Update on Protein Engineering and Directed Evolution

Applications of Protein Engineering and Directed

Evolution in Plant Research1[OPEN]

Martin K.M. Engqvist,a,2,3 and Kersten S. Rabe b

a
Department of Biology and Biological Engineering, Chalmers University of Technology, Division of Systems
and Synthetic Biology, Gothenburg, Sweden
b
Institute for Biological Interfaces (IBG 1), Karlsruhe Institute of Technology (KIT), Group for Molecular
Evolution, Karlsruhe, Germany

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ORCID IDs: 0000-0003-2174-2225 (M.K.M.E.); 0000-0001-7909-8191 (K.S.R.).

Protein engineering and directed evolution are Protein Engineering

powerful technologies for probing protein sequence-
function relationships. These methods have been used Protein engineering is the process by which a re-
to engineer both plant-derived proteins and exoge- searcher modifies a protein sequence through substi-
nous proteins heterologously expressed in plants. In tution, insertion, or deletion of nucleotides in the
this review, we aim to further increase the interdis- encoding gene, with the goal of obtaining a modified
ciplinary crossover between the disciplines of protein that is more suitable for a particular application
protein engineering and plant biology by first in- or purpose than the unmodified protein. The focus on
troducing protein engineering in some detail. This application sets protein engineering apart from the
introduction is key to understanding current limi- broader term “targeted mutagenesis.” Targeted muta-
tations to protein engineering when applied to genesis, or site-directed mutagenesis, is a method
plants. Subsequently, we provide an overview of the whereby a specific site within a gene sequence is altered
recent methodological progress in, and novel appli- (Hutchison et al., 1978). Such alterations can be
cations of, protein engineering and directed evolu- performed for engineering purposes, as in protein
tion in plant research. engineering, or for examining the effect of specific
mutations in a gene.
Directed protein evolution—a method that was
awarded the Nobel prize in chemistry in 2018—is a
A PRIMER ON PROTEIN ENGINEERING
specific conceptual and methodological approach
Proteins and Their Properties within protein engineering (Chen and Arnold, 1993;
Evolution has shaped the functions and properties of
proteins found in nature such that they contribute to
beneficial phenotype in living organisms. However, ADVANCES
these functions and roles are just a fraction of those
biologically possible. By modifying the sequence of • A rapidly increasing number of sequenced plant
individual proteins, one can go beyond what nature genomes combined with improved codon
has evolved and gain completely new functions or optimization algorithms and cheap gene
properties (Brustad and Arnold, 2011). Such modified synthesis opens the door to large-scale
proteins can be used to improve the phenotype of engineering of diverse plant proteins in
living organisms or have industrial or medical ap- heterologous hosts.
plications (Kumar and Singh, 2013; Porter et al., • CRISPR/Cas9 has proven to be a powerful
2016). The processes and frameworks for modifying technology for engineering plant genomes,
protein sequences fall in the domain of protein including targeted mutagenesis of plant genes.
engineering. • The CRISPR/Cas9 system can be used for non-
transgenic introduction of mutants in perennial
1
M.K.M.E. acknowledges funding through the Chalmers Univer- crops, mitigating the need for the time-
sity of Technology Life Science Engineering Area of Advance. K.S.R. consuming process of backcrossing edited
acknowledges financial support by the Helmholtz program “BioIn- plants.
terfaces in Technology and Medicine.”
2
• Improvements in library generation,
Author for contact: martin.engqvist@chalmers.se. amplification and transformation enable the
3
Senior author.
expression and screening of DNA libraries
M.K.M.E. and K.S.R. conceptualized and wrote the paper.
[OPEN]
Articles can be viewed without a subscription. directly in vivo in C. reinhardtii chloroplasts.
www.plantphysiol.org/cgi/doi/10.1104/pp.18.01534

Plant PhysiologyÒ, March 2019, Vol. 179, pp. 907–917, www.plantphysiol.org Ó 2019 American Society of Plant Biologists. All Rights Reserved. 907
Engqvist et al.

