University of Groningen Sustainable membrane biosynthesis ...biosynthesis pathway the essential...

University of Groningen

Sustainable membrane biosynthesis for synthetic minimal cellsExterkate, Marten

DOI:10.33612/diss.98704569

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Citation for published version (APA):Exterkate, M. (2019). Sustainable membrane biosynthesis for synthetic minimal cells. RijksuniversiteitGroningen. https://doi.org/10.33612/diss.98704569

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https://doi.org/10.33612/diss.98704569

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https://doi.org/10.33612/diss.98704569

CHAPTER 3Cardiolipin Biosynthesis by the Promiscuous ClsA in a Synthetic Cellular Membrane

Marten Exterkate†, Niels W.A. de Kok†, Ruben L.H. Andringa#, Iris C. Verhoek†, Adriaan J.

Minnaard#, and Arnold J.M. Driessen†*

† Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University

of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands

# Department of Chemical Biology, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG

Groningen, The Netherlands

*Correspondence: Arnold J. M. Driessen, [email protected]

ABSTRACT

The bottom-up construction of a synthetic cell comprises many different processes,

including expansion of the membrane. In a previously developed in vitro phospholipid

biosynthesis pathway the essential phospholipid species PE and PG of the Escherichia coli

cytoplasmic membrane could be produced at high levels yielding sustainable membrane

growth. Here the pathway was extended with the production of cardiolipin by the E. coli

cardiolipin synthase A (ClsA), thereby mimicking a native membrane composition. Further

analysis of ClsA revealed a remarkable promiscuity of this enzyme towards a wide variety

of substrates. In the reverse reaction that utilizes cardiolipin, numerous primary alcohols

other than glycerol could be incorporated, which resulted in the synthesis of phospholipids

with a wide variety in polar headgroups.

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CHAPTER 3

INTRODUCTION

The development of a synthetic minimal cell, that is built bottom-up from lifeless components,

is a complex process in which many challenges are encountered 1,2. Although in such a cell

only basic cellular functions will be implemented, still numerous cellular components have

to be assembled that need to interact in a functional manner. One of the basic engineering

challenges will be the generation of a boundary membrane consisting of phospholipids

(mimics) that separates the compartment interior from the exterior environment 3. Besides

functioning as the general barrier, such membranes additionally need to support a wide variety

of membrane processes, (e.g. transport and energy transduction) 4,5. As often these processes

require the presence of specific phospholipid species, the boundary layer of a synthetic

minimal cell should have a well-defined lipid composition 3. The cytoplasmic membrane of

Escherichia coli can serve as a suitable template, as it is relatively simple and well-studied 5. It consists mostly of the three main phospholipid species: phosphatidylethanolamine (PE,

70-75%), phosphatidylglycerol (PG 20-25%) and cardiolipin (CL 0-10%, depending on the

growth phase 6. As a first step toward a self-reproducing boundary layer of a synthetic cell,

we recently reconstituted the E. coli phospholipid biosynthetic pathway, thereby realizing

the self-assembly of enzymatically synthesized phospholipids into a growing membrane 7.

In this system, simple fatty acid and glycerol 3-phosphate building blocks are converted by

a cascade of 8 purified (membrane) proteins into the main E. coli lipid species PE and PG.

In this system, cardiolipin was missing and although this phospholipid is not essential for

the viability of E. coli, it is believed to facilitate several membrane related processes, among

which activation of the respiratory complex and formation of intracellular membranes 8,9.

In addition, cardiolipin appears to fulfil a key role in osmo-tolerance of membranes 10–12.

Therefore, the introduction of cardiolipin will further extent the functional integration of a

boundary layer in a synthetic cell.

Cardiolipin is an anionic phospholipid that is universally present in all domains of life.

It consists of a glycerol headgroup, coupled to two pairs of acyl-chains via two glycerol-

phosphate moieties, thus carrying a divalent negative charge. In bacteria CL is believed

to be located in lipid-domains at the cell poles and division site 13–15. The molecule has an

inverted conical structure, caused by the small anionic head group relative to the bulky

hydrophobic acyl chains. Due to this structural feature, accumulation of CL possibly induces

membrane curvature, but this process is not critical as CL is not an essential constituent

of the E. coli membrane 16. In bacteria, CL is most commonly synthesized by transferring

the phosphatidyl-group, originating from a PG molecule, to another PG, whereby glycerol

leaves as a by-product. In E. coli, three enzymes are capable of producing CL: ClsA, ClsB

(YbhO) and ClsC 12,17 . Interestingly, the substrate specificity of ClsC differs from the other

two enzymes, as ClsC utilizes the phosphatidyl group of a PE instead of another PG as

donor to PG, which produces an ethanolamine leaving group 12. Moreover, ClsB does not

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3

CARDIOLIPIN BIOSYNTHESIS BY THE PROMISCUOUS ClsA IN A SYNTHETIC CELLULAR MEMBRANE

only produce cardiolipin, but can also synthesizes PG, by exchanging the headgroup of

PE for glycerol 18. Hence, depending on the substrates, an enzymatic reaction of ClsB can

result in the production of either ethanolamine or glycerol as possible leaving group. As in

E. coli, ClsA is responsible for the synthesis of the majority of CL 16,19, this enzyme is the

most promising candidate for cardiolipin synthesis in a synthetic cell.

