An Engineered Constitutive Promoter Set with Broad Activity Range for

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An engineered constitutive promoter set with broad activity range for Cupriavidus necator H16 Abayomi Oluwanbe Johnson, Miriam Gonzalez-Villanueva, Kang Lan Tee, and Tuck Seng Wong ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00136 • Publication Date (Web): 27 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

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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

An engineered constitutive promoter set with broad activity range for Cupriavidus necator H16 Abayomi Oluwanbe Johnson, Miriam Gonzalez-Villanueva, Kang Lan Tee* and Tuck Seng Wong* Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, United Kingdom.

*Address correspondence to: Dr. Kang Lan Tee Email: [email protected] Tel: +44 (0)114 222 7591 Fax: +44 (0)114 222 7501 Or Dr. Tuck Seng Wong Email: [email protected] Tel: +44 (0)114 222 7591 Fax: +44 (0)114 222 7501

ACS Paragon Plus Environment

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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

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1

Abstract

2

Well-characterized promoters with variable strength form the foundation of

3

heterologous pathway optimization. It is also a key element that bolsters the

4

success of microbial engineering and facilitates the development of biological

5

tools like biosensors. In comparison to microbial hosts such as Escherichia

6

coli and Saccharomyces cerevisiae, the promoter repertoire of Cupriavidus

7

necator H16 is highly limited. This limited number of characterized promoters

8

poses a significant challenge during the engineering of C. necator H16 for

9

biomanufacturing and biotechnological applications. In this article, we first

10

examined the architecture and genetic elements of the 4 most widely used

11

constitutive promoters of C. necator H16 (i.e., PphaC1, PrrsC, Pj5 and Pg25), and

12

established a narrow 6-fold difference in their promoter activities. Next, using

13

these 4 promoters as starting points and applying a range of genetic

14

modifications (including point mutation, length alteration, incorporation of

15

regulatory

16

alteration), we created a library of 42 constitutive promoters; all of which are

17

functional in C. necator H16. Although these promoters are also functional in

18

E. coli, they show different promoter strength and hierarchical rank of

19

promoter activity. Subsequently, the activity of each promoter was individually

20

characterized, using L-arabinose-inducible PBAD promoter as a benchmark.

21

This study has extended the range of constitutive promoter activities to 137

22

folds, with some promoter variants exceeding the L-arabinose-inducible range

23

of PBAD promoter. Not only has the work enhanced our flexibility in

24

engineering C. necator H16, it presented novel strategies in adjusting

25

promoter activity in C. necator H16 and highlighted similarities and differences

26

in transcriptional activity between this organism and E. coli.

genetic

element,

promoter

hybridization

and

configuration

27 28

Keywords

29

Cupriavidus necator H16, Ralstonia eutropha H16, gene expression,

30

constitutive promoter, synthetic biology, metabolic engineering

31


2

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1

Introduction

2

Cupriavidus

3

chemolithoautotrophic soil bacterium, most widely known for its ability to

4

accumulate polyhydroxyalkanoates (PHA).1 This metabolically versatile

5

organism is capable of utilizing a wide range of energy and carbon sources

6

(including H2 and CO2) to support growth and achieving high cell density.2

7

These intrinsic properties have cemented its potential applications in

8

biological CO2 capture and utilization3-4 as well as commercial-scale

9

production of diverse bio-products5 including polymers,6-9 hydrocarbons10-14

10

and amino acids.15 Its potential will rapidly come into fruition, aided by the

11

development of molecular tools. These include genome engineering methods

12

to permanently alter its metabolic phenotype,16 expression vectors to

13

assemble

14

mutagenesis18

15

plasmids.19

necator

H16

heterologous and

(or

Ralstonia

pathways,8,

transformation

17

method

eutropha

H16)

transposon-based to

introduce

is

a

random

recombinant

16 17

Maximal product yield and titre are key requirements in biomanufacturing. To

18

this end, metabolic pathway optimization is vital in eliminating metabolic

19

bottlenecks that compromise cellular productivity and metabolic phenotypes

20

that are detrimental to cell viability. Proven strategies of tuning gene

21

expression of a metabolic pathway include varying plasmid copy number,

22

gene dosage and promoter strength, among others. Leveraging on promoter

23

strength for pathway optimization is the most straightforward strategy, which

24

involves tuning promoter activity at both transcriptional and translational

25

levels.

26 27

L-arabinose-inducible PBAD promoter and anhydrotetracycline-inducible Ptet

28

promoter are most widely applied to tune expression of genes, gene clusters

29

or operons in C. necator H16.20-23 With a PBAD promoter, high inducer

30

concentration (up to 1 g/L) is required to achieve high expression yield. The

31

leaky Ptet promoter, on the other hand, is induced by a weak antibiotic that is

32

undesirable and its promoter strength is comparatively weaker. These factors

33

greatly limit the use of these two promoters for large-scale fermentation. The

34

more recently developed 3-hydroxypropionic acid-inducible systems24 and the ACS Paragon Plus Environment

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p-cumate- and IPTG-inducible Pj5 promoters25 are promising alternatives.

2

Nonetheless, achieving scalable and tunable gene expression of a multi-gene

3

pathway by solely relying on inducible expression systems is severely limited

4

by the poor modularity of inducer-based systems (or potential risk of

5

unwanted inducer crosstalk) and limited number of C. necator H16-compatible

6

inducible promoters. Further, the use of inducers such as L-arabinose or

7

anhydrotetracycline on a large scale is commercially uneconomical.

8 9

In this regard, engineering constitutive promoters with a broad range of

10

activities is a more facile means to modularly adjust gene expressions of a

11

multi-gene pathway to the desired levels or ratios. In addition to facilitating

12

static metabolic control, constitutive promoters are used to engineer more

13

efficient inducible promoters23 and to construct metabolite-sensing genetic

14

circuits that in turn facilitate dynamic metabolic control in microorganisms.26

15

Examples of constitutive promoters for use in C. necator H16 include Plac and

16

Ptac promoters, native C. necator H16 promoters such as PphaC1 promoter and

17

coliphage T5 promoter and its variants such as Pj5, Pg25, Pn25 and Pn26

18

promoters.17, 21, 27 Despite these precedent studies, there are knowledge gaps

19

that hinder constitutive promoter utilization and engineering for C. necator

20

H16, which are (1) lack of a universal definition of promoter architecture, (2)

21

lack of a universal reference scale for hierarchical ranking of constitutive

22

promoter activities and (3) limited examples of rational promoter engineering.

23

The latter is in stark contrast to promoter engineering reported for E. coli and

24

yeast.

25 26

In this study, we first examined the architecture of four notable C. necator

27

H16-compatible constitutive promoters: the native PphaC1 promoter, a semi-

28

synthetic PrrsC promoter, and two coliphage T5 promoters Pj5 and Pg25. We

29

then evaluated their activities using in vivo fluorescence measurement of red

30

fluorescent protein (RFP) expression to establish an understanding of the

31

relationship between promoter architecture and activity. Guided by these

32

structure-function relationships, we next proceeded to rational engineering of

33

these 4 parental promoters. Our engineering strategies include combinations

34

of point mutation, length alteration, incorporation of regulatory genetic ACS Paragon Plus Environment

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element, promoter hybridization and configuration alteration. This resulted in a

2

collection of 42 promoters displaying a range of promoter activities. Of these,

3

there are composite promoter variants that are stronger than the Pj5 promoter;

4

the latter has previously been acclaimed to be the strongest known

5

constitutive promoter for gene expression in C. necator H16.17 This new

6

promoter library is envisaged to further propel the biotechnological

7

applications of C. necator H16.