Arnold, 1998). The conceptual approach recognizes that Arnold, 2003; Wong et al., 2006; Labrou, 2010; Packer
we have a limited capability to predict the impact of and Liu, 2015). In this update article, we only deal with
individual amino acid substitutions on protein prop- the first three methods, since they enable the targeting
erties, but measuring the effect of those same substitu- of mutations to a specific locus. Regardless of the
tions can be readily achieved. The methodological method used, the goal is to generate a sequence library,
approach involves generating a large set of diverse i.e. a large collection of diverse sequences, which in-
protein sequences, with some representing a potential clude potential solutions to the engineering goal.
solution to the engineering goal, and then experimen-
tally screening the resulting proteins for desirable Error-Prone PCR
properties and functions. In a striking parallel to
mathematics, the problem in protein engineering re- Error-prone PCR (Fig. 1A) relies on the introduction
sembles the P Þ NP problem; whereby finding a solu- of random mutations throughout the amplified DNA

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tion to a problem is hard, but verifying the solution is sequence by means of DNA polymerase errors (Leung
easy (Pierce and Winfree, 2002). et al., 1989). The error rate in this method can be in-
In directed evolution, sequence diversification and creased using specific polymerase mutants or by in-
screening are often repeated multiple times, with ad- troducing low concentrations of MnCl2 into the PCR
ditional amino acid substitutions accumulating in each reaction. Error-prone PCR can be used to test the effect
round and each round providing a protein sequence of mutations throughout the entire gene sequence
closer to that of the protein engineering target. Directed (Fig. 1A). Consequently, improved variants identified
evolution relies on methods from molecular biology to from an error-prone PCR library often carry mutations
perform sequence diversification and methods from at unexpected positions. Furthermore, the number of
biochemistry, analytical chemistry, and microbiology amino acid substitutions sampled at any given position
to screen the resulting proteins for desired properties. of the sequence is limited by the inability to introduce
concomitant random mutations at more than one base
in a three-base codon sequence (Zhao et al., 2017). Im-
Methods for Sequence Diversification provements on the original method, such as sequence
saturation mutagenesis, address some of these limita-
Many methods for DNA sequence diversification tions (Wong et al., 2004).
have been developed since directed evolution was first
conceptualized (Fig. 1). Most of these methods fall into Site Saturation Mutagenesis
the following categories: error-prone PCR, site satura-
tion mutagenesis, DNA shuffling or chimeragenesis, Site saturation mutagenesis targets one or a few
and random mutagenesis using chemical agents, specific codons from the gene sequence (Fig. 1B) and
physical agents, or hypermutator strains (Hiraga and introduce all possible amino acid substitutions at those

Figure 1. Procedures for the diversification

of genetic sequences. A limited number of
random codon exchanges can be intro-
duced via error-prone PCR (A). In this
method, both the positions within the se-
quence and the nature of the amino acid
modification are undefined. When employ-
ing site saturation mutagenesis (B), the posi-
tions of modification within the sequence are
predetermined, and all possible amino acid
modifications can be realized. A much
higher rate of mutated amino acids relative to
the original sequence can be achieved by
utilizing chimeragenesis (C). In this method,
several parental DNA sequences are being
recombined, resulting in protein variants that
contain different parts from different parents’
DNA sequences.

908 Plant Physiol. Vol. 179, 2019

 Directed Evolution in Plant Research

sites (Zheng et al., 2004). This method is typically PCR combining desirable properties from two or more parent
based (Aiyar et al., 1996), but instead of relying on proteins, but also for generating proteins with properties
polymerase errors, the mutations are introduced using not found in either parent (Hiraga and Arnold, 2003).
primers containing nucleotide mismatches at the tar- Chimeragenesis implies a predefined reassembly of the
geted sites. Typically, pools of primers with the same genetic information and builds on the earlier approach of
binding site are used, with each individual primer en- DNA shuffling (Stemmer, 1994a, 1994b). Chimeragenesis
coding one specific amino acid substitution. has been further developed to encompass computational
The advantage of using site saturation mutagenesis is methods for designing chimeric protein libraries through
that a small number of sites within a gene can be pre- SCHEMA (Meyer et al., 2003; Silberg et al., 2004) and
cisely targeted, and for these sites, all possible amino novel methods for recombination (Coco et al., 2001; Sun
acid substitutions can be sampled. Hence, this method et al., 2003; Smith et al., 2013).
is suitable if one knows which positions in the amino

