A standardized synthetic Eucalyptus transcription factor and promoter

Subscriber access provided by Iowa State University | Library

Technical Note

A standardized synthetic Eucalyptus transcription factor and promoter panel for re-engineering secondary cell wall regulation in biomass and bioenergy crops Steven Hussey, Jacqueline Grima-Pettenati, Alexander A Myburg, Eshchar Mizrachi, Siobhan M Brady, Yasuo Yoshikuni, and Samuel Deutsch ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00440 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1 2

A standardized synthetic Eucalyptus transcription factor and promoter panel for re‐engineering secondary cell wall regulation in biomass and bioenergy crops

3 4

5 6

Steven G. Hussey1, Jacqueline Grima-Pettenati2, Alexander A. Myburg1, Eshchar Mizrachi1, Siobhan M. Brady3, Yasuo Yoshikuni4, Samuel Deutsch4

7 8 9

1

10 11

2

12 13

3

Department of Plant Biology and Genome Center, University of California, Davis, California 95616, United States

14 15

4

US Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598, United States

16

Abstract

17

Re‐engineering transcriptional networks regulating secondary cell wall formation may allow the

18

improvement of plant biomass in widely grown plantation crops such as Eucalyptus. However, there

19

is currently a scarcity of freely available standardized biological parts (e.g. Phytobricks) compatible

20

with Type IIS assembly approaches from forest trees, and there is a need to accelerate transcriptional

21

network inference in non‐model biomass crops. Here we describe the design and synthesis of a

22

versatile three‐panel biological parts collection of 221 secondary cell wall‐related Eucalyptus grandis

23

transcription factor coding sequences and 65 promoters that are compatible with GATEWAY, Golden

24

Gate, MoClo and GoldenBraid DNA assembly methods and generally conform to accepted Phytobrick

25

syntaxes. This freely available resource is intended to accelerate synthetic biology applications in

26

multiple plant biomass crops and enable reconstruction of secondary cell wall transcriptional

27

networks using high‐throughput assays such as DNA Affinity Purification sequencing (DAP‐seq) and

28

enhanced yeast one‐hybrid (eY1H) screening.

29

Keywords: secondary cell wall, Phytobrick, Eucalyptus, transcriptional network

30

Transcription factors (TFs) regulating secondary cell wall (SCW) deposition and their associated gene

31

promoters comprise a molecular toolbox for re‐engineering SCW deposition1. By understanding the

32

regulatory architecture of the transcriptional networks they form, novel network linkages may be

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute (GRI), University of Pretoria, Private Bag X28, Pretoria, South Africa 0002

Laboratoire de Recherche en Sciences Végétales (LRSV), Université Toulouse, UPS, CNRS, BP 42617, F-31326, Castanet-Tolosan, France

1

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 8

33

engineered or transgenes expressed in a cell type‐specific fashion such that plant biomass may be

34

tailored for renewable biomaterials production and ever‐diversifying biorefinery applications.

35

Achieving this via synthetic biology (SynBio) is challenged by a limited availability of plant standard

36

biological parts compatible with one‐step multipartite DNA assembly approaches and cost‐effective

37

methods for elucidating SCW transcriptional networks in non‐model crops. Following the

38

standardization of biological parts in prokaryotic SynBio (e.g. BioBrickTM), the plant SynBio community

39

is beginning to adopt the Phytobrick standard characterised by Type IIS assembly methods such as

40

Golden Gate, MoClo and GoldenBraid, coupled with a standard lexicon2. These assembly standards aim

41

to assemble complex multipartite DNA constructs using a minimal set of restriction endonuclease in a

42

single reaction vessel, beginning with separate standardized plasmids. Here, we describe the design

43

and synthesis of a versatile three‐panel biological parts collection of 221 SCW‐related Eucalyptus

44

grandis TF coding sequences (CDSs) and 65 SCW‐related promoters that are compatible with various

45

DNA assembly standards and generally conform to accepted plant SynBio syntaxes.

46

Commercial Eucalyptus plantations comprise the most widely planted hardwoods globally, and the

47

genus has become a useful model for studying woody biomass accumulation3. This motivates the need

48

to understand the transcriptional control of Eucalyptus wood formation using high‐throughput assays

49

such as DNA Affinity Purification sequencing (DAP‐seq)4 and enhanced yeast one‐hybrid analysis

50

(eY1H)5. The collection, distributed by the University of Pretoria, is freely accessible to the academic

51

community through a US Department of Energy (DOE) Joint Genome Institute (JGI) material transfer

52

agreement.

