrn; the gene for the large

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subunit of Rubisco, PrbcL; and the gene for the D1 protein of photosystem II, PpsbA) and a

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handful of 5’- and 3’-UTRs (Jin and Daniell, 2015). In addition, the bacterial hybrid promoter Ptrc

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(Newell et al., 2003) and several bacteriophage-derived expression elements (McBride et al.,

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1994; Kuroda, 2001; Yang et al., 2013) have been successfully used for plastid transgene

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expression. A greater variety of parts available for controlled expression of plastid transgenes is

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desirable for several reasons. First, multiple use of the same genetic element within the

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chloroplast genome is problematic due to the risk of unwanted homologous recombination

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between sequence stretches as short as 50 bp (Dauvillee et al., 2004; Rogalski et al., 2006).

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Second, synthetic genetic circuits commonly require precise tuning of the activity of their

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constitutive parts for optimal function (Brophy and Voigt, 2014).

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For synthetic biology applications in plastids to catch up in versatility and complexity

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with those already demonstrated in bacteria, gene expression elements covering a wider activity

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range will be required. Natural plastid genomes represent an obvious source of such elements.

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The small size and low coding capacity of chloroplast genomes (in most seed plants,

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approximately 130 genes in a ~150 kb genome) should allow refactoring of all coding and

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regulatory regions into standardized genetic parts. The sequences of over 800 chloroplast

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genomes have been determined (Daniell et al., 2016), and the functions of most of their (widely

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conserved) genes are known (Scharff and Bock, 2014). Plastid genetic elements contained

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within this wealth of sequence data can be domesticated according to a recently proposed

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common syntax for plant synthetic biology (Patron et al., 2015). This scheme promises to

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facilitate sharing of genetic resources among the community and, although developed for a

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eukaryotic system, is also compatible with GoldenBraid-based modular cloning of chloroplast

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transformation vectors (Vafaee et al., 2014). Plastid parts containing internal recognition sites

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for type IIS restriction enzymes (e.g., BsaI, BsmBI, BbsI) that cannot be synonymously changed

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(e.g., because they constitute essential sequence motifs in a promoter or UTR sequence) may

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alternatively be assembled using long-overlap-based methods such as Gibson Assembly (Gibson

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et al., 2009).

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Gene Expression Devices

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Gene expression devices send or receive signals in the form of levels of gene expression. A basic

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device of this kind may be composed of four parts: a promoter, a ribosome-binding site, a coding

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sequence and a terminator. This device architecture is commonly used for the quantification of

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part performance to inform the rational design of genetic circuits. Hundreds of prokaryotic

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gene-expression elements (including promoters, ribosome-binding sites and terminators) have

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been characterized in bacterial hosts using reporter gene-based assays (Salis et al., 2009;

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Cambray et al., 2013; Chen et al., 2013; Kosuri et al., 2013; Mutalik et al., 2013), and standards

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have been formulated for quantifying their activities (Canton et al., 2008; Kelly et al., 2009;

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Rudge et al., 2016). To reduce the context dependence of part activity, standardized flanking

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sequences (Mutalik et al., 2013), strong terminators (Chen et al., 2013) and enzymatic cleavage

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of UTRs (Lou et al., 2012; Qi et al., 2012) have been successfully employed as insulators in

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bacteria. In plastids, not more than two dozen combinations of regulatory elements (i.e., 5 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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promoters, 5’-UTRs and 3’-UTRs) have been systematically characterized for their impact on

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transgene expression using GFP (Barnes et al., 2005; Caroca et al., 2013), GUS (Eibl et al., 1999;

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Herz et al., 2005; Gerasymenko et al., 2017) or other reporter proteins (Ruhlman et al., 2010;

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Zhang et al., 2012).