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acid sequence are important for a certain protein
property (Zheng et al., 2004; Pedotti et al., 2009). Initial Methods for Screening
methods developed for site saturation mutagenesis in-
troduced all 64 possible codons at a site, equivalent to Regardless of the method used for sequence diver-
using NNN of the International Union of Pure and sification, an efficient search for improved variants
Applied Chemistry standardized ambiguous nucleo- must be conducted using screening methods (Fig. 2).
tide alphabet (Cornish-Bowden, 1985). Many of these Screens can be performed using a wide array of
codons are redundant and thus needlessly increase the methods that broadly fall into two categories: assaying
extent of subsequent screening. To reduce this burden, protein properties in vitro or measuring protein effects
many methods have been developed whereby a subset in vivo. Both methods require an accurate and precise
of codons are used (Reetz and Wu, 2008; Jochens and readout of the protein property one wishes to engineer.
Bornscheuer, 2010), including computational tools for If the measurements are imprecise, improved variants
choosing codons for arbitrary selections of amino acids will be overlooked (false negatives), and nonimproved
(Mena and Daugherty, 2005; Firth and Patrick, 2008; variants will be incorrectly scored as improved (false
Engqvist and Nielsen, 2015). positives). Such incorrect scoring will greatly increase
the difficulty of finding improved variants that match
Chimeragenesis
the engineering goal.

Chimeragenesis entails creating new protein se- In Vitro Methods

quences (chimeras) through concatenating parts of
amino acid sequences derived from homologous pro- In vitro screens typically employ heterologous hosts,
teins (Fig. 1C). This approach can be successful in such as Escherichia coli or Saccharomyces cerevisiae or

Figure 2. Procedures for library screening. Ge-

netic libraries, generated via methods depicted in
Figure 1, have to be analyzed in order to identify
improved protein variants employing methods,
which depend on the individual protein case and
the screen available. As such, the library can be
expressed in a heterologous host and analyzed
in vitro, here exemplified by a screen in multiwell
format (A). If screening in vivo in a non-
photosynthetic heterologous host, fitness or sur-
vival of the mutant strains (B) or a colorimetric
detection of improved mutants (C) can be utilized.
These two approaches can also be employed in
the photosynthetic hosts (D and E).

Plant Physiol. Vol. 179, 2019 909

Engqvist et al.

alternatively in vitro translation, to produce protein We have considered literature relating to the engi-
products from the sequence library (Fig. 2A). The pro- neering and use of plant proteins in nonplant orga-
tein products are assayed for improvement in the target nisms or for in vitro use and have selected different,
property using colorimetric assays, analytical mea- prominent use cases. In addition to this, we have sur-
surements of substrate consumption or product for- veyed the literature relating to the engineering of pro-
mation, target affinity assays, or a range of other teins either derived from plant or nonplant organisms
methods (Aharoni et al., 2005). In addition to providing for the use in plants, algae, or cyanobacteria. Finally, we
accurate readouts of the engineered protein property, briefly explore recent methodological developments for
such screens must ensure a link between data obtained performing protein engineering and directed evolution
from the screen and a genotype, allowing the identifi- in planta.
cation of beneficial mutations. When performed in a To limit the scope of this review, we have chosen not
multiwell format, the connection between phenotype to cover the introduction of a small number of targeted