8 9

Results and Discussion

10 11

Defining a promoter and quantifying its activity

12

Standardizing the definition of a promoter is deemed necessary and

13

particularly relevant to this study for 4 obvious reasons: (1) to objectively

14

benchmark the activities of wildtype and engineered promoters, (2) to critically

15

assess promoter structure-function relationships, (3) to measure the

16

effectiveness of various promoter engineering strategies, and (4) to compare

17

promoters reported by various research groups. In this study, we describe a

18

promoter as a constellation of three distinct genetic elements as shown in

19

Figure 1A: (Part 1) a core promoter sequence comprising -35 box, -10 box (or

20

the Pribnow box), +1 transcriptional start, spacer of 16-18 bp between the -35

21

and the -10 boxes as well as the spacer between the -10 box and +1 site,

22

(Part 2) an upstream element (UP) that refers to the entire DNA sequence

23

upstream of the core promoter sequence, and (Part 3) a downstream element

24

spanning the nucleotide after the +1 transcriptional start and the nucleotide

25

before the translation initiation codon. Therefore, the 5’-untranslated region

26

(5’-UTR) consists of the +1 transcriptional start and the downstream element,

27

with the latter typically containing cis-acting regulatory elements such as the

28

ribosome binding site (RBS). Putting it simply, a promoter is a defined stretch

29

of sequence upstream of the translational start. This definition is not

30

uncommon in genome annotation, particularly when the promoter boundary is

31

unclear or ambiguous. This definition also pre-supposes that the functional

32

characteristics of a given promoter are composite effect of all genetic

33

elements within the pre-defined promoter architecture. To quantify promoter

34

activity in C. necator H16, RFP was used as a reporter protein (Figure 1B). ACS Paragon Plus Environment

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Briefly, DNA fragments corresponding to a promoter and rfp gene were

2

cloned, in tandem, into a broad host range pBBR1MCS plasmid backbone

3

harbouring a chloramphenicol resistance gene (Figures 1B & S1).

4 5

Defining the boundaries and architectures of 4 parental promoters

6

Since the 4 parental promoters, PphaC1, PrrsC, Pj5 and Pg25, form the basis of

7

this entire study, clearly defining their boundaries and examining their

8

architectures are essential, such that all subsequently engineered promoters

9

can be compared against these 4 ‘standards’. All 4 parental promoters contain

10

-35 box and -10 box that are almost identical to the hexameric promoter

11

consensus sequences recognized by the E. coli housekeeping sigma factor

12

σ70 (Figures 2A & 2B).28 PphaC1 is known to be a relatively strong native

13

promoter.21, 23, 27 Based on the data reported by Fukui et al., the strength of

14

PphaC1 is ~58% that of Ptac.21 It has been widely studied and applied for

15

improved PHA-based biopolymer production in C. necator H16.21, 27 However,

16

PphaC1 promoters of different lengths are used in various studies, making

17

objective comparison impossible. In this study, PphaC1 promoter is defined as a

18

466-bp DNA sequence upstream of the translation start of the phaC1 gene.

19

Previous studies of this promoter affirmed the presence of a 7-bp

20

“AGAGAGA” Shine-Dalgarno (SD) sequence within its 5’-UTR. This native

21

RBS is located 11 bp upstream of the translational start.27 The PrrsC promoter

22

used in this study is a combination of a 210-bp DNA sequence upstream of

23

the +1 transcriptional start of the native rrsC gene, the first 5 bp of the native

24

5’-UTR and a 26-bp synthetic genetic element. The latter comprises a 20-bp

25

RBS found in the pBBR1c-RFP PBAD promoter (see Supplementary

26

Information) flanked by an upstream 6-bp BglII restriction site. The synthetic

27

RBS contains a purine-rich 5-bp “AGGAG” SD sequence known to markedly

28

improve translation efficiency. This PrrsC is almost identical to the one used in

29

Li and Liao,23 with only minor difference in the downstream element that

30

contains an RBS. Finally, the Pg25 and Pj5 promoters used in this study are

31

both 75-bp DNA sequences, identical to those previously reported by Gentz

32

and Bujard.29

33


6

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Narrow range of promoter activities between the 4 parental promoters

2

Fluorescence measurement of RFP expression revealed a narrow range of

3

promoter activities (defined as relative fluorescence unit normalized by optical

4

density; see Methods & Materials) between the 4 parental promoters (Figure

5

2C). The ratio of promoter activities between the strongest (Pg25) and the

6

weakest (PphaC1) is only 6.3. The promoter activity of PrrsC is 1.7-fold higher

7

than that of PphaC1. This difference may be attributed to the synthetic RBS in

8

PrrsC being more effective in promoting translation compared to the native

9

RBS in PphaC1. The coliphage T5 promoters, Pj5 (75 bp) and Pg25 (75 bp), are

10

much shorter in length compared to PphaC1 (466 bp) and PrrsC (241 bp). They

11

have previously been reported as some of the strongest constitutive

12

promoters in E. coli.29 The strong transcriptional activity was also verified in C.

13

necator H16.17 The A/T rich sequence of many coliphage T5 promoters,

14

particularly in their upstream elements, has been implicated in accounting for

15

their high transcriptional activity.29 Indeed, the upstream elements of both Pj5

16

and Pg25 promoters show high A/T contents (65% for Pj5 and 85% for Pg25),

17

and the difference in the A/T content may partly be responsible for the higher

18

activity of Pg25 relative to Pj5 (Figure 2B). In addition, Pg25 possesses within its

19

5’-UTR the purine-rich 5-bp SD sequence used in most commercially

20

available pET and pBAD vectors, which is lacking in Pj5 (Figure 2A). Important

21

to point out, Gruber et al. had an opposite observation that the strength of Pj5

22

was stronger than that of Pg25.17 The promoter sequences used by Gruber et

23

al. were different compared to those used in this study, although the core

24

promoter sequences remain identical (Figure S2). Promoters in Gruber et al.

25

were essentially a core promoter sequence flanked by NotI and EcoRI

26

restriction sites. Further, they both contain an RBS of T7 gene 10,

27

downstream to the core promoter sequence. These differences could explain

28

the discrepancy between the two studies. It further emphasizes the necessity

29

to define a promoter for objective comparison of promoters used, created or

30

engineered by various researchers.

31 32

Our initial examination of the 4 parental promoters highlighted two critical

33

points which guided our subsequent promoter engineering: (1) conservation of

34

-35 and -10 boxes is important for maintaining high transcriptional activity in ACS Paragon Plus Environment

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C. necator H16, (2) promoter length or A/T content or cis-acting genetic

2

regulatory element (such as a synthetic RBS) could potentially influence

3

transcriptional activity in C. necator H16 significantly.