53

//fabinet.up.ac.za/index.php/fmg‐projects/synthetic‐biology.

54

Candidate SCW‐related TFs from E. grandis were identified based on homology to known Arabidopsis

55

SCW regulators1 using the annotation files of the E. grandis v2.0 genome sequence

56

(//phytozome.jgi.doe.gov/) and evidence implicating them in E. grandis SCW regulation from studies

57

of MYB and NAC protein families and yeast two‐hybrid screens6‐8

58

prioritised to select a total of ~303 Kb of sequence based on preferential expression in developing

59

xylem (among seven tissues; //eucgenie.org/), association with Eucalyptus biomass and bioprocessing

60

traits (Dataset 1 from Mizrachi et al.9) and differential expression during tension wood formation10. The

61

prioritised candidates thus had at least one evidence line linking them to wood formation in Eucalyptus.

62

CDSs of 261 prioritised TFs from the E. grandis v2.0 genome (//phytozome.jgi.doe.gov/) were then

63

“domesticated”, entailing the synonymous substitution of the sequences to enhance synthetic gene

More

information

on

the

constructs

2


can

be

. The

obtained

from

candidates were

Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


64

synthesis and remove internal BsaI, BpaI and BsmBI sites required for Type IIS assembly. Two TF panels

65

were successfully synthesized, with 40 TF candidates failing gene synthesis. The first comprises a

66

Phytobrick parts panel of 173 constructs compatible with all Type IIS and GATEWAY approaches,

67

containing standard syntax sequences2 flanking each CDS followed by a convergent BsaI restriction site

68

for Golden Gate assembly, Gibson‐cloned into pCR8TM/GW/TOPO® (InvitrogenTM) via a “chew back”

69

linker (Fig. 1A). We included bordering attL1 and attL2 sites to enable GATEWAY recombination to attR‐

70

enabled destination vectors, with spacers between the BsaI and attL sequences such that destination

71

vector‐encoded N‐terminal tags remain in frame. The second panel, intended for DAP‐seq analysis

72

primarily, comprises 48 CDSs cloned into pIX‐HALO in‐frame with an N‐terminal HALO (haloalkane

73

dehalogenase) tag for protein purification using Promega Magne® HaloTag® Beads during the DAP‐seq

74

assay4 (Fig. 1B). While standard syntax sequences and BsaI assembly sites are not included in this panel

75

due to the presence of the tag, the constructs are otherwise domesticated and can easily be converted

76

into Phytobricks by PCR amplification of the CDS with primers containing BpiI recognition sites and

77

compatible overhangs, followed by one‐step digestion‐ligation into a Universal Acceptor Plasmid2.

78

The promoter panel features 65 SCW‐related structural genes (n = 50) and TFs (n = 15) in

79

pCR8TM/GW/TOPO® (Fig. 1C), successfully produced from 90 prioritized candidates (~180 Kb) selected

80

using similar evidence lines as for the TF panels, as well as a set of what are likely bona fide cellulose,

81

hemicellulose and lignin biosynthetic genes in Eucalyptus3. Since the sequences include the 5’ UTR, we

82

implemented the GGAG prefix and AATG suffix2, allowing the promoter standards to be appended to

83

any Phytobrick CDS. Due to the frequent occurrence of BsaI and other illegal sites in these sequences,

84

sequence substitution of which could affect promoter function, the AarI heptanucleotide recognition

85

sequence was adopted due to its considerably lower frequency relative to the hexanucleotide

86

signatures of BsaI, BpaI and BsmBI recognition sequences. AarI cleavage yields tetranucleotide

87

overhangs similar to BsaI and is hence Type IIS assembly‐compatible. A limitation of this strategy is that

88

BsaI‐digested Phytobrick CDS fragments may need to be gel‐purified prior to digestion‐ligation with a

89

particular promoter. The few illegal AarI sites found in the wild‐type promoter sequences were

90

eliminated, for all but seven constructs, by substitution with naturally occurring E. grandis SNPs

91

(Myburg et al., unpublished). To enable compatibility of the promoter panel with GATEWAY cloning, in

92

which case the AATG suffix opens the reading frame upstream of destination vector‐encoded

93

reporters, spacers were added such that the AarI recognition sequence, chew back linker and resulting

94

attB2 site following LR recombination collectively encode a 44‐residue peptide in‐frame with the

3



95

reporter. We also avoided repetitive microsatellites flanking the target sequences. This approach

96

yielded construct lengths of 1,995±72 bp.