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and genotype is given by mapping the assay plate po- mutations but focus instead on approaches requiring
sition to the plate position of the cells on which the the screening of many variants. Additionally, there is a
assay was performed. In veritable high-throughput large body of literature relating to directed evolution of
methods such as phage display (Smith, 1985), yeast enzymes to break down plant biomass (Álvarez et al.,
surface display (Gai and Wittrup, 2007), or artificial 2016; Kumar et al., 2016b), but these will not be
microdroplet compartments (Tawfik and Griffiths, covered here.
1998), this connection is provided through physical The protein engineering methodologies applied in
colocalization of DNA and protein products. plants and their outstanding problems differ widely
depending on the area of application. To facilitate the
structured discussion of these methods and difficulties,
In Vivo Methods we divide the topic in two sections. This division is
In vivo screens make use of a wide variety of phe- made based on which organism is used for the screen-
notypes as a readout of the engineered protein prop- ing process. The first section deals with using heterol-
erties and have to be carefully selected according to the ogous hosts to screen protein variants for subsequent
function of the engineered protein (Fig. 2, B–E). applications in plants. The second section deals specifi-
Screening by selection is an elegant and powerful ap- cally with using plants or algae to screen protein variants,
proach that establishes a connection between properties without the use of nonphotosynthetic heterologous hosts.
of the engineered protein and the survival of an orga- For both of these sections, we outline in what situation the
nism (Fig. 2, B, D, and E). Selection has been used ex- approach may be applicable, give examples of past ap-
tensively, particularly in studying antibiotic resistance plications, and specify known difficulties and novel
(Orencia et al., 2001). Other in vivo screens directly methodological advances.
measure the color or fluorescence (Zlokarnik et al.,
1998; McIsaac et al., 2014) of the engineered protein to
identify improved variants (Fig. 2C). Yet other screens The Use of Heterologous Hosts for Protein Engineering in
monitor the ability of an organism to consume or pro- Plant Biotechnology
duce a specific compound, either colorimetrically
(Zhang et al., 1997), fluorometrically (Jeschek et al., Use Cases for Heterologous Hosts
2016), or analytically (Coelho et al., 2013). Cell surface
display can be used to probe specific properties, such as Engineering proteins for applications in plants is a
the engineered affinity for a ligand (Xiao et al., 2015). A key method in plant biotechnology. However, much of
major benefit of in vivo screens is that they often lend this engineering has focused on improving a small
themselves to extremely high throughout, allowing an number of plant traits, such as glyphosate resistance
investigator to screen large sequence libraries, particu- (Pollegioni et al., 2011) or Rubisco performance (Wilson
larly in cases where survival can be used as a readout or and Whitney, 2017). Furthermore, this engineering is
where cell sorting can be employed. In in vivo screens, usually performed within heterologous hosts to lever-
the connection between the engineered target property age established microbial methods. According to cur-
and genotype is a natural consequence of physical rent legislation, plants containing DNA that has been
colocalization of protein and DNA inside the organism, manipulated outside of the host are considered ge-
in a manner analogous to phage display. netically modified organisms. Bringing genetically-
modified-organism plants to market is coupled to a
lengthy regulatory process and large capital investments
(Bradford et al., 2005; Qaim, 2009). Combined, these
METHODOLOGIES AND LIMITATIONS IN PLANT two factors have resulted in companies targeting so-
PROTEIN ENGINEERING called “blockbuster traits” in crops. Blockbuster traits
Review Scope are those that have a very large market value, a neces-
sary requirement to recoup the investment required to
Protein engineering and directed evolution are develop and deregulate the engineered plants. Below,
methods; here, we review their use in plant research. we highlight approaches for engineering exogenous
910 Plant Physiol. Vol. 179, 2019
 Directed Evolution in Plant Research

genes for crop pest and herbicide resistance, two of the promising protein variants in a plant system. A recent
most prominent blockbuster traits. In addition, we re- example of this was performed by Das et al. (2017). The
view the use of heterologous hosts to engineer Rubisco. authors found that feeding chickpea (Cicer arietinum)
leaves expressing the Cry1Aabc protein, which had
Engineering of Bacterial Toxins and Their Applications been generated by chimeragenesis, to larvae of the
in Plants gram pod borer (Helicoverpa armigera Hubner) led to
significantly increased mortality when compared to
Optimization of Bacillus thuringiensis toxin has been a control leaves not expressing the protein. In this study,
focus of insect-specific pest control strategies. This op- transformation efficiencies of 0.076% were reported,
timization has been carried out in a variety of ways, for underscoring the benefit of initial studies outside of the
example, through truncation, domain swapping, pep- plant to find promising protein candidates. This argu-
tide addition, and amino acid mutation (Deist et al., ment is further strengthened by the fact that not all