4 5

Expanding the range of promoter activities through rational engineering

6

Informed by our understanding of the 4 ‘standard” promoters, we proceed to

7

increase the range of promoter activities beyond the existing 6.3 folds

8

between PphaC1, PrrsC, Pj5 and Pg25. Our objectives are 3-fold: (1) creating both

9

weaker (‘tuning down’) and stronger (‘tuning up’) promoter variants to further

10

expand the promoter activity range, (2) generating promoter variants that

11

exceed or at least cover the entire L-arabinose inducible range of PBAD

12

promoter, and (3) developing a set of promoters with gradual increase in

13

activity (i.e., having promoters with activities evenly distributed across the

14

entire promoter activity scale).

15 16

To this end, we applied a range of rational engineering approaches. These

17

strategies, summarized in Table S1 and Figure S3, can be loosely classified

18

into 5 categories: (A) point mutation, (B) length alteration, (C) incorporation of

19

regulatory genetic element, (D) promoter hybridization and (E) configuration

20

alteration. Category A, point mutation, includes base substitution, single-base

21

insertion and single-base deletion. Category B, length alteration, refers to

22

truncation or extension of a promoter from either terminus and insertion or

23

deletion of a stretch of random DNA sequence. Incorporating cis-acting

24

translational regulatory elements such as T7 stem-loop and RBS are grouped

25

within category C. Category D, promoter hybridization, encompasses both

26

creating hybrid promoters and incorporating cis-acting transcriptional

27

regulatory element such as an operator. Category E, configuration alteration,

28

involves transcriptional amplification using a secondary promoter that is

29

placed in divergent configuration to a primary promoter. In other words,

30

composite promoters are placed in category E. Each category is further

31

divided into sub-categories (e.g., C1 for T7 stem-loop and C2 for RBS in

32

Category C) and sub-sub-categories (e.g., B1a for truncation of 25 bp

33

upstream of -35 box and B1b for truncation of 50 bp upstream of -35 box in

34

Category B) to pinpoint specific modification made. Using a combination of ACS Paragon Plus Environment

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1

the aforementioned strategies, we created in total 38 promoter variants as

2

summarized in Table 1.

3 4

Promoter nomenclature

5

Based on the classification of our promoter engineering strategies, we

6

devised a promoter nomenclature system to systematically name all 38

7

engineered promoters (Table 1). The system is designed to provide 3 pieces

8

of key information: parental promoter, modifications made and promoter

9

architecture, using the standard format of Pparent[M1M2M3O]. In this nomenclature

10

system, capital P signifies a promoter. Italic subscript

11

parental promoter from which the engineered promoter is derived. All

12

modifications made are summarised in bracketed subscript

13

modifications arranged in sequence of appearance to reflect the engineered

14

promoter architecture. As an example, Pj5[C1C2] is a promoter variant

15

engineered from the parental promoter Pj5 by inserting a T7-stem loop

16

(denoted by C1) as well as an RBS (denoted by C2) in its 5’-UTR. The T7-stem

17

loop was placed upstream of the RBS, as indicated by

18

C2.

C1

parent

indicates the

[M1M2M3O],

with the

that comes before

19 20

Mutations in -35 box tuned transcriptional activity down

21

Li and Liao previously reported PphaC1-G3 promoter, in which its -35 box was

22

mutated from “TTGACA” to “TTCGGC” (Figure 3A).23 This promoter variant

23

was shown to retain 15% of the activity of its PphaC1 parent, when

24

characterized using enhanced green fluorescent protein (eGFP) as a

25

reporter.23 To validate our initial hypothesis that conserved -35 and -10 boxes

26

are necessary in maintaining high transcriptional activity in C. necator H16,

27

we recreated PphaC1-G3 and this variant was named PphaC1[A1] in our

28

nomenclature system. RFP fluorescence measurement ascertained that

29

PphaC1[A1] retains 16% of the activity of PphaC1 (Figure 3B). This data also

30

indicated that both reporter proteins, RFP and eGFP, give similar outcome in

31

transcriptional activity quantification. RFP and eGFP are commonly used in

32

synthetic biology for characterization of biological parts (e.g., promoter,

33

terminator).30-31 Li and Liao also created a promoter library with mutated -35

34

box (TTNNNN). All promoter variants screened showed lower activity in ACS Paragon Plus Environment

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Page 10 of 34

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comparison to the wildtype promoter.23 Therefore, mutations in -35 box likely

2

tune the transcriptional activity down.

3 4

A minimal PphaC1 promoter with enhanced activity

5

PphaC1 (466 bp) is the longest promoter among the 4 parental promoters. To

6

find the minimal functional sequence, we created 5’→3’ truncated variants of

7

PphaC1: 25 bp truncation in PphaC1[B1a], 50 bp in PphaC1[B1b], 100 bp in PphaC1[B1c],

8

and 124 bp in PphaC1[B1d] (Figure 3A). Interestingly, removing the entire

9

upstream element (124 bp) in PphaC1[B1d] resulted in highest promoter activity,

10

which is 2-fold higher compared to that of its PphaC1 parent (Figure 3B). This

11

suggested that cis-acting elements exist within the upstream element of PphaC1

12

and contribute to transcriptional suppression.

13

Synthetic RBS and RBS repeat increased transcriptional activity

14

Our initial study with the 4 parental promoters, along with a previous study

15

conducted by Bi et al,20 motivated us to further investigate the effects of cis-

16

acting translational regulatory elements on promoter activity. We focused

17

specifically on the 26-bp synthetic genetic element derived from PrrsC

18

(containing a 20-bp RBS found in the pBBR1c-RFP PBAD promoter flanked by

19

an upstream 6-bp BglII restriction site; herein denoted as synthetic RBS) and

20

the 37-bp T7 stem-loop reported by Bi et al.20 We observed a 4.7-fold

21

increase in promoter activity when a synthetic RBS was added to Pj5 (the

22

Pj5[C2] variant) (Figure 3B). On the contrary, there was no significant change in

23

promoter activity when a T7 stem-loop was added to Pj5 (the Pj5[C1] variant). A

24

drop in promoter activity was observed when a T7 stem-loop was added to

25

Pj5[C2] (the Pj5[C1C2] variant). As such, synthetic RBS is an effective means to

26

amplify promoter activity. We then added the same synthetic RBS to Pg25 and

27

created a variant Pg25[C2] that also showed ~50% increase in promoter activity.

28

A smaller promoter activity increase in Pg25[C2] could be attributed to a pre-

29

existing effective SD sequence (“AGGAG”) in Pg25.

30 31

Repeat of -35 and -10 boxes increased transcriptional activity

32

-35 and -10 boxes are highly conserved regions in prokaryotic promoters,

33

essential for the binding of RNA polymerases. To test if creating more binding


10

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1

sites for RNA polymerase would further increase transcriptional activity, we

2

created Pg25[D1] variant. In this variant, we duplicated the DNA sequence

3

spanning -35 box and -10 box. In essence, this hybrid promoter is a tandem

4

repeat of two Pg25 promoters and we observed ~40% increase in promoter

5

activity (Figure 3). Again, incorporating a synthetic RBS (Pg25[D1C2]) gave an

6

additive effect, resulting in further increase in promoter activity.