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Compared to part characterization in microbes, that in plastids involves several notable

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challenges. First, relatively long timescales are required to generate transplastomic organisms

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ready for characterization. While only a few days are needed for transformation of the microbial

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models Escherichia coli or Saccharomyces cerevisiae by a genetic part, several months of

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selection are needed to recover homoplasmic plastid transformants (i.e., transplastomic cells or

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plants that are devoid of residual copies of the wild-type plastid genome). In theory, the

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establishment of homoplasmy could be accelerated through inducible expression of

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endonucleases that selectively target the wild-type chloroplast genome, but it remains to be

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tested how much time this approach can save. Alternatively, measurement fidelity can be traded

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for high-throughput, transient assays to quantify part performance within days of particle

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bombardment of algal cells or plant tissues. Such assays will require (i) high transient

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transformation frequencies, (ii) high sensitivity, and (iii) a robust way of normalizing the

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primary reporter signal to the copy number of transformed plastomes. The latter could be

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achieved by using a ratiometric approach (Rudge et al., 2016; Boehm et al., 2018). If a suitable

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reporter system can be developed, the activities of hundreds of plastid parts could rapidly be

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measured in algal cells or plant protoplasts using microtiter plate-based assays (Schaumberg et

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al., 2016) or microfluidic devices (Yu et al., 2018).

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Second, the plastome exhibits abundant read-through transcription due to inefficient

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termination (Stern and Gruissem, 1987; Rott et al., 1996; Legen et al., 2002; Shi et al., 2016).

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Consequently, part behavior is, by default, poorly insulated from its specific genetic context:

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both upstream promoters and downstream antisense promoters may significantly affect the

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expression level of a target gene (Quesada-Vargas et al., 2005; Sharwood et al., 2011). However,

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some sequences such as the endogenous tRNA genes trnS and trnH (Stern and Gruissem, 1987)

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or the heterologous E. coli threonine attenuator (thra; Chen and Orozco, 1988) have been shown

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to terminate plastid transcription with at least 85% efficiency. Use of insulators based on these

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parts or new synthetic terminators can potentially enhance the robustness of gene expression

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levels generated by plastid synthetic biology devices.

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Third, plastid transgene expression has been shown to be primarily determined by post-

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transcriptional control and protein stability rather than by the accumulation of mRNA (Eberhard

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et al., 2002; Birch-Machint et al., 2004; Bellucci et al., 2005; Kahlau and Bock, 2008; Valkov et al.,

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2009; Zoschke and Bock, 2018). Chloroplast transcripts are subject to a series of complex

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processing steps which include intercistronic cleavage, 5’- and 3’-end maturation, intron splicing

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and mRNA editing (Stern et al., 2010). These steps are largely mediated by nucleus-encoded and

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organelle-targeted factors, including a large family of modular proteins known as

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pentatricopeptide repeat (PPR) proteins that site-specifically bind to one or several pre-mRNAs

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(Barkan and Small, 2014). Plastid gene expression levels can, therefore, vary considerably

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between different transgenes even if the same promoter and 3’-UTR are used, limiting the

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informative value of part characterization based on standard reporter protein assays. While the

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amino acid sequence of the N-terminus is thought to substantially influence protein stability in

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the chloroplast (Apel et al., 2010; De Marchis et al., 2012), our general knowledge of plastid

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proteostasis remains limited. A better understanding of the molecular determinants of plastid

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protein (in)stability may in the future allow the design of protective amino acid sequences

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(Elghabi et al., 2011) that level the stabilities of different plastid-expressed proteins and make

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transgene expression from the plastid genome more predictable.

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Metabolic Devices

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Metabolic devices send or receive signals in the form of levels of metabolites. Accordingly, a

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synthetic metabolic pathway represents a metabolic device carrying out a specific series of

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enzyme-catalyzed reactions. A variety of metabolic devices have been successfully implemented

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in plastids for the production of molecules such as polyhydroxybutyrate (Bohmert-Tatarev et al.,

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2011), carotenoids (Wurbs et al., 2007; Hasunuma et al., 2008; Apel and Bock, 2009), fatty acids 7 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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(Madoka et al., 2002; Craig et al., 2008), artemisinic acid (Fuentes et al., 2016), vitamin E (Lu et

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al., 2013) and dhurrin (Gnanasekaran et al., 2016). These applications have been reviewed in

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more detail elsewhere (Bock, 2015; Fuentes et al., 2018). While heterologous pathways

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composed of 20 genes or more have been expressed in bacteria and yeast (Temme et al., 2012;

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Galanie et al., 2015; Li et al., 2018), no more than seven transgenes have to date been

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simultaneously expressed from the plastome (Krichevsky et al., 2010). The complexity of

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plastid-based metabolic devices has primarily been limited by a scarcity of available expression

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signals (see Gene Expression Devices) rather than by the physical size of the introduced DNA

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(Adachi et al., 2007). Recently, the complexity and number of pathway variants accessible to

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experimental interrogation has been expanded through combinatorial supertransformation of

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transplastomic recipient lines (COSTREL). Using this approach, an up to 77-fold increase in

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artemisinic acid production has been demonstrated in transplastomic tobacco plants

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combinatorially supertransformed by five additional nuclear transgenes (Fuentes et al., 2016).