Here we report on the biosynthesis of CL and related phospholipid species by ClsA as

part of an in vitro reconstituted phospholipid biosynthesis pathway to generate native-like

E. coli membranes with substantial levels of CL. Furthermore, the data shows that ClsA

exhibits a remarkable promiscuity that can be used to incorporate a multitude of polar

headgroups into phospholipids.

RESULTS

MEMBRANE ORGANIZATION AND EXPRESSION OF ClsA

Cardiolipin synthase A (ClsA) is an integral membrane protein. Although its structure has

not been determined, a membrane topology prediction based on the average hydropathy-

profile of the amino acid sequences of a family of bacterial ClsA proteins reveals the

presence of two hydrophobic regions at the N-terminus (corresponding to amino acids

10-29 and 36-58 in ClsA). These most likely represent transmembrane segments that

are linked C-terminally to a large globular domain (Fig.1A), which is believed to associate

with the membrane. This region contains two HXK motifs, which are universally present

in cardiolipin synthesizing enzymes, and a common characteristic for enzymes of the

phospholipase D superfamily 20. ClsA has a predicted mass of 54 kDa, but when a crude

extract containing overexpressed ClsA is analyzed on SDS-PAGE, it migrates as a 46 kDa

protein 17,21. Remarkably, in vitro transcribed and translated ClsA shows the expected mass

of 54 kDa, suggesting post-translational processing of ClsA 22. Further assessment revealed

that a N-terminal truncated (Δ2–60) ClsA variant remained membrane associated and

active in vitro, whereas a C-terminal truncated ClsA is no longer functional 23. Therefore,

His-tags were introduced at the N-terminus yielding His ClsA, and the aforementioned

truncate His-ClsA(Δ2-60) (Fig.1B).

The genes were cloned into a pET-based overexpression vector, transformed into E.

coli, and overexpressed for purification. ClsA and ClsA(Δ2-60) were found to be associated

with the membrane, and could be extracted by membrane solubilization with the detergent

n-dodecyl-β-d-maltoside (2%, w/v). Subsequently, Ni-NTA agarose affinity chromatography

was performed, and the elution fractions were analyzed with SDS-PAGE, resulting in

purified ClsA(Δ2-60) whereas His-ClsA was enriched (Fig.1C). Noteworthy, both proteins

exhibit a similar mass on SDS-PAGE of about 46 kDa, suggesting truncation of the wild-

type ClsA. Indeed, western blotting with a His-tag antibody confirmed the presence of the

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CHAPTER 3

N-terminal His-tag for ClsA(Δ2-60) while ClsA was not stained (Fig. S1A). To elucidate the

processing site of ClsA, a proteomics analysis was performed using LC-MS on in-gel tryptic

digests of both proteins (Fig.S1B). Peptides covering the complete amino acid sequence

of ClsA(Δ2-60) could be identified, whereas no peptides were found corresponding to the

N-terminal region of ClsA, which is consistent with the notion that N-terminal cleavage of

ClsA occurred. To examine the functionality of both proteins, in vitro activity assays were

performed in the presence of the detergent solubilized substrate PG. Whereas the truncate

ClsA(Δ2-60) was largely inactive, the in vivo processed ClsA showed a high cardiolipin

biosynthesis activity (Fig.1D). Control samples from a strain not overexpressing ClsA were,

as expected, inactive. Hence, the enriched ClsA fraction was further used in this study.

Noteworthy, introduction of a His-tag at the C-terminus of ClsA (ClsA-His) also yielded a

N-terminal truncated protein, but after purification, ClsA-His was completely inactive (data

not shown).