7 8

Operator insertion reduced transcriptional activity drastically

9

Hybrid promoters are crucial genetic elements in the construction of

10

biosensors. Using a malonyl-CoA biosensor26 as an example, we inserted the

11

fapO operator sequence “TTAGTACCTGATACTAA” in PrrsC promoter to

12

create two variants: PrrsC[D4] (fapO inserted between -35 and -10 boxes) and

13

PrrsC[D6] (fapO inserted within the 5’-UTR) (Figure 3A). fapO operator is

14

conserved in Gram-positive bacteria such as Bacillus subtilis and acts as a

15

cis-regulatory unit for transcriptional regulation of fatty acid biosynthesis. For

16

both promoter variants, we observed a drastic activity reduction to ~10% of

17

their PrrsC parent (Figure 3B). The position of operator and the copy number of

18

operator could therefore significantly change the transcriptional activity of the

19

resultant hybrid promoters. Our data corroborated a previous study by Li and

20

Liao, where tetO operators were inserted to create PrrsC hybrid promoters.23

21 22

Divergent

promoters,

arranged

in

back-to-back,

23

transcriptional activity

24

The distance between and the relative transcriptional directions of adjacent

25

genes are known to be important in some organisms. Neighbouring genes

26

arranged in head-to-head (HH) orientation, for instance, could be co-regulated

27

and this has been proven experimentally.32 For gene pairs in HH

28

arrangement, promoters that effect divergent transcription can be organized in

29

3 possible ways: back-to-back, overlapping or face-to-face (Figure 4A).33 In a

30

recent attempt to construct a malonyl-CoA biosensor for C. necator H16

31

(publication in preparation), we discovered the significance of divergent

32

transcription in this organism. Therefore, we created 21 composite promoters

33

to systematically investigate divergent transcription in C. necator H16. Each

34

composite promoter is made up of two promoters arranged in back-to-back (or ACS Paragon Plus Environment

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Page 12 of 34

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divergent to each other). The promoter driving the RFP expression is termed

2

the primary promoter, while the counterpart is called the secondary promoter.

3 4

When PphaC1 (denoted as modification

5

(modification

6

generally served as transcriptional amplifiers, increasing the transcriptional

7

activity of the primary promoters (Figures 4B-G). The promoter activities of

8

these three secondary promoters follow the order of Pj5[A1C1C2] > Pg25 > PphaC1

9

(eq.

E3

>

E2

E3)

>

E1).

E1),

Pg25 (modification

E2)

and Pj5[A1C1C2]

was applied individually as secondary promoters, they

Interestingly, transcriptional amplification depends on the

10

transcriptional activities of both the primary and the secondary promoters. For

11

the same primary promoter, activity enhancement typically decreases with the

12

increased activity of the secondary promoter (Figures 4B-D; herein described

13

as secondary promoter effect). Also, for the same secondary promoter,

14

activity enhancement decreases with the increased activity of the primary

15

promoter (Figures 4E-G; herein described as primary promoter effect). We

16

postulate that secondary promoter effect is attributed to the fact that weaker

17

secondary

18

transcriptional machinery or factors. If a secondary promoter of very high

19

activity is used, the competition is so strong that it diminishes the activity of

20

the primary promoter (data not shown). The primary promoter effect observed

21

is perhaps more intuitive and easier to comprehend. If the primary promoter

22

displays high activity, it is more difficult to further improve its activity using an

23

amplifier. Important to point out, transcriptional enhancement resulted from

24

divergent promoters is not universal to all prokaryotic systems. Generally, we

25

did not observe such behaviours when we tested our composite promoters in

26

E. coli (data not shown).

promoter

competes

less

with

the

primary

promoter

for

27 28

Promoter characterization using PBAD as reference scale

29

This study resulted in a set of 42 constitutive promoters, including the 4

30

parental promoters. The ratio of promoter activities between the strongest

31

(Pj5[E1A3C2]) and the weakest (PphaC1[A1], eq. PphaC1-G3 reported by Li and Liao23)

32

is 137. These promoters showed incremental increase in activity across the

33

entire scale (Figure 5A). To promote the widespread use of these promoters,

34

we benchmarked each of them using L-arabinose inducible PBAD promoter as ACS Paragon Plus Environment

12

Page 13 of 34 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


1

a reference scale. Figure S4 illustrates the dose-dependent induction of PBAD

2

promoter, using L-arabinose concentration from 0.001% (w/v) to 0.200%

3

(w/v). Expression maxima was reached at 0.200% (w/v) L-arabinose. As

4

depicted in Figure 5B, our engineered promoters covered the entire L-

5

arabinose inducible range (indicated by scattered data points). We

6

categorized all promoters into 5 activity levels to aid promoter selection: Level

7

1 (with promoter activity between 0 a.u. – 2000 a.u.), Level 2 (2000 a.u. –

8

4000 a.u.), Level 3 (4000 a.u. - 6000 a.u.), Level 4 (6000 a.u. – 8000 a.u.) and

9

Level 5 (> 8000 a.u.). With a PBAD promoter, one could only achieve

10

expression levels between 1 – 4. Through promoter engineering, we obtained

11

seven Level 5 variants (Pj5[A3C2], Pj5[C2], Pj5[E1C1C2], Pj5[E2C2], Pj5[E2A3C2], Pj5[E1C2]

12

and Pj5[E1A3C2]) with promoter activities exceeding that of PBAD (Table S2). For

13

easy classification of all engineered promoters, we developed a numerical

14

coding system (Table 1) and assigned a digital identifier to each promoter.

15

This will allow us to develope a C. necator H16-specific promoter database

16

(work in progress). Each promoter code is in the format of [X-Y-Z], with X

17

representing activity level, Y representing relative activity to PphaC1[A1] and Z

18

representing promoter length. While conceptualizing our engineered promoter

19

nomenclature and coding systems, we have endeavoured to make them

20

universal such that they can be applied to other promoters yet to be

21

developed.

22 23

Summary of rational promoter engineering for C. necator H16

24

Figure 6 provides an overview of the rational promoter engineering strategies

25

discussed in this article. It shows the effect of a specific modification by

26

looking at the promoter activity difference before and after that particular

27

modification. Creating mutation(s) within -35 box and inserting operator

28

sequence(s) resulted in drastic reduction in promoter activity (represented by

29

red data points), while inserting a T7 stem-loop caused almost no change in

30

promoter activity. On the contrary, inserting a synthetic RBS and applying a

31

transcriptional amplifier (specifically PphaC1 or Pj5[A1C1C2]) gave the highest

32

increase in promoter activity (>100%). In fact, those promoters that are

33

stronger than PBAD promoter (indicated as blue data points) were mostly

34

created using either one of these strategies or combination of them. All the ACS Paragon Plus Environment

13


Page 14 of 34

1

other strategies provided marginal promoter activity increase (from 10% to

2

50%). Also clearly reflected in Figure 5B, creating divergent promoters is the

3

most effective way of broadening the range of promoter activity. Worthy of

4

note, relative promoter activity change is dependent on the parental promoter,

5

judging on the work presented in this article.

6 7

The use of engineered constitutive promoters in C. necator H16

8

The use of strong constitutive promoters could potentially result in (a)

9

bacterial growth impairment due to high metabolic burden and/or (b) protein

10

excretion/leakage due to high protein expression level. To study these effects,

11

we selected representative promoters from each activity level (Table S3) and

12

conducted further characterization. We observed similar growth for most of

13

the strains (Figure 7), with growth rates (µmax) falling between 0.21 h-1 and

14

0.24 h-1 (Table S3). For promoter Pj5[E2C2], which is a Level 5 promoter, we

15

noticed a slight drop in growth rate with µmax of 0.18 h-1 (Table S3).