97

Compared to microbial prokaryotic SynBio, plant SynBio is lagging with respect to the availability of

98

open‐source standard biological parts. Here we have described what is to the best of our knowledge

99

the largest publically available standard biological parts collection of a forest tree yet produced. These

100

synthetic DNA constructs are chiefly intended to provide forest biotechnologists with a molecular

101

toolkit to modify and study wood formation in forest trees and enable the reconstruction of the SCW

102

transcriptional network using high‐throughput assays. However, the toolkit also has the potential to

103

support SynBio innovation in other plant biomass crops. Moving forward, we propose that even basic

104

cloning experiments should attempt to create Phytobrick standards of DNA sequences wherever

105

possible to advance the plant biotechnology community as a whole to a state of SynBio readiness.

106

107

References

108

1. Hussey, S. G., Mizrachi, E., Creux, N. M., and Myburg, A. A. (2013) Navigating the transcriptional

109

roadmap regulating plant secondary cell wall deposition. Front Plant Sci 4, 325.

110

2. Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., Raitskin, O.,

111

Leveau, A., Farré, G., Rogers, C., et al. (2015) Standards for plant synthetic biology: a common syntax

112

for exchange of DNA parts. New Phytol 208, 13‐19.

113

3. Myburg, A. A., Grattapaglia, D., Tuskan, G. A., Hellsten, U., Hayes, R. D., Grimwood, J., Jenkins, J.,

114

Lindquist, E., Tice, H., Bauer, D., et al. (2014) The genome of Eucalyptus grandis. Nature 510, 356‐362.

115

4. Bartlett, A., O'Malley, R. C., Huang, S. C., Galli, M., Nery, J. R., Gallavotti, A., and Ecker, J. R. (2017)

116

Mapping genome‐wide transcription‐factor binding sites using DAP‐seq. Nat Protoc 12, 1659‐1672.

117

5. Gaudinier, A., Zhang, L., Reece‐Hoyes, J. S., Taylor‐Teeples, M., Pu, L., Liu, Z., Breton, G., Pruneda‐

118

Paz, J. L., Kim, D., Kay, S. A., et al. (2011) Enhanced Y1H assays for Arabidopsis. Nat Methods 8, 1053‐

119

1055.

120

6. Hussey, S. G., Saidi, M. N., Hefer, C. A., Myburg, A. A., and Grima‐Pettenati, J. (2015) Structural,

121

evolutionary and functional analysis of the NAC domain protein family in Eucalyptus. New Phytol 206,

122

1337‐1350. 4


Page 4 of 8

Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


123

7. Soler, M., Camargo, E. L. O., Carocha, V., Cassan‐Wang, H., Clemente, H. S., Savelli, B., Hefer, C. A.,

124

Paiva, J. A. P., Myburg, A. A., and Grima‐Pettenati, J. (2015) The Eucalyptus grandis R2R3‐MYB

125

transcription factor family: evidence for woody growth‐related evolution and function. New

126

Phytologist 206, 1364‐1377.

127

8. Soler, M., Plasencia, A., Larbat, R., Pouzet, C., Jauneau, A., Rivas, S., Pesquet, E., Lapierre, C.,

128

Truchet, I., and Grima‐Pettenati, J. (2017) The Eucalyptus linker histone variant EgH1.3 cooperates

129

with the transcription factor EgMYB1 to control lignin biosynthesis during wood formation. New

130

Phytol 213, 287‐299.

131

9. Mizrachi, E., Verbeke, L., Christie, N., Fierro, A. C., Mansfield, S. D., Davis, M. F., Gjersing, E.,

132

Tuskan, G. A., Van Montagu, M., Van de Peer, Y., et al. (2017) Network‐based integration of systems

133

genetics data reveals pathways associated with lignocellulosic biomass accumulation and processing.

134

Proc Natl Acad Sci U S A 114, 1195‐1200.

135

10. Mizrachi, E., Maloney, V. J., Silberbauer, J., Hefer, C. A., Berger, D. K., Mansfield, S. D., and

136

Myburg, A. A. (2015) Investigating the molecular underpinnings underlying morphology and changes

137

in carbon partitioning during tension wood formation in Eucalyptus. New Phytol 206, 1351‐1363.