Amino acid residue

Hyd

roph

obic

ity

E. coli

Bacterial family

ClsA

HisHis

ClsA ClsA(Δ2-60)A B

C

40

55

70

100

130

35

25

EV ClsAClsA

(Δ2-60) D

Control Emptyvector

ClsA ClsA(Δ2-60)0.0

0.2

0.4

0.6

0.8

1.0

1.2

Lipi

d sp

ecie

s(n

orm

laiz

ed io

n co

unt)

PG CL PA

MW(kDa)

A

C D

B

Figure 1. ClsA (Ec_ClsA) and ClsA(Δ2-60). (a) Hydropathy profile alignment of E. coli ClsA (red line)

with the averaged hydropathy profile of their bacterial protein family (black line). (b) Schematic

representation of ClsA and the designed mutant ClsA(Δ2-60). (c) Coomassie stained SDS-PAGE gel of

purified empty vector (EV) control, ClsA and ClsA(Δ2-60) by Ni-NTA chromatography. (d) In vitro activity

of purified ClsA and ClsA(Δ2-60). Lipid species were analyzed by LC-MS, normalized for the internal

standard and plotted on the y-axis.

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CARDIOLIPIN SYNTHASE ACTIVITY REPRESENTS A GLYCEROL-DEPENDENT DYNAMIC

EQUILIBRIUM BETWEEN PG AND CL

In the ClsA mediated synthesis of cardiolipin, LC-MS analysis shows that about 80-

85% of the PG is utilized, of which the majority is converted into cardiolipin (Fig.2A). In

addition, some phosphatidic acid (PA) is produced which could arise from an unsuccessful

transfer of the phosphatidyl-group, thereby directly hydrolyzing the phosphate-group. To

promote formation of CL and prevent formation of PA, 100 mM of glycerol was included

in the reaction. Although the production of PA was decreased slightly, the presence

of glycerol resulted in higher concentrations of PG (Fig.2A), which could be caused by

glycerol-mediated inhibition of ClsA. Alternatively, glycerol may stimulate the formation

of PG in the reverse transesterification reaction upon hydrolysis of CL. Therefore, also the

reverse reaction starting with cardiolipin was performed, in which ClsA converted CL into

predominantly PG in the presence of 100 mM glycerol (Fig.2B), whereas in the absence of

glycerol mostly PA was formed. This demonstrates that ClsA not only synthesizes CL from

two PG molecules, but also can utilize CL to form PG and PA in a glycerol-dependent manner.

Noteworthy, in the absence of glycerol, only a small amount of PG is observed, which is not

proportional with the amount of PA. This suggests that the released PG originating from the

hydrolysis of CL is most likely directly re-utilized for production of CL, eventually leading

to the accumulation of PA that cannot be further converted. Indeed, when palmitoleoyl-

oleoyl phosphatidic acid (POPA; C16:0-C18:1) was added to a reaction containing di-

oleoyl phosphatidylglycerol (DOPG; C18:1-C18:1), the ClsA-mediated reaction resulted

in the formation of only di-oleoyl-di-oleoyl cardiolipin (DODOCl) and DOPA, whereas no

palmitoleoyl-oleoyl phosphatidylglycerol (POPG) or palmitoleoyl-oleoyl-palmitoleoyl-oleoyl

cardiolipin (POPOCL) could be observed. This demonstrates that PA cannot be utilized by

ClsA (Fig.S2).

The ability to distinguish a single lipid species, such as PG, based on variations in the

acyl-chain composition was further exploited to examine the Cl biosynthetic equilibrium

reaction and to address the transesterification step. Incubation of ClsA with an equimolar

ratio of POPG and DODOCL, in the presence of glycerol, resulted in the expected production

of POPOCL, DOPG and DOPA, but also in the production of POPA (Fig.2C), thereby

demonstrating ClsA mediated conversions occur in both directions (Fig.2D). Moreover, a

cardiolipin species with the mixed DOPO acyl-chain configuration could be identified as

well. (Fig.2C). This molecule is the synthesis product of a POPG combined with a DOPG,

which originated from the hydrolytic or transesterification reaction of DODOCL, thereby

emphasizing the dynamic character of this equilibrium.

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CHAPTER 3

2 PG CL PG+PAGlycerol

Glycerol H2O

A

B

C

POPG

; D

OPG

(nor

mal

ized

ion

coun

t)

Control ClsA0.0

0.5

1.0

0.0

0.05

0.1

POPG POPOCL POPA DOPG DODOCL DOPA PODOCL

Oth

er li

pid

spec

ies

(nor

mal

ized

ion

coun

t)

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

Control ClsA ClsA +glycerol

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

PG CL PA

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


0.0

0.2

0.4

0.6

0.8

PG CL PA

D


Glycerol H2O

A

B

C

POPG

; D

OPG

(nor

mal

ized

ion

coun

t)

Control ClsA0.0

0.5

1.0

0.0

0.05

0.1


Oth

er li

pid

spec

ies

(nor

mal

ized

ion

coun

t)

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

PG CL PA

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


0.0

0.2

0.4

0.6

0.8

PG CL PA

D


Glycerol H2O

A

B

C

POPG

; D

OPG

(nor

mal

ized

ion

coun

t)