16

Comparing fluorescence of cell culture and of spent medium (Figure 8)

17

confirmed that there was no protein excretion/leakage. Fluorescence of spent

18

medium was maintained at the level of ~1000 a.u, throughout the bacterial

19

cultivation. This value was almost identical to that of the control (C. necator

20

H16 harbouring pBBR1MCS-1). To study the time-dependent increase in

21

fluorescence signal, we fitted the cell culture fluorescence vs time data to a 4-

22

parameter dose-response curve (Figure S5 and Table S4) for all promoters

23

from Level 2 and above. The fluorescence increase was mainly caused by

24

bacterial growth. If we divided the fluorescence measured (Figure 8) by the

25

OD600 (Figure 7), the ratio was kept almost constant at cultivation times above

26

12 h (Figure S6), further verifying our approach in promoter activity

27

quantification in 96-well plate by taking the RFU/OD600 at t = 48 h.

28 29

Conclusion

30

This article (1) reported and characterized a set of 42 constitutive promoters

31

with a broad range of promoter activity, which are derived from the 4 most

32

widely used constitutive promoters for C. necator H16 (PphaC1, PrrsC, Pj5 and

33

Pg25), (2) introduced a nomenclature system and a coding system for


14

Page 15 of 34 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


1

engineered promoters, (3) sketched out the relationship between promoter

2

architecture and its resultant activity, (4) highlighted similarities (conservation

3

of -35 and -10 boxes) and differences (composite promoters) in transcriptional

4

activity between C. necator H16 and E. coli, and (5) provided guidelines for

5

rational promoter engineering. We strongly believe our constitutive promoter

6

toolbox that exceeds the activity range of the inducible PBAD promoter will

7

serve the biotechnology community working on C. necator H16, be it strain

8

engineering for industrial biomanufacturing or developing advanced molecular

9

biology tools for this organism.

10


15


1

Page 16 of 34

Materials & Methods

2 3

Materials

4

All DNA modifying enzymes were purchased from either New England Biolabs

5

(Hitchin, UK) or Agilent (Craven Arms, UK). Nucleic acid purification kits were

6

purchased from Qiagen (Manchester, UK). All oligonucleotides were

7

synthesized by Eurofins (Ebersberg, Germany).

8 9

Strains

10

Escherichia coli DH5α was used for all molecular cloning, plasmid

11

propagation

12

purchased from DSMZ, Braunschweig, Germany) was used for all

13

experiments described in this article.

and

maintenance.

Cupriavidus

necator

H16

(DSM-428,

14 15

Promoter engineering and sequences

16

All

17

Information) and constructed using standard molecular biology techniques. All

18

engineered promoters were verified by restrictive analysis and/or DNA

19

sequencing and their sequences were provided in the Supplementary

20

Information.

plasmids

were

derived

from

pBBR1c-RFP

(see

Supplementary

21 22

Bacterial cultivation and transformation

23

C. necator H16 was cultivated at 30°C in nutrient broth (NB: 5 g/L peptone, 1

24

g/L beef extract, 2 g/L yeast extract, 5 g/L NaCl; pH 7.0 ± 0.2 @ 25°C)

25

supplemented with 10 µg/mL of gentamicin. Cells were transformed with

26

plasmids using the electroporation protocol described by Tee et al,19 plated on

27

NB agar supplemented with 10 µg/mL of gentamicin and 25 µg/mL of

28

chloramphenicol, and incubated at 30°C for 40–60 h. E. coli DH5α was

29

transformed with plasmids using the standard CaCl2 method, plated on TYE

30

agar (10 g/L tryptone, 5 g/L yeast extract, 8 g/L NaCl, 15 g/L agar)

31

supplemented with 25 µg/mL of chloramphenicol and incubated overnight at

32

37°C.

33


16

Page 17 of 34 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


1

Promoter activity quantification using fluorescence assay

2

Transformants of C. necator H16, carrying either an RFP-null or an RFP-

3

expressing vector, were pre-cultured in 96-well microtitre plate containing 200

4

µL/well of NB supplemented with 10 µg/mL of gentamicin and 25 µg/mL of

5

chloramphenicol at 30°C for 40 h. This pre-culture was used to inoculate a

6

fresh clear-bottom 96-well microtitre plate [Greiner Bio-One (Stonehouse,

7

UK)] containing 200 µL/well of NB supplemented with 10 µg/mL of gentamicin,

8

25 µg/mL of chloramphenicol as well as 0–0.2% (w/v) L-arabinose (when

9

required) to induce RFP expression. The plate was cultivated at 30°C for a

10

total of 48 h. OD600 and fluorescence (Ex 584 nm, Em 607 nm; bottom read)

11

were measured using SpectraMax M2e microplate/cuvette reader [Molecular

12

Devices (Wokingham, UK)] after 12 h of cultivation and repeated at 6 h

13

intervals. Relative fluorescence unit (RFU) was calculated by normalizing

14

fluorescence value with the fluorescence value of C. necator H16 carrying an

15

RFP-null vector (negative control). The RFU value therefore represents the

16

fluorescence fold increase owing to RFP expression. RFU/OD600 value was

17

then calculated as the ratio of RFU and OD600 value of the respective strain.

18

The ratio was used to account for potential metabolic burden due to high

19

protein expression level, affecting bacterial growth. Promoter activity (PA) was

20

defined as the RFU/OD600 value after 48 h of cultivation. The ratio of

21

RFU/OD600 was more or less a constant at cultivation time more than 12 h.

22

All experiments were done in triplicate.

23 24

Fold change and relative promoter activity change

25

Fold change and relative promoter activity change were calculated using the

26

formulae below: ℎ =

ℎ =

− ! × 100%

!

27 28

Effects of engineered constitutive promoters on bacterial growth and

29

protein excretion


17


Page 18 of 34

1

Selected plasmids were freshly transformed into C. necator H16 and single

2

colonies were picked to prepare overnight cultures. Falcon tubes, containing 6

3

mL of fresh mineral salts medium (MSM)34 supplemented with 10 g/L sodium

4

gluconate

5

chloramphenicol, were inoculated at a starting OD600 of 0.2. Cells were

6

cultivated at 30°C and sampled at regular time intervals. OD600 of each

7

sample was measured using BioPhotometer Plus [Eppendorf (Stevenage,

8

UK)]. For all samples collected, the fluorescence (Ex 584 nm, Em 607 nm;

9

bottom read) of the cell culture (90 µL) and of the spent medium (90 µL) was

10

measured using SpectraMax M2e microplate/cuvette reader [Molecular

11

Devices (Wokingham, UK)].

(carbon

source),

10

µg/mL

gentamicin

and

25

µg/mL

12 13

Supporting Information

14

Experimental procedures; plasmid map of pBBR1c-RFP; alignment of Pj5 and

15

Pg25 promoters used in this study and in Gruber et al. (2014); graphical

16

representation of rational promoter engineering strategies; L-arabinose-dose

17

dependent induction of PBAD promoter; curve fitting of cell culture fluorescence

18

vs time data; fluorescence of cell culture normalized by OD600 value; promoter

19

sequences; sub-categories of each promoter engineering strategy; promoter

20

activity table; specific growth rates of C. necator H16 carrying plasmids

21

containing various promoters.