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2014). The most prevalent engineering goals addressed transgenic plants carrying new genes also express the
in these optimization approaches include increasing corresponding protein. For instance, in transgenic to-
toxin potency to combat increasing pest resistance to- bacco (Nicotiana tabacum NC89) protected against
ward the toxins and expanding their applicability to a Cyr1Ac-resistant cotton bollworm, only 13%–38% of
wider range of pests. the regenerated plants expressed the target protein (Li
In a recent example, the toxin Cry1Ab, which does et al., 2018a).
not significantly affect the insect pest Nilaparvata lugens
(rice brown planthopper), was engineered to yield a Engineering Enzymes for Glyphosate Tolerance and Their
variant that, when fed as a purified protein, increases Applications in Plants
the mortality of the pest (Shao et al., 2016). In this work,
the key to increased pest mortality was to retain the Glyphosate [N-(phosphonomethyl)Gly] is the best-
toxin in the insect. To achieve this, the authors identi- selling herbicide to date, and much research has been
fied peptides that were reported to bind to the gut of the devoted to generating transgenic plants that are not
rice brown planthopper in an earlier study employing susceptible to its effects (Sammons and Gaines, 2014).
phage-display technology and randomized peptide li- Glyphosate inhibits the enzyme 5-enolpyruvyl-
braries (Shao et al., 2013). When these gut-binding shikimate-3-phosphate synthase (EPSPS), which is
peptides were rationally introduced into loops on the part of the essential shikimate pathway leading to the
surface of the Cry1Ab protein and the resulting proteins production of the aromatic amino acids Phe, Tyr, and
were fed to the pest, an increased mortality was ob- Trp, and this inhibition results in plant death. Two
served because the toxin was able bind to and interact main strategies have been employed to generate
with Nilaparvata lugens, thus broadening the scope of glyphosate-tolerant transgenic plants: engineering EPSPS
affected pests. to remain active in the presence of glyphosate or intro-
Similarly, the toxin resistance of the insect pest Tri- ducing genes encoding enzymes that remove glyphosate
choplusia ni (Tn) was addressed (Badran et al., 2016). For by breaking it down.
this purpose, the authors chose the widely used Cry1Ac Engineering the EPSPS for activity in the presence of
toxin and enabled its binding to protein receptors in the glyphosate typically involves screening large libraries
insect gut cell membrane that usually do not interact of genetic variants. In plants, this approach is hampered
with the toxin. Specifically, they evolved the toxin to by limited transformation efficiency. Therefore, initial
bind to TnCAD, an insect cell membrane cadherin- libraries of EPSPS variants, generated by DNA shuf-
like receptor, employing a phage-based technology, fling (Tian et al., 2013) or error-prone PCR (Mao et al.,
which has recently been introduced and enables the 2017), are typically tested in E. coli by selecting for
continuous selection of efficient binding peptides (phage- colony growth in the presence of glyphosate at inhibi-
assisted continuous evolution). Phage-assisted continu- tory concentrations. Improved protein variants are then
ous evolution was used to continuously mutate and select characterized and verified by generating transgenic
variants of the Cry1Ac toxin that efficiently bind TnCAD. plants, such as rice (Oryza sativa; Tian et al., 2013, 2015)
After 500 generations (approximately 22 d), several toxin and Arabidopsis (Arabidopsis thaliana; Tian et al., 2015;
variants with binding constants to TnCAD in the nano- Mao et al., 2017).
molar range were identified, whereas for wild-type An example for the removal of glyphosate is the use
Cry1Ac, no binding was observed. When one of these and engineering of bacterial Gly oxidases. Gly oxidases
protein variants was fed to Tn insects, a 335-fold increased cleave the carbon-nitrogen bond in glyphosate, allow-
rate of mortality was observed. ing libraries generated by error-prone PCR, site-directed
The two examples outlined above showcase efficient mutagenesis, and DNA shuffling to be screened in
strategies to obtain early indications whether a protein E. coli using glyphosate as the sole nitrogen source
evolution strategy is successful, without the need to (Zhan et al., 2013). Enzyme variants obtained in this
express the proteins in plants. This is especially im- approach showed up to a 160-fold increase in substrate
portant if thousands of variants need to be tested, affinity and a 326-fold enhancement in catalytic effi-
which is prohibitive in plants, due to low transforma- ciency against glyphosate. Nicolia et al. (2014) have
tion rates. However, it is important to subsequently test used a rational engineering approach employing site
Plant Physiol. Vol. 179, 2019 911
Engqvist et al.

saturation and site-directed mutagenesis to show that proof of principle was achieved using an improved non-
transgenic alfalfa (Medicago sativa) plants do indeed photosynthetic Rubisco from Methanococcoides burtonii
show an increased tolerance to glyphosate when (Wilson et al., 2016).
expressing optimized Gly oxidase variants.
Difficulties and Recent Developments in Using
Engineering Rubisco for Improved Carboxylation Properties Heterologous Hosts
Rubisco catalyzes the incorporation of CO2 into ri- There are several challenges when expressing plant
bulose-1,5-bisphosphate (RuBP), a key reaction in car- proteins in heterologous hosts. Some of these chal-
bon fixation in plants. In a competing reaction, Rubisco lenges relate to problems with the RNA transcript se-
also catalyzes the incorporation of O2 into RuBP. The quence, such as poor codon usage (Chaney and Clark,
metabolites produced in this competing reaction are 2015) and the formation of hairpin structures (Cambray