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There is no in-principle limitation to the number of transgenes that can be simultaneously

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introduced into the plant nucleus using combinatorial transformation (Naqvi et al., 2009).

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However, handling hundreds to thousands of plants resulting from combinatorial

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transformation with several dozen transgenes will require an effective screening pipeline.

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In plastid-based metabolic devices containing multicistronic operons, intercistronic

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expression elements (IEEs) can be used to facilitate correct processing of polycistronic

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transcripts into monocistronic mRNAs and their efficient translation (Fig. 2A; Zhou et al., 2007).

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To avoid defects in mRNA stabilization upon repeated use of the same IEE, more complex future

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metabolic devices may feature a variety of different such elements and/or additionally

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overexpress their cognate RNA-binding proteins (Legen et al., 2018).

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Genetic Circuits

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Genetic circuits mimic logical functions commonly found in their electronic counterparts. A

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genetic circuit can be used to control the activity of other devices (such as the gene expression

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devices or metabolic devices discussed above) in response to external stimuli. A wide range of 8 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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genetic circuits implementing Boolean logic functions such as YES, NOT, AND, OR, NAND, NOR, XOR

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and N-IMPLY has been reported for bacteria, yeast and mammalian cells (Miyamoto et al., 2013).

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In plastids, only the simplest logic function YES has been implemented in the form of chemically

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inducible transgene expression.

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Chloroplast transcription is natively controlled by two different types of RNA

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polymerases in seed plants. The nucleus-encoded RNA polymerase (NEP) is a chloroplast-

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targeted bacteriophage-type single subunit enzyme, while the plastid-encoded RNA polymerase

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(PEP) is a eubacteria-type multisubunit enzyme (Barkan, 2011; Börner et al., 2015). The

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promoter specificity of PEP is modulated by nucleus-encoded and plastid-targeted sigma factors

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in response to light, hormones and biotic as well as abiotic stresses. However, due to their

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important role in plant growth, development and survival (and the pervasive transcription of

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essentially all plastid genes), NEP and PEP are poorly suited as stringent controllers of synthetic

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genetic circuits in plastids.

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As an alternative to transgene control by the endogenous transcription machineries,

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plastid transgene expression has been controlled through nucleus-encoded and plastid-targeted

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bacteriophage RNA polymerases or processing factors that are responsive to chemical inducers

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such as salicylic acid (Magee et al., 2004), ethanol (Lössl et al., 2005), copper (Surzycki et al.,

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2007) or thiamine (Ramundo et al., 2013). To avoid (pollen-transmissible) nuclear transgenes

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and increase transgene containment, inducible expression systems encoded solely in the plastid

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genome are particularly desirable. Plastid-only inducible circuits responsive to isopropyl β-D-1-

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thiogalactopyranoside (IPTG; Mühlbauer and Koop, 2005) or theophylline (Verhounig et al.,

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2010; Emadpour et al., 2015) have been shown to be functional, yet fall short of binary behavior

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due to the pronounced transcriptional leakiness present in plastids (see Gene Expression

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Devices). To achieve a signal-to-noise ratio sufficient for the implementation of more complex

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logic gates, future plastid-based genetic circuits may employ synthetic RNA-binding proteins of

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the PPR class (see Gene Expression Devices; Coquille et al., 2014; Gully et al., 2015) to selectively

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control the maturation of target mRNAs in the chloroplast (Fig. 2B; Stern et al., 2010; Barkan and

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Small, 2014).

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Systems

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Beyond hard-wired logic gates, synthetic biologists have explored dynamic feedback

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mechanisms to enhance the efficiency of engineered metabolic pathways in bacteria and yeast

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(Venayak et al., 2015; Del Vecchio et al., 2016). Translation of this approach to plastids is

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currently hampered by our limited quantitative understanding of chloroplast gene expression,

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though new tools for analysis of the metabolic network shared between the chloroplast and its

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host cell are emerging (Gloaguen et al., 2017). Metabolic engineering in plastids may further be

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supported by expression of synthetic subcompartments for substrate concentration, metabolite

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channeling and the prevention of unwanted reactions between subcompartmentalized and

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endogenous plastid metabolites and enzymes (Winkel, 2004; Ort et al., 2015; Hanson et al.,

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2016). Synthetic subcompartments have already been introduced in bacteria and yeast (Bonacci

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et al., 2012; Lau et al., 2018), and carboxysomal shell proteins transiently expressed in leaves of

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Nicotiana benthamiana have been shown to be capable of assembling into carboxysome-like

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structures within chloroplasts (Lin et al., 2014), encouraging further efforts in this area.