138 139

5



attL1

G G T CT CgA A TG C C A GA GcT T AC

TGAGC TTcGAGACC 35 nt ACTCG AAgCTCTGG

CDS

attL2

HALO

BsaI

295 AA

TGA

B

BsaI 51 nt

ATG

A

ATG

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 8

CDS

SP6pro

pCR8/GW/TOPO

pIX‐HALO

Phytobrick panel

DAP‐seq panel

173 parts

48 parts

AarI

C attL1

C A C CT G Cac tcG G A G 49 nt G T G GA C Gtg agC C T C

A A T G gc tgG C A G GT G t

2 kb T T A C cg acC G T C CA C a Promoter AarI

35 nt

attL2

pCR8/GW/TOPO

Promoter panel 65 parts

140

141

Figure 1. Design of standardized synthetic E. grandis SCW‐related transcription factors and

142

promoters. Hammer symbols represent domesticated sequences, dotted lines indicate restriction

143

cleavage sites, coloured bases indicate standard syntax sequences and lowercase sequences indicate

144

spacer nucleotides. (A) Transcription factor Phytobricks contain attL GATEWAY recombination sites

145

(orange), chew back linkers, BsaI Type IIS recognition sites and standard syntax sequences (purple

146

text). The start codon of the domesticated coding sequence (green) remains in frame with N‐terminal

147

tags in GATEWAY destination vectors, while the panel is primarily intended for Golden Gate, MoClo 6


Page 7 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


148

and GoldenBraid assembly. (B) The DNA Affinity Purification Sequencing (DAP‐seq) panel of

149

transcription factors is available as a C‐terminal fusion to the HALO purification tag, intended for in

150

vitro transcription and translation via the SP6 phage promoter in pIX‐HALO. While not standardized

151

by a universal syntax, the coding sequences are sequence‐domesticated and can thus be subcloned

152

as standard parts into a universal acceptor plasmid. (C) The secondary cell wall promoter panel

153

features 2 kb promoter sequences (including 5’ UTRs) compatible with GATEWAY and AarI‐mediated

154

Golden Gate cloning. Standard prefix and suffix syntax sequences (blue text) allow for two‐step Type

155

IIS assembly to any Phytobrick panel CDS in (A). AmpR, ampicillin resistance gene; CDS, coding

156

sequence; SpecR, spectinomycin resistance gene.

157

158

Acknowledgements

159

SH, JP‐G, EM and AM compiled and prioritized candidate genes, where SH, AM and EM are co‐PIs and

160

JP‐G is a grant collaborator. SH drafted the manuscript and led the synthetic panel design in

161

consultation with SD, YY and Nicola Patron (Earlham Institute, UK). SMB, a grant collaborator

162

supported by an HHMI Faculty Scholar fellowship, provided expertise in eY1H. We thank Ronan

163

O’Malley (Sequencing Technologies Group Leader, DOE Joint Genome Institute) for advice on DAP‐

164

seq. Gene synthesis was funded by the DNA Synthesis Science Programme of the DOE Joint Genome

165

Institute, in partnership with Integrated DNA Technologies. The work conducted by the U.S.

166

Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by

167

the Office of Science of the U.S. Department of Energy (Contract No. DEAC02‐05CHI/231). Profiling

168

and selection of gene candidates was supported by the Department of Science and Technology

169

(Strategic Grant for the Eucalyptus Genomics Platform), the National Research Foundation of South

170

Africa (Bioinformatics and Functional Genomics Programme, Grants IUD 86936 and 97911), the

171

Technology and Human Resources for Industry Programme (grant UID 80118) and by Sappi and

172

Mondi through the Forest Molecular Genetics Programme (University of Pretoria). The authors

173

declare no conflict of interest.

174

175

7



For Table of Contents Use Only

176 177 178 179 180

Page 8 of 8

A standardized synthetic Eucalyptus transcription factor and promoter panel for re‐engineering secondary cell wall regulation in biomass and bioenergy crops

181 182

Steven G. Hussey1, Jacqueline Grima-Pettenati2, Alexander A. Myburg1, Eshchar Mizrachi1, Siobhan M. Brady3, Yasuo Yoshikuni4, Samuel Deutsch4

183

184

8


A standardized synthetic Eucalyptus transcription factor and promoter

Recommend Documents