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salvaged through the photorespiratory pathway, a et al., 2018). Further challenges relate to how other
wasteful process in which CO2 is lost (Peterhansel and proteins interact with the target protein to improve
Maurino, 2011). Improving the carboxylation proper- folding, such as chaperones (Aigner et al., 2017) and
ties of Rubisco thus has the potential to significantly enzymes performing posttranslational modifications
improve crop yield. on the protein product (Hou et al., 2012; Mattanovich
Research relating to this important enzyme has an et al., 2012). A protein that is poorly expressed or in-
almost 50-year history. In 1971, Rubisco was identified correctly folded is difficult or impossible to engineer.
as the direct cause for photorespiration (Bowes et al., Much work has been invested in solving the codon
1971), and already in 1980, the first attempts were made bias problem, which can lead to poor expression or
to engineer more efficient carbon fixation by screening incorrect folding of plant proteins in heterologous
for suppressor mutants in plants harboring a deficient hosts. Living organisms have widely different usage
photorespiratory pathway (Somerville and Ogren, preferences for codons that encode the same amino
1980). Rubisco itself was first targeted through site- acids (Chaney and Clark, 2015). The specific prefer-
directed mutagenesis in 1984 (Gutteridge et al., 1984). ences for each organism can be obtained by computa-
The first report describing the directed evolution of tionally analyzing their genome sequence (Athey et al.,
Rubisco, using an in vivo Rhodoobacter capsulatus screen- 2017). Codon optimization, a process where less-
ing system, was published fifteen years later (Smith and frequent codons in the coding sequence are replaced
Tabita, 2003). Subsequently, E. coli was developed as a by more frequent synonymous codons, has long been
Rubisco screening system (Parikh et al., 2006; Mueller- used to address this issue (Burgess-Brown et al., 2008;
Cajar et al., 2007; Antonovsky et al., 2016). Welch et al., 2009; Maertens et al., 2010). However, in
E. coli screening systems typically depend on the many cases, codon optimization improves expression
heterologous expression of phosphoribulokinase, which but fails to yield correctly folded protein. These failures
produces RuBP. RuBP is toxic to bacteria, and Rubisco may be due to the fact that some proteins require sec-
activity can therefore be used to alleviate the toxicity of tions of less-frequent codons for translation to slow
this compound and ensure survival of the host organism. down, thereby allowing time for the emerging protein
One issue in these screens is that false positives are chain to fold properly (Marin, 2008; Zhang et al., 2009;
obtained at high frequencies (Greene et al., 2007; Cai Deane and Saunders, 2011; Zhang and Ignatova, 2011;
et al., 2014) due to natural transposon-mediated si- Rosenblum et al., 2013).
lencing of phosphoribulokinase (Wilson and Whitney, Several recent computational approaches have been
2017). In a clever approach, the problem of false posi- developed to improve codon optimization methods.
tives was combated by expressing a phosphoribulokinase- Some of these approaches provide a tool without ex-
neomycin phosphotransferase fusion protein and perimentally testing its efficacy (Rodriguez et al., 2018).
including the additional selection pressure of antibiotic In other cases, investigators do experimentally test their
resistance (Wilson et al., 2018). In an approach similar to predictions, sometimes verifying the tool (Tian et al.,
the one taken in E. coli, the soil bacterium Ralstonia 2017) and sometimes finding that the tool has more
eutropha has also been developed for in vivo screening limited efficacy (Mignon et al., 2018). There are also
of Rubisco variants (Satagopan and Tabita, 2016). experimental methods leveraging directed evolution to
Even though these in vivo screening methods show improve protein folding in vivo, as reviewed recently
great promise, their impact for improving crop yields (Sachsenhauser and Bardwell, 2018).
remains to be realized. This may soon change, however,
as it is now possible, using coexpression of five plant-
derived chaperones, to obtain functional plant Rubisco
in E. coli (Aigner et al., 2017). Heterologous expression The Use of Photosynthetic Organisms for Protein
of land plant Rubisco represents a major advance, as Engineering in Plant Biotechnology
established mutagenesis procedures and selection sys- Use Cases for In Planta Protein Engineering
tems can be leveraged for improving its catalytic prop-
erties. Improved Rubisco variants will subsequently need Engineering proteins by screening sequence libraries
to be reintroduced into plants, a process for which a recent directly in plants, instead of using heterologous hosts, is
912 Plant Physiol. Vol. 179, 2019
 Directed Evolution in Plant Research