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Among the most complex systems proposed for implementation in plastids are entire

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synthetic genomes, inspired by recent successes in microbial synthetic genomics (Hutchison et

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al., 2016; Richardson et al., 2017). A minimum-size plastid genome composed of the smallest

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possible number of components will be of great value for two reasons: it will advance our

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understanding of the regulatory network underlying plastid function, and it will serve as a

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template for engineering synthetic plastomes to be used in biotechnological applications. We

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have previously proposed a design for a synthetic minimal plastome of N. tabacum that is free of

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all genes non-essential under heterotrophic growth conditions (Fig. 3), intergenic spacers,

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introns, and isoaccepting tRNA genes that are dispensable or become dispensable after genome-

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wide modification of codon usage (Scharff and Bock, 2014). Such a synthetic chloroplast genome

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can be assembled from linear DNA fragments in yeast (O’Neill et al., 2012) and, prior to plant 10 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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transformation, can be amplified in vitro using rolling circle amplification (Jansen et al., 2005).

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The major hurdle to the successful implementation of fully synthetic plastomes in planta is the

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high probability of homologous recombination between the (largely non-recodeable) rRNA and

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tRNA genes and their counterparts in the resident plastid genome, leading to chimeric genomes

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of unpredictable structure and function (O’Neill et al., 2012). In addition, the effects of synthetic

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lethality (i.e., the combined knock-out of two non-essential genes being lethal; e.g., Ehrnthaler et

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al., 2014) cannot currently be excluded to occur in a synthetic minimal plastome.

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Despite numerous technical advances made over the past 30 years, the number of algal and

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plant species whose plastids can reliably be transformed has remained small (Bock, 2015).

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Transplantation of transgenic plastids from a species amenable to transformation to a species

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recalcitrant to transformation represents an attractive alternative to painstakingly developing

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specialized transformation protocols for the latter. Plastomes can be horizontally transferred

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across graft junctions with relative ease (Stegemann and Bock, 2009; Stegemann et al., 2012;

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Thyssen et al., 2012; for review, see Bock, 2017) and this process has been exploited for

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transplanting a plastid-encoded synthetic metabolic device into a currently non-transformable

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species (Lu et al., 2017). The graft-mediated horizontal transfer of transgenic plastid genomes

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may not be feasible between distantly related species due to the close co-evolution of nuclear

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and plastid genomes, and the probability of nuclear-cytoplasmic incompatibilities that increases

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with phylogenetic distance and can cause deleterious phenotypes (Schmitz-Linneweber et al.,

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2005; Greiner and Bock, 2013). However, the transfer will certainly facilitate the expansion of

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transplastomic technologies from model species and cultivars used in research to related species

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and elite cultivars grown commercially.

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FIGURE LEGENDS

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11 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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Figure 1. Biological properties and existing technical capacities for synthetic biology of plastids

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compared to bacteria, yeast and the plant nucleus. The number of asterisks roughly illustrates

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the relative degree of (top) presence of a biological feature, (middle) availability of a tool or

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technique, and (bottom) current implementation of a type of application across the different

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chassis.

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Figure 2. Design of plastid-based metabolic devices. A, Intercistronic expression elements (IEEs;

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Zhou et al., 2007) can be used to design synthetic operons composed of n genes of interest

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(GOIs) under the control of a single promoter. Alternatively, each transgene can be controlled by

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its own promoter. SD: Shine-Dalgarno sequence; SMG: selectable marker gene. B, Expression of a

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GOI can be controlled by a synthetic 5’-UTR that is specifically stabilized by a designer PPR

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protein (that recognizes a different binding sequence than all other RNA-binding proteins

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present in the plastid).

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Figure 3. Physical map of the N. tabacum chloroplast genome with all genes classified by

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essentiality. Genes shown in blue are essential for both heterotrophic and autotrophic growth.