22 23

Author Contributions

24

T.S.W. and K.L.T. conceived and supervised the project. A.O.J. and M.G.

25

performed the experiments and analysed the data. T.S.W., K.L.T. and A.O.J.

26

wrote the manuscript. All authors discussed the content of the manuscript and

27

provided critical revisions on the manuscript.

28 29

Acknowledgement

30

We thank the Department of Chemical and Biological Engineering, ChELSI

31

and EPSRC (EP/E036252/1) for financial support. AOJ and MGV are

32

supported

33

scholarships, respectively.

by

the

University

of

Sheffield

and


CONACYT

(Mexico)

18

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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

References 1. Pohlmann, A.; Fricke, W. F.; Reinecke, F.; Kusian, B.; Liesegang, H.; Cramm, R.; Eitinger, T.; Ewering, C.; Pötter, M.; Schwartz, E.; Strittmatter, A.; Voss, I.; Gottschalk, G.; Steinbüchel, A.; Friedrich, B.; Bowien, B., Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16. Nat Biotechnol 2006, 24 (10), 1257-62. 2. Volodina, E.; Raberg, M.; Steinbüchel, A., Engineering the heterotrophic carbon sources utilization range of Ralstonia eutropha H16 for applications in biotechnology. Crit Rev Biotechnol 2016, 36 (6), 978-991. 3. Jajesniak, P.; Omar Ali, H. E. M.; Wong, T. S., Carbon dioxide capture and utilization using biological systems: Opportunities and challenges. J Bioprocess Biotech 2014, 4, 155. 4. Peplow, M., Industrial biotechs turn greenhouse gas into feedstock opportunity. Nat Biotechnol 2015, 33 (11), 1123-5. 5. Brigham, C. J.; Zhila, N.; Shishatskaya, E.; Volova, T. G.; Sinskey, A. J., Manipulation of Ralstonia eutropha carbon storage pathways to produce useful bio-based products. Subcell Biochem 2012, 64, 343-66. 6. Lutke-Eversloh, T.; Steinbüchel, A., Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha. FEMS Microbiol Lett 2003, 221 (2), 191-6. 7. Steinbüchel, A.; Pieper, U., Production of a copolyester of 3hydroxybutyric acid and 3-hydroxyvaleric acid from single unrelated carbon sources by a mutant of Alcaligenes eutrophus. Appl Microbiol Biotechnol 1992, 37 (1), 1-6. 8. Voss, I.; Steinbüchel, A., Application of a KDPG-aldolase genedependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16. Metab Eng 2006, 8 (1), 66-78. 9. Valentin, H. E.; Zwingmann, G.; Schönebaum, A.; Steinbüchel, A., Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophus. Eur J Biochem 1995, 227 (1-2), 43-60. 10. Chen, J. S.; Colon, B.; Dusel, B.; Ziesack, M.; Way, J. C.; Torella, J. P., Production of fatty acids in Ralstonia eutropha H16 by engineering betaoxidation and carbon storage. PeerJ 2015, 3, e1468. 11. Crépin, L.; Lombard, E.; Guillouet, S. E., Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production. Metab Eng 2016, 37, 92-101. 12. Marc, J.; Grousseau, E.; Lombard, E.; Sinskey, A. J.; Gorret, N.; Guillouet, S. E., Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production. Metab Eng 2017, 42, 74-84. 13. Müller, J.; MacEachran, D.; Burd, H.; Sathitsuksanoh, N.; Bi, C.; Yeh, Y. C.; Lee, T. S.; Hillson, N. J.; Chhabra, S. R.; Singer, S. W.; Beller, H. R., Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones. Appl Environ Microbiol 2013, 79 (14), 4433-9. 14. Lu, J.; Brigham, C. J.; Gai, C. S.; Sinskey, A. J., Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha. Appl Microbiol Biotechnol 2012, 96 (1), 283-97. 15. Lütte, S.; Pohlmann, A.; Zaychikov, E.; Schwartz, E.; Becher, J. R.; Heumann, H.; Friedrich, B., Autotrophic production of stable-isotope-labeled ACS Paragon Plus Environment

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arginine in Ralstonia eutropha strain H16. Appl Environ Microbiol 2012, 78 (22), 7884-90. 16. Park, J. M.; Jang, Y. S.; Kim, T. Y.; Lee, S. Y., Development of a gene knockout system for Ralstonia eutropha H16 based on the broad-host-range vector expressing a mobile group II intron. FEMS Microbiol Lett 2010, 309 (2), 193-200. 17. Gruber, S.; Hagen, J.; Schwab, H.; Koefinger, P., Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16. J Biotechnol 2014, 186, 74-82. 18. Raberg, M.; Heinrich, D.; Steinbüchel, A., Analysis of PHB Metabolism Applying Tn5 Mutagenesis in Ralstonia eutropha. In Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks., McGenity, T.; Timmis, K.; Nogales, B., Eds. Springer: Berlin, Heidelberg, 2015; pp 120-148. 19. Tee, K. L.; Grinham, J.; Othusitse, A. M.; Gonzalez-Villanueva, M.; Johnson, A. O.; Wong, T. S., An Efficient Transformation Method for the Bioplastic-Producing "Knallgas" Bacterium Ralstonia eutropha H16. Biotechnol J 2017, 12 (11). 20. Bi, C.; Su, P.; Müller, J.; Yeh, Y. C.; Chhabra, S. R.; Beller, H. R.; Singer, S. W.; Hillson, N. J., Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production. Microb Cell Fact 2013, 12, 107. 21. Fukui, T.; Ohsawa, K.; Mifune, J.; Orita, I.; Nakamura, S., Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16. Appl Microbiol Biotechnol 2011, 89 (5), 1527-36. 22. Guzman, L. M.; Belin, D.; Carson, M. J.; Beckwith, J., Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 1995, 177 (14), 4121-30. 23. Li, H.; Liao, J. C., A synthetic anhydrotetracycline-controllable gene expression system in Ralstonia eutropha H16. ACS Synth Biol 2015, 4 (2), 101-6. 24. Hanko, E. K. R.; Minton, N. P.; Malys, N., Characterisation of a 3hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator. Sci Rep 2017, 7 (1), 1724. 25. Gruber, S.; Schwendenwein, D.; Magomedova, Z.; Thaler, E.; Hagen, J.; Schwab, H.; Heidinger, P., Design of inducible expression vectors for improved protein production in Ralstonia eutropha H16 derived host strains. J Biotechnol 2016, 235, 92-9. 26. Johnson, A. O.; Gonzalez-Villanueva, M.; Wong, L.; Steinbüchel, A.; Tee, K. L.; Xu, P.; Wong, T. S., Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories. Metab Eng 2017, 44, 253-264. 27. Arikawa, H.; Matsumoto, K., Evaluation of gene expression cassettes and production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necator. Microb Cell Fact 2016, 15 (1), 184. 28. Kutuzova, G. I.; Frank, G. K.; Makeev, V.; Esipova, N. G.; Polozov, R. V., [Fourier analysis of nucleotide sequences. Periodicity in E. coli promoter sequences]. Biofizika 1997, 42 (2), 354-62.