currently advisable only for a small set of use cases In a similar approach, petD—the gene which encodes
where specific circumstances make it necessary. Such the core protein subunit of the cytochrome b6f com-
circumstances typically involve plants having some plex—was engineered through the in vivo screening of
property that is required to evaluate the engineered an in vitro-generated error-prone PCR library. The li-
proteins’ performance—a property that is difficult to brary was integrated directly at the petD locus inside
replicate in vitro or in a heterologous host. Examples for chloroplasts of a petD-deficient C. reinhardtii strain,
use cases where engineering in plants is preferable in- followed by a screen for photoautotrophic growth
clude engineering of plant signaling pathways, engi- (Dumas et al., 2018). In this study, the goal was not to
neering enzymes acting on plant metabolites that are engineer a more efficient protein but rather to probe the
difficult to obtain, and engineering plant-microbe in- robustness and plasticity of this transmembrane com-
teractions. The use of in planta screening of variants of a plex subunit through mutagenesis and screening. In
single gene essentially provides a more focused and principle, it should also be possible to apply methods

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powerful approach for interrogating plant physiology such as these to plastids of land plants (Dumas et al.,
through mutagenesis, as compared to genome-wide 2018). The two approaches have important limitations,
mutagenesis. however. The first is that they rely on selection systems
requiring mutant strains that are impaired in the func-
Engineering Plant Immune Effectors tional phenotype being selected for. Such strains may
be difficult to obtain. A second limitation is the low
Recently, there has been an increased interest in fur- transformation efficiencies, which limits the number of
ther understanding and modulating the innate immune distinct library sequences that can be introduced. For
response of plants (Bent and Mackey, 2007; Grant et al., further reading regarding plastid synthetic biology, we
2013; Kourelis et al., 2016; Sun et al., 2017). A key part of refer interested readers to the companion paper by
the molecular system to defend against pathogens is the Boehm and Bock (2019) as well as the companion paper
intracellular immune receptors, which belong to the on engineering the photosynthetic light reactions by
nucleotide-binding Leu-rich-repeat-containing protein (Leister, 2019).
family. Nucleotide-binding Leu-rich-repeat-containing
proteins bind to specific effectors found in pathogens Difficulties and Recent Developments for In Planta
and trigger a defense reaction. Random mutagenesis of Protein Engineering
these proteins can modify or broaden the spectrum of
potential pathogens being detected, as has been dem- A main difficulty for in planta protein engineering is
onstrated with initial diversification of the genes by low transformation rates. Improvements to current
error-prone PCR (Segretin et al., 2014; Steinbrenner transformation methods or the development of novel
et al., 2015; Sueldo et al., 2015) or site saturation mu- ones (Altpeter et al., 2016) will be key for expanding the
tagenesis (Helft et al., 2016) and subsequent transfor- use of plants for protein engineering. Alternatively,
mation employing Argobacterium tumefaciens. Such in vivo mutagenesis could provide a viable option for
studies can only be performed inside the plant host species wherein transformation rates are low. The
system, as the response (often cell death) can only be power of this approach comes from the fact that the
observed there. sequence diversity is generated directly inside the tar-
get organism, thus negating constraints on transfor-
Engineering Genes Encoded in the Plastid Genome mation efficiency. For example, a nitrogen-regulated
mutator strain has been developed in the cyanobacte-
Genes-encoding proteins participating in both the rium Synechococcus sp. to alleviate transformation bot-
photosynthetic dark and light reactions have been tar- tlenecks (Emlyn-Jones et al., 2003). A drawback of
geted by mutagenesis and screening in the photosyn- current methods for in vivo mutagenesis is that muta-
thetic unicellular alga Chlamydomonas reinhardtii. For tions cannot be targeted to a specific locus. Novel
engineering the dark reactions, Zhu and colleagues methods to perform targeted in vivo mutagenesis in
screened a DNA-shuffled C. reinhardtii Rubisco large plants are needed. Indeed, in vivo site saturation mu-
subunit library through chloroplast transformation of a tagenesis has already been successfully performed in
Rubisco large subunit-deficient C. reinhardtii strain human (Homo sapiens) cell lines (Findlay et al., 2014; Ma
(Zhu et al., 2010). A three-tiered selection/screening et al., 2017). The key technological advance in these
procedure was used, involving selection for autotro- methods is to couple CRISPR/Cas9-induced double-
phic growth on minimal media, followed by selection strand breaks with multiplex homology-directed repair.
by competitive growth and subsequent identification of Whether this approach can be adapted to plants is an
improved variants. This allowed the investigators to open question.
identify multiple clones with increased carboxylase Even though in vivo saturation mutagenesis has not
activity (Zhu et al., 2010). Some of the specific claims yet been performed in plants, the use of CRISPR/Cas9
relating to the improved Rubisco properties have been for plant genome editing was achieved as early as in
challenged (Wilson and Whitney, 2017), but the study 2013 (Li et al., 2013; Nekrasov et al., 2013; Shan et al.,
remains an important proof of principle for screening 2013). This technology has since been used in a wide
sequence libraries directly in chloroplasts. variety of applications to improve crop plants. For
Plant Physiol. Vol. 179, 2019 913
Engqvist et al.