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Genes shown in green are essential for autotrophic growth only. Light green indicates borderline

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cases where knock-out plants survive under carefully controlled growth conditions. Genes

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shown in grey are non-essential under both heterotrophic and autotrophic growth conditions, in

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that their knock-out causes no or only a mild phenotype (Scharff and Bock, 2014). Origins of

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replication are highlighted in red. Grey arrows indicate the direction of transcription for the two

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DNA strands. The map was drawn using the OrganellarGenomeDRAW (OGDRAW) software

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(Lohse et al., 2013) based on the complete plastome sequence of N. tabacum (Shinozaki et al.,

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1986; GenBank accession number Z00044.2). Abbreviations: LSC, large single-copy region; IRA,

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inverted repeat A; IRB, inverted repeat B; SSC, small single-copy region.

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LITERATURE CITED

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Adachi T, Takase H, Tomizawa K (2007) Introduction of a 50 kbp DNA fragment into the plastid genome. Biosci Biotechnol Biochem 71: 2266–2273 Andrianantoandro E, Basu S, Karig DK, Weiss R (2006) Synthetic biology: new engineering rules for an emerging discipline. Mol Syst Biol 2: 2006.0028 Apel W, Bock R (2009) Enhancement of carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitamin A conversion. Plant Physiol 151: 59–66 Apel W, Schulze WX, Bock R (2010) Identification of protein stability determinants in chloroplasts. Plant J 63: 636–650 Barkan A (2011) Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold. Plant Physiol 155: 1520–1532 Barkan A, Small I (2014) Pentatricopeptide repeat proteins in plants. Annu Rev Plant Biol 65: 415–442 Barnes D, Franklin S, Schultz J, Henry R, Brown E, Coragliotti A, Mayfield SP (2005) Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol Genet Genomics 274: 625–636 Bellucci M, De Marchis F, Mannucci R, Bock R, Arcioni S (2005) Cytoplasm and chloroplasts are not suitable subcellular locations for β-zein accumulation in transgenic plants. J Exp Bot 56: 1205–1212 Birch-Machint I, Newell CA, Hibberd JM, Gray JC (2004) Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability. Plant Biotechnol J 2: 261–270 Bock R (2015) Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annu Rev Plant Biol 66: 211–241 Bock R (2017) Witnessing genome evolution: experimental reconstruction of endosymbiotic and horizontal gene transfer. Annu Rev Genet 51: 1–22 Boehm CR, Grant PK, Haseloff J (2018) Programmed hierarchical patterning of bacterial populations. Nat Commun 9: 776 Boehm CR, Pollak B, Purswani N, Patron N, Haseloff J (2017) Synthetic botany. Cold Spring Harb Perspect Biol a023887 Bohmert-Tatarev K, McAvoy S, Daughtry S, Peoples OP, Snell KD (2011) High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for the production of polyhydroxybutyrate. Plant Physiol 155: 1690–1708 Bonacci W, Teng PK, Afonso B, Niederholtmeyer H, Grob P, Silver PA, Savage DF (2012) Modularity of a carbon-fixing protein organelle. Proc Natl Acad Sci U S A 109: 478–483 Börner T, Aleynikova AY, Zubo YO, Kusnetsov VV (2015) Chloroplast RNA polymerases: role in chloroplast biogenesis. Biochim Biophys Acta - Bioenerg 1847: 761–769 Boynton J, Gillham N, Harris E, Hosler J, Johnson A, Jones A, Randolph-Anderson B, Robertson D, Klein T, Shark K, et al (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240: 1534–1538 Brophy JAN, Voigt CA (2014) Principles of genetic circuit design. Nat Methods 11: 508–520 Cambray G, Guimaraes JC, Mutalik VK, Lam C, Mai QA, Thimmaiah T, Carothers JM, Arkin AP, Endy D (2013) Measurement and modeling of intrinsic transcription terminators. Nucleic Acids Res 41: 5139–5148 Cameron DE, Bashor CJ, Collins JJ (2014) A brief history of synthetic biology. Nat Rev Microbiol 12: 381–390 13 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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Canton B, Labno A, Endy D (2008) Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26: 787–793 Caroca R, Howell KA, Hasse C, Ruf S, Bock R (2013) Design of chimeric expression elements that confer high-level gene activity in chromoplasts. Plant J 73: 368–379 Chen LJ, Orozco EM (1988) Recognition of prokaryotic transcription terminators by spinach chloroplast RNA polymerase. Nucleic Acids Res 16: 8411–8431 Chen Y-J, Liu P, Nielsen AA, Brophy JA, Clancy K, Peterson T, Voigt CA (2013) Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10: 659–664 Coquille S, Filipovska A, Chia T, Rajappa L, Lingford JP, Razif MFM, Thore S, Rackham O (2014) An artificial PPR scaffold for programmable RNA recognition. Nat Commun 5: 1–9 Craig W, Lenzi P, Scotti N, De Palma M, Saggese P, Carbone V, McGrath Curran N, Magee AM, Medgyesy P, Kavanagh TA, et al (2008) Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance. Transgenic Res 17: 769–782 Daniell H, Lin C-S, Yu M, Chang W-J (2016) Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol 17: 134 Dauvillee D, Hilbig L, Preiss S, Johanningmeier U (2004) Minimal extent of sequence homology required for homologous recombination at the psbA locus in Chlamydomonas reinhardtii chloroplasts using PCR-generated DNA fragments. Photosynth Res 79: 219–224 Eberhard S, Drapier D, Wollman F-A (2002) Searching limiting steps in the expression of chloroplast-encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii. Plant J 31: 149–160 Ehrnthaler M, Scharff LB, Fleischmann TT, Hasse C, Ruf S, Bock R (2014) Synthetic lethality in the tobacco plastid ribosome and its rescue at elevated growth temperatures. Plant Cell 26: 765–776 Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop H-U (1999) In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency. Plant J 19: 333–345 Elghabi Z, Karcher D, Zhou F, Ruf S, Bock R (2011) Optimization of the expression of the HIV fusion inhibitor cyanovirin-N from the tobacco plastid genome. Plant Biotechnol J 9: 599– 608 Emadpour M, Karcher D, Bock R (2015) Boosting riboswitch efficiency by RNA amplification. Nucleic Acids Res 43: e66 Endy D (2005) Foundations for engineering biology. Nature 438: 449–453 Fesenko E, Edwards R (2014) Plant synthetic biology: a new platform for industrial biotechnology. J Exp Bot 65: 1927–1937 Fuentes P, Armarego-Marriott T, Bock R (2018) Plastid transformation and its application in metabolic engineering. Curr Opin Biotechnol 49: 10–15 Fuentes P, Zhou F, Erban A, Karcher D, Kopka J, Bock R (2016) A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop. Elife 5: 1–26 Galanie S, Thodey K, Trenchard IJ, Filsinger Interrante M, Smolke CD (2015) Complete biosynthesis of opioids in yeast. Science 349: 1095–1100 Gerasymenko IM, Sheludko YV, Klebanovych AA, Rudas VA, Shakhovsky AM, Klein TM, Kuchuk NV. (2017) Comparison of effectiveness of 5′-regulatory sequences in transplastomic tobacco chloroplasts. Transgenic Res 26: 65–75 Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345 Gloaguen P, Bournais S, Alban C, Ravanel S, Seigneurin-Berny D, Matringe M, Tardif M, Kuntz M, Ferro M, Bruley C, et al (2017) ChloroKB: a web application for the integration of knowledge related to chloroplast metabolic network. Plant Physiol 174: 922–934 Gnanasekaran T, Karcher D, Nielsen AZ, Martens HJ, Ruf S, Kroop X, Olsen CE, Motawie MS, 14 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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Quesada-Vargas T, Ruiz ON, Daniell H (2005) Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation. Plant Physiol 138: 1746–1762 Ramundo S, Rahire M, Schaad O, Rochaix J-D (2013) Repression of essential chloroplast genes reveals new signaling pathways and regulatory feedback loops in Chlamydomonas. Plant Cell 25: 167–186 Richardson SM, Mitchell LA, Stracquadanio G, Yang K, Dymond JS, DiCarlo JE, Lee D, Huang CLV, Chandrasegaran S, Cai Y, et al (2017) Design of a synthetic yeast genome. Science 355: 1040–1044 Ro DK, Paradise EM, Quellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, et al (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440: 940–943 Rogalski M, Ruf S, Bock R (2006) Tobacco plastid ribosomal protein S18 is essential for cell survival. Nucleic Acids Res 34: 4537–4545 Rott R, Drager RG, Stern DB, Schuster G (1996) The 3’ untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators. Mol Gen Genet 252: 676–683 Rudge TJ, Brown JR, Federici F, Dalchau