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29. Gentz, R.; Bujard, H., Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. J Bacteriol 1985, 164 (1), 70-7. 30. Guo, Y.; Dong, J.; Zhou, T.; Auxillos, J.; Li, T.; Zhang, W.; Wang, L.; Shen, Y.; Luo, Y.; Zheng, Y.; Lin, J.; Chen, G. Q.; Wu, Q.; Cai, Y.; Dai, J., YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res 2015, 43 (13), e88. 31. Phelan, R. M.; Sachs, D.; Petkiewicz, S. J.; Barajas, J. F.; BlakeHedges, J. M.; Thompson, M. G.; Reider Apel, A.; Rasor, B. J.; Katz, L.; Keasling, J. D., Development of Next Generation Synthetic Biology Tools for Use in Streptomyces venezuelae. ACS Synth Biol 2017, 6 (1), 159-166. 32. Gherman, A.; Wang, R.; Avramopoulos, D., Orientation, distance, regulation and function of neighbouring genes. Hum Genomics 2009, 3 (2), 143-56. 33. Beck, C. F.; Warren, R. A., Divergent promoters, a common form of gene organization. Microbiol Rev 1988, 52 (3), 318-26. 34. Schlegel, H. G.; Kaltwasser, H.; Gottschalk, G., [A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies]. Arch Mikrobiol 1961, 38, 209-22.

22


21


1

Page 22 of 34

Figure legend

2 3

Figure 1: (A) Promoter definition used in this study. (B) High-throughput

4

characterization of engineered promoters using a fluorescence-based assay.

5

(5’-UTR: 5’-untranslated region; bp: base pair; CamR: chloramphenicol

6

resistance gene; Rep: replication gene; RFP: red fluorescent protein)

7 8

Figure 2: (A) Boundaries and architectures of the 4 parental promoters,

9

PphaC1, PrrsC, Pj5 and Pg25, used in this study. (B) Comparison of the 4 parental

10

promoters to an Escherichia coli σ70 promoter. (C) Activities of the 4 parental

11

promoters.

12 13

Figure 3: (A) Architectures of parental promoters and their engineered

14

variants. (B) Activities of parental promoters and their engineered variants.

15 16

Figure 4: (A) For gene pairs in HH arrangement, promoters that effect

17

divergent transcription can be organized in 3 possible ways: back-to-back,

18

overlapping or face-to-face. (B) Composite promoters engineered using Pg25

19

as parental promoter. (C) Composite promoters engineered using PrrsC as

20

parental promoter. (D) Composite promoters engineered using Pj5 as parental

21

promoter. (E) Composite promoters with PphaC1 as secondary promoter. (F)

22

Composite promoters with Pg25 as secondary promoter. (G) Composite

23

promoters with Pj5[A1C1C2] as secondary promoter. In graphs E to F, blue, red

24

and orange symbols represent primary promoter activity, composite promoter

25

activity and fold change, respectively.

26 27

Figure 5: (A) Hierarchical ranking of all 42 constitutive promoters reported in

28

this study. (B) The range of promoter activity was expanded from 6 folds to

29

137 folds, after applying combination of promoter engineering strategies (A =

30

point mutation, B = length alteration, C = incorporation of regulatory genetic

31

element, D = promoter hybridization and E = configuration alteration).

32

Promoters derived from PphaC1, PrrsC, Pj5 and Pg25 were coloured in blue, green,

33

pink and red, respectively. Promoters were categorized into 5 activity levels:


22

Page 23 of 34 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


1

Level 1 (with promoter activity between 0 a.u. – 2000 a.u.), Level 2 (2000 a.u.

2

– 4000 a.u.), Level 3 (4000 a.u. - 6000 a.u.), Level 4 (6000 a.u. – 8000 a.u.)

3

and Level 5 (> 8000 a.u.). Each promoter was benchmarked against PBAD

4

promoter, induced using various concentrations of L-arabinose, from 0.001%

5

(w/v) to 0.200% (w/v).

6 7

Figure 6: Relative promoter activity change upon application of promoter

8

engineering strategies (A = point mutation, B = length alteration, C =

9

incorporation of regulatory genetic element, D = promoter hybridization and E

10

= configuration alteration). Modifications that resulted in loss of promoter

11

activity were indicated as red data points. Promoters with activities higher

12

than PBAD promoter were indicated as blue data points.

13 14

Figure 7: Growth curves of C. necator H16 harbouring either pBBR1MCS-1

15

(control; black line) or plasmids containing various engineered constitutive

16

promoters [Pg25 (red line), PphaC1[B1d] (blue line), PrrsC[E1D4] (brown line), Pg25[E3]

17

(green line), Pg25[D1C2] (pink line), Pj5[E1A1C1C2] (orange line) and Pj5[E2C2] (purple

18

line)].

19 20

Figure 8: Fluorescence of cell culture (black columns) and of spent medium

21

(grey columns) of C. necator H16 harbouring either pBBR1MCS-1 (control) or

22

plasmids

23

PphaC1[B1d], PrrsC[E1D4], Pg25[E3], Pg25[D1C2], Pj5[E1A1C1C2] and Pj5[E2C2]).

containing

various

engineered

constitutive


promoters

(Pg25,

23

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

Page 24 of 34

Table 1: Summary of 42 parental promoters and their variants engineered using a combination of promoter engineering strategies (A = point mutation, B = length alteration, C = incorporation of regulatory genetic element, D = promoter hybridization and E = configuration alteration). Numbers in bracket represent promoter digital identifier, in the format of [Activity level – Relative activity to PphaC1[A1] – Promoter length].

Parental promoter

PphaC1 [1-6-466]

A (Point mutation)

PphaC1[A1] [1-1-466]

B (Length alteration)

C (Incorporation of regulatory genetic element)

E (Configuration alteration) Transcriptional amplifier (Secondary promoter) PphaC1

Pg25

Pj5[A1C1C2]

PphaC1[B1a] [1-8-441] PphaC1[B1b] [1-10-416] PphaC1[B1c] [1-6-366] PphaC1[B1d] [1-12-342]

PrrsC [1-11-241]

Pj5 [1-25-75]

D (Promoter hybridization)

PrrsC[D6] [1-1-258] PrrsC[D4] [1-1-241] Pj5[C1] [1-26-136] Pj5[C1C2] [4-91-162] Pj5[A1C1C2] [3-64-162]

PrrsC[E1] [2-34-713] PrrsC[E1D6] [2-31-730] PrrsC[E1D4] [1-14-713]

Pj5[E1C1C2] [5-120-634] Pj5[E1A1C1C2] [4-104-634]

PrrsC[E3] [1-22-434] PrrsC[E3D6] [1-21-451] PrrsC[E3D4] [1-9-434]

Pj5[E2C1C2] [4-106-268] Pj5[E2A1C1C2] [4-84-268]

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Pj5[A1D6aC1C2] [3-70-179] Pj5[A1D6bC1C2] [3-68-196] Pj5[C2] [5-117-101] Pj5[A3C2] [5-116-100]

Pg25 [2-39-75]

Pj5[E1A1D6aC1C2] [4-112-651] Pj5[E1A1D6bC1C2] [4-111-668] Pj5[E1C2] [5-134-573] Pj5[E1A3C2] [5-137-572] Pg25[E1] [3-61-547]

Pj5[E2A1D6aC1C2] [4-90-285] Pj5[E2A1D6bC1C2] [4-87-302] Pj5[E2C2] [5-125-207] Pj5[E2A3C2] [5-128-206] Pg25[E2] [2-46-181]