set of plant traits. Further developments in transfor-

OUTSTANDING QUESTIONS mation technologies, the use of CRISPR/Cas9 for tar-
geted mutagenesis, and possibly the development of
• Can the transformation efficiency of various crop technologies for in planta library generation are ex-
plants be sufficiently improved to enable large- pected to yield more protein engineering approaches in
scale screening of diverse libraries of gene plant biotechnology (see Outstanding Questions).
variants? However, any new technologies resulting from such
• Can protocols for CRISPR-based in vivo site- developments must also be accompanied by favorable
saturation mutagenesis, which have been used in regulatory frameworks or they will likely result in
human cell lines, be modified and successfully limited use for plant improvement.
applied in plants? One underdeveloped application area for protein
• To what extent will protein engineering be engineering lies in engineering plant-microbiome in-

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applied for studying plant–microbe interactions teractions. We believe that engineering such interac-
in the microbiome, and to what extent can the tions will be a key component in the future of plant
insights gained be leveraged to improve the biotechnology. A holistic approach is needed, encom-
yield in commercial crops? passing soil amendment, microbial engineering, and
plant engineering, to sufficiently raise crop yields
• What legal framework will be used to regulate
(Dessaux et al., 2016). Whereas protein engineering for
plants engineered through various methods for
plants has been the main focus of this review, plant-
in vivo targeted mutagenesis?
microbe interactions can also be modified using gene-
editing and systems biology tools (Kumar et al., 2016a).
Techniques to perform host-mediated microbiome en-
gineering already exist (Mueller and Sachs, 2015), but
example, CRISPR/Cas9 has been used for the domes- protein engineering is not commonly used for this
tication of new crop plants, as recently showcased in purpose. Using protein engineering to achieve these
work on the orphan crop groundcherry (Physalis prui- goals should not only focus on crop improvement and
nosa; Lemmon et al., 2018) and wild tomato (Solanum product development but also serve as a powerful tool
pimpinellifolium; Li et al., 2018b; Zsögön et al., 2018). to further understand the basis of plant-microbe inter-
Similarly, CRISPR/Cas9 was used for simultaneously actions. We look forward to future developments in
modifying different homeologous gene copies in this area.
Brassica napus to improve the agronomic trait shatter
resistance (Braatz et al., 2017). In another important ACKNOWLEDGMENTS
example, the CRISPR/Cas9 system was used to achieve The authors would like to dedicate this article to Prof. Frances Arnold to
nontransgenic mutations in perennial heterozygous mark the occasion of her winning the Nobel prize in chemistry in 2018 for her
plants. The method leverages agrobacterial transfor- work on directed evolution of enzymes, as well as to show our gratitude for her
mation and transient expression to perform edits with mentorship and support. The authors declare no conflicts of interest.
an overall nontransgenic mutation rate of 8.2% (Chen Received December 10, 2018; accepted December 25, 2018; published January 9,
et al., 2018). Genomes in leaf disks, shoots, roots, or 2019.
cotyledons can be edited using this method. This rep-
resents an important advance, as plant regeneration
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