N, Phillips A, Ajioka JW, Haseloff J (2016) Characterization of intrinsic properties of promoters. ACS Synth Biol 5: 89–98 Ruhlman T, Verma D, Samson N, Daniell H (2010) The role of heterologous chloroplast sequence elements in transgene integration and expression. Plant Physiol 152: 2088–2104 Ryan Georgianna D, Mayfield SP (2012) Exploiting diversity and synthetic biology for the production of algal biofuels. Nature 488: 329–335 Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27: 946–950 Scharff LB, Bock R (2014) Synthetic biology in plastids. Plant J 78: 783–798 Schaumberg KA, Antunes MS, Kassaw TK, Xu W, Zalewski CS, Medford JI, Prasad A (2016) Quantitative characterization of genetic parts and circuits for plant synthetic biology. Nat Methods 13: 94–100 Schmitz-Linneweber C, Kushnir S, Babiychuk E, Poltnigg P, Herrmann RG, Maier RM (2005) Pigment deficiency in nightshade/tobacco cybrids is caused by the failure to edit the plastid ATPase alpha-subunit mRNA. Plant Cell 17: 1815–1828 Sharwood RE, Halpert M, Luro S, Schuster G, Stern DB (2011) Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA. RNA 17: 2165–2176 Shi C, Wang S, Xia E-H, Jiang J-J, Zeng F-C, Gao L-Z (2016) Full transcription of the chloroplast genome in photosynthetic eukaryotes. Sci Rep 6: 30135 Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, et al (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5: 2043–2049 Smolke CD (2009) Building outside of the box: IGEM and the BioBricks Foundation. Nat Biotechnol 27: 1099–1102 Stegemann S, Bock R (2009) Exchange of genetic material between cells in plant tissue grafts. Science 324: 649–651 Stegemann S, Keuthe M, Greiner S, Bock R (2012) Horizontal transfer of chloroplast genomes between plant species. Proc Natl Acad Sci U S A 109: 2434–2438 Stern DB, Goldschmidt-Clermont M, Hanson MR (2010) Chloroplast RNA metabolism. Annu Rev Plant Biol 61: 125–155 Stern DB, Gruissem W (1987) Control of plastid gene expression: 3′ inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription. Cell 51: 1145–1157 Surzycki R, Cournac L, Peltier G, Rochaix J-D (2007) Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas. Proc Natl Acad Sci U S A 104: 17 Downloaded from on September 4, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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17548–17553 Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci U S A 87: 8526–8530 Temme K, Zhao D, Voigt CA (2012) Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc Nat Acad Sci USA 109: 7085–7090 Thyssen G, Svab Z, Maliga P (2012) Cell-to-cell movement of plastids in plants. Proc Natl Acad Sci U S A 109: 2439–2443 Vafaee Y, Staniek A, Mancheno-Solano M, Warzecha H (2014) A modular cloning toolbox for the generation of chloroplast transformation vectors. PLoS One 9: e110222 Valkov VT, Scotti N, Kahlau S, MacLean D, Grillo S, Gray JC, Bock R, Cardi T (2009) Genomewide analysis of plastid gene expression in potato leaf chloroplasts and tuber amyloplasts: transcriptional and posttranscriptional control. Plant Physiol 150: 2030–2044 Del Vecchio D, Dy AJ, Qian Y (2016) Control theory meets synthetic biology. J R Soc Interface 13: 20160380 Venayak N, Anesiadis N, Cluett WR, Mahadevan R (2015) Engineering metabolism through dynamic control. Curr Opin Biotechnol 34: 142–152 Verhounig A, Karcher D, Bock R (2010) Inducible gene expression from the plastid genome by a synthetic riboswitch. Proc Natl Acad Sci U S A 107: 6204–6209 Winkel BSJ (2004) Metabolic channeling in plants. Annu Rev Plant Biol 55: 85–107 Wurbs D, Ruf S, Bock R (2007) Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J 49: 276–288 Yang H, Gray BN, Ahner BA, Hanson MR (2013) Bacteriophage 5′ untranslated regions for control of plastid transgene expression. Planta 237: 517–527 Yu Z, Boehm CR, Hibberd JM, Abell C, Haseloff J, Burgess SJ, Reyna-Llorens I (2018) Dropletbased microfluidic analysis and screening of single plant cells. PLoS One 13: e0196810 Zhang J, Ruf S, Hasse C, Childs L, Scharff LB, Bock R (2012) Identification of cis-elements conferring high levels of gene expression in non-green plastids. Plant J 72: 115–128 Zhou F, Karcher D, Bock R (2007) Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons. Plant J 52: 961–972 Zoschke R, Bock R (2018) Chloroplast translation: structural and functional organization, operational control and regulation. Plant Cell 30: 745-770