Pg25[E3] [2-45-268]

Pg25[D1] [2-53-130] Pg25[C2] [2-57-101] Pg25[D1C2] [3-75-156]

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A

18 bp spacer

-10

GCGCGTGC GTTGCAAGGC

AACAAT

18 bp spacer

-10

GCGCGTGC GTTGCAAGGC

AACAAT

18 bp spacer

-10

GCGCGTGC GTTGCAAGGC

AACAAT

18 bp spacer

-10

GCGCGTGC GTTGCAAGGC

AACAAT

18 bp spacer

-10

GCGCGTGC GTTGCAAGGC

AACAAT

-35

17 bp spacer

-10

5’-UTR

TTGACA

CAGGTGGAAATTTAGAA

TATACT

AAACCTAATGGATCGACCTT

RBS AGAGAGA

GGACTC

RBS AGAGAGA

GGACTC

Synthetic RBS TTTAAGAAGGAGATATACAT

T7 Stem loop GGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTG

TTTAAGAAGGAGATATACAT

1000

-35

17 bp spacer

-10

RBS

TTGATA

AAATTTTCCAATACTAT

TATAAT

ATTAAAGAGGAGAAATTAAC

0

Synthetic RBS

Pg25[C2]

Pg25[D1]

Pg25


-35

17 bp spacer

-10

TTGCCA

AAATTTTCCAATACTAT

TATAAT

Pg25

Synthetic RBS

Pg25[D1C2]

Pg25[D1]

-35

PrrsC

PrrsC[D4]

17 bp spacer

-10

TTGCCA

GTCGGTCCTGCGTCCCT

TAATATT

-35

17 bp fapO unit

-10

TTGCCA

TTAGTACCTGATACTAA

TAATATT

-35

PrrsC[D6]


TTGCCA

17 bp spacer GTCGGTCCTGCGTCCCT

RBS CGCCCCCTCGC


RBS CGCCCCCTCGC

-10 TAATATT

Promoter

CGCCCCCTCGC


17 bp fapO unit

RBS

TTAGTACCTGATACTAA



PrrsC[D6]

Pg25

Pj5[C1]

3000 2000

Synthetic RBS

Pj5[C1C2]

4000

PrrsC[D4]

Pj5

5000

PrrsC

Pj5[C1]

6000

Pg25[D1C2]

Pj5

7000

Pg25[D1]

TTGACA

8000

Pg25[C2]

-35

AGAGAGA

GGACTC

Pg25

TTGACA

RBS

Pj5[C1C2]

-35

9000

Pj5[C1]

24 bp UP

AGAGAGA

GGACTC

Pj5[C2]

TTGACA

RBS

Pj5

-35

AGAGAGA

GGACTC

PphaC1[B1d]

74 bp UP

RBS

PphaC1[B1c]

-35

AGAGAGA

GGACTC

PphaC1[B1b]

-35

TTGACA

P

P

AACAAT

PphaC1[B1a]

99 bp UP

P

P

GCGCGTGC GTTGCAAGGC

TTCGGC

P

P

-10

PphaC1[A1]

124 bp UP

RBS

18 bp spacer

TTGACA

P

P

-35

Page 28 of 34

B

PphaC1

124 bp UP

RFU/OD600 [a.u.]

1 2 3 4 5 6 7 8 9 10 phaC1 11 12 13 14 15 16 17 18 phaC1[A1] 19 20 21 22 23 24 25 phaC1[B1a] 26 27 28 29 30 31 32 phaC1[B1b] 33 34 35 36 37 38 39 40 phaC1[B1c] 41 42 43 44 45 46 47 phaC1[B1d] 48 49 50 51 52 53j5 54 55 56 57 58 59 60j5[C2]


P

rr

P sC

6]

[D

4]

[D

Promoter ACS Paragon Plus Environment

Promoter

0 0.9

Promoter

g2 5

1.0

P

2000

sC

1.1

rr

4000

P

1.2

6]

6000

[D

1.3

sC

8000

rr

j5 [C

P

2]

3C 2]

j5 [A

P

2] 1C 2]

j5 [C

P

1C 2] 1C

6a C

j5 [A 1D

6b C

15000

10000

4000

3000 20

15

2000 10

1000 5

0 0

Fold change [-]

Promoter

P

1.4

4]

10000

[D

F P

0

sC

1000

rr

2]

E1

1D

1C

2000

j5 [A

P

j5 [A 1C

P

E3

RFU/OD600 [a.u.]

[-]

RFU/OD600 [a.u.]

sC

3000

Fold change [-]

rr

6]

[D

P

sC

C

P

2]

j5 [C

P

2]

2]

3C

j5 [A

P

1C

j5 [C

2]

1C

rr

P

4]

[D

A

P

0 6a C

0

1D

5

2]

2000

1C

10

C

Promoter

j5 [A

15

6b

20

P

25

2]

30

1D

10000

1C

35

1C

E

j5 [A

4000 sC

0

P

6000 rr

1000

j5 [A

8000 P

2000

RFU/OD600 [a.u.]

3000

g2 5

E1 ]

g2 5[

P

4000

RFU/OD600 [a.u.]

E2 ]

g2 5[

P

5000

P

12000

Fold change [-]

E3 ]

g2 5[

P

g2 5

P

B

P

j5 g j5 [A1 25 [A C P 1D 1C2 6 j5 ] [A bC 1D 1C 6a 2] P C1C 2 j5 [C ] P 1C 2 j5 [A ] 3 P C2] j5 [C 2]

P

P

sC

sC

rr

P

rr

RFU/OD600 [a.u.]

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

P

RFU/OD600 [a.u.]

Page 29 of 34 ACS Synthetic Biology

Back-to-back

Overlapping

Face-to-face

D [-]

E2

E1

5000

0

Promoter

G 25

A

3000

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

RFU/OD600 [a.u.]

6000

Promoter 10000

L5 8000

0.200 0.050 0.020

L4 6000

L3 4000

0.010

L2

0.006 0.005

2000

L1 0


- Parents A

B

C

D

0.001

.

E

Promoter engineering strategies

PBAD ruler

L-arabinose [%]

B

Page 30 of 34

9000

0

Promoter activity [a.u.]

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


12000

Relative promoter activity change [%]

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

1


Pj5[A1C1C2] as transcriptional amplifier [E]

Pg25 as transcriptional amplifier [E]

PphaC1 as transcriptional amplifier [E]

-35 and -10 boxes repeat [D]

Operator insertion [D]

RBS repeat [C]

Synthetic RBS insertion [C]

T7 stem-loop insertion [C]

5'-truncation [B]

Mutation in -35 box [A]

-

Page 31 of 34 ACS Synthetic Biology

10000

1000

100 100% 50%

10 10%


18

OD600 [-]

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

Page 32 of 34

pBBR1MCS-1 Pg25

16 14

PphaC1[B1d]

12

PrrsC[E1D4]

10

Pg25[E3]

8

Pg25[D1C2]

6

Pj5[E1A1C1C2]

4

Pj5[E2C2]

2 0

0

10

20 ACS Paragon 30Plus Environment40 Time [h]

50

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ACS Synthetic Biology Page 34 of 34 1 2 3 4 5 6


An Engineered Constitutive Promoter Set with Broad Activity Range for

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