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ADVANCES • • • • • •

The function of most plastid genes is known. Plastid transformation vectors can be cloned using a modular toolbox. Inducible plastid gene expression has been demonstrated in the absence of nuclear transgenes. Heterologous metabolic pathways composed of up to nine enzymes have been implemented in plastids. Synthetic plastomes can be designed, assembled and amplified. Engineered plastomes can be introduced into non-transformable species using horizontal genome transfer.

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OUTSTANDING QUESTIONS • • • • • •

How do we best characterize plastid genetic parts or devices in a consistent and efficient manner? How can individual plastid genetic parts or devices be functionally insulated from one another? Can we engineer RNA-binding proteins to tightly control plastid gene expression? How can we obtain quantitative parameters for modeling plastid gene expression? Can synthetic feedback loops and subcompartments enhance future plastid-based metabolic engineering efforts? Can synthetic genomes be introduced into plastids and booted up without undergoing recombination with the resident genome?

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Bacteria

Yeast

Plant (nucleus)

Plant (plastid)

Biological features Genomic simplicity Physiological simplicity Homologous recombination Polycistronic gene expression Gene expression strength Absence of transgene silencing Biological transgene containment Photoautotrophic growth

** *** ** *** ** *** * **

* ** *** * ** ** * *

* * * * * * * ***

*** ** *** *** *** *** ** ***

Available tools and techniques Ease of genetic manipulation Speed of genetic manipulation Genetic parts Genetic part characterization Genetic part assembly Genetic part insulation Gene expression control Genetic & metabolic network models

*** *** *** *** *** *** *** ***

*** *** ** ** *** ** ** **

** ** * * ** ** ** *

* * * * ** * * *

Demonstrated applications Synthetic metabolic pathways *** *** ** Synthetic genetic circuits *** ** * Synthetic subcompartments Downloaded from on September ** 4, 2018 - Published **by www.plantphysiol.org * Copyright © 2018 American Society of Plant Biologists. All rights reserved. Synthetic genomes ** * *

*** * * **

A n

Promoter

5’-UTR

GOI(1)

3’-UTR(1) IEE(1)

SD(i)

GOI(i)

3’-UTR(i) IEE(i)



B

i=2

Designer PPR protein

Promoter(1)

Designer 5’-UTR

SD(n+1)

Downloaded from on September 4, 2018 - Published by www.plantphysiol.org GOI 3’-UTR(1) SMG 5’-UTR(2) Promoter(2) Copyright © 2018 American Society of Plant Biologists. All rights reserved.







3’-UTR(2)

SMG

3’-UTR(n+1)

Ni cot i anat abacum

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Parsed Citations Adachi T, Takase H, Tomizawa K (2007) Introduction of a 50 kbp DNA fragment into the plastid genome. Biosci Biotechnol Biochem 71: 2266–2273 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

Andrianantoandro E, Basu S, Karig DK, Weiss R (2006) Synthetic biology: new engineering rules for an emerging discipline. Mol Syst Biol 2: 2006.0028 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

Apel W, Bock R (2009) Enhancement of carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitamin A conversion. Plant Physiol 151: 59–66 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

Apel W, Schulze WX, Bock R (2010) Identification of protein stability determinants in chloroplasts. Plant J 63: 636–650 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

Barkan A (2011) Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold. Plant Physiol 155: 1520–1532 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

Barkan A, Small I (2014) Pentatricopeptide repeat proteins in plants. Annu Rev Plant Biol 65: 415–442 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

Barnes D, Franklin S, Schultz J, Henry R, Brown E, Coragliotti A, Mayfield SP (2005) Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol Genet Genomics 274: 625–636 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

Bellucci M, De Marchis F, Mannucci R, Bock R, Arcioni S (2005) Cytoplasm and chloroplasts are not suitable subcellular locations for βzein accumulation in transgenic plants. J Exp Bot 56: 1205–1212 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

Birch-Machint I, Newell CA, Hibberd JM, Gray JC (2004) Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability. Plant Biotechnol J 2: 261–270 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title

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