Control ClsA0.0

0.5

1.0

0.0

0.05

0.1


Oth

er li

pid

spec

ies

(nor

mal

ized

ion

coun

t)

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

PG CL PA

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


0.0

0.2

0.4

0.6

0.8

PG CL PA

D


Glycerol H2O

A

B

C

POPG

; D

OPG

(nor

mal

ized

ion

coun

t)

Control ClsA0.0

0.5

1.0

0.0

0.05

0.1


Oth

er li

pid

spec

ies

(nor

mal

ized

ion

coun

t)

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

PG CL PA

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


0.0

0.2

0.4

0.6

0.8

PG CL PA

DC D

BA

Figure 2. ClsA activity in vitro in the presence or absence of glycerol starting with the substrate(s)

(a) PG, (b) CL and (c) POPG together with CL. Lipid species were analyzed by LC-MS, normalized for

the internal standard and plotted on the y-axis. (d) Schematic representation of the ClsA mediated

glycerol-dependent dynamic equilibrium.

ENZYMATIC RECYCLING OF CL-DERIVED PA IN THE PHOSPHOLIPID BIOSYNTHETIC

PATHWAY

The production of PA during ClsA mediated cardiolipin synthesis is an undesired

phenomenon, as it is a dead-end product in the reversed reaction that will accumulate.

However, PA is a common intermediate in phospholipid biosynthesis, and in principle

would be recycled into the pathway by the enzyme CdsA that utilizes PA and CTP to yield

CDP-DAG, a central precursor in phospholipid synthesis. CDP-DAG can then be converted

into PG through the intermediate PGP via the enzymes PgsA and PgpA, which requires

one molecule of glycerol 3-phosphate. This concept was tested by the co-reconstitution

of ClsA with CdsA, PgsA and PgpA. In this reaction, the PA was completely converted into

PG and cardiolipin (Fig.3A), whereas in the absence of ClsA, PA was completely converted

into PG. Since in both reactions, no PA could be detected, complete recycling of the PA that

originated from the hydrolysis of CL must have occurred.

The aforementioned reaction was expanded with the other enzymes of the in vitro

phospholipid synthesis pathway yielding PA starting from simple building blocks 7. Herein,

oleic acid is converted by the enzyme FadD into acyl-CoA. This acyl-chain donor is utilized

75

3


by the enzymes PlsB and PlsC, which attach an acyl-chain onto respectively the 1- and

the 2-position of glycerol-3-phosphate (G3P), resulting in the formation of PA. As shown

before, combining all the enzymes used in the aforementioned pathway in vitro, resulted

in almost complete conversion of fatty acid into PG (Fig.3B). However, some cardiolipin

production can be observed as well, possibly due to co-purification. In the presence of only

CdsA, PgsA and PgpA, no cardiolipin was observed, indicating that ClsA would co-purify

with either FadD, PlsB or PlsC. Nevertheless, by addition of ClsA, a significantly larger

fraction of PG is converted into cardiolipin, thereby confirming successful incorporation of

ClsA into the in vitro phospholipid biosynthesis pathway. Furthermore, PG-CL ratios are

similar starting from the building block PA or oleic acid, indicating full compatibility of ClsA

with the pathway (Fig.3).

A

B

Control PG synthesis CL synthesis0

1

2

3

4

5

6

7

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

Oleic acid PG CL

Control PG synthesis CL synthesis0.0

0.2

0.4

0.6

0.8

1.0

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

PA PG CL

A

B

Control PG synthesis CL synthesis0

1

2

3

4

5

6

7

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

Oleic acid PG CL

Control PG synthesis CL synthesis0.0

0.2

0.4

0.6

0.8

1.0

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

PA PG CL

A B

Figure 3. Introduction of Cardiolipin synthesis in the in vitro phospholipid biosynthesis pathway starting

from either (a) the intermediate PA, or (b) oleic acid. Lipid species were analyzed by LC-MS, normalized

for the internal standard and plotted.

ClsA EXHIBITS A HIGH PROMISCUITY TOWARD PRIMARY ALCOHOLS

In the reverse CL hydrolysis reaction catalyzed by ClsA, transesterification results in the

incorporation of a glycerol headgroup onto PA yielding PG. To examine the specificity of

ClsA towards different alcohols, molecules structurally related to glycerol were tested

as a substrate in the CL hydrolysis reaction (Fig.4A). Conversion of CL in the presence

of 1-propanol resulted in the production of PG and phosphatidyl-1-propanol (p-1-prOH),

whereas 2-propanol appeared a poor substrate in this reaction. This indicates that a terminal

OH-group is essential. Likewise, 1,2-propanediol and 1,3-propanediol could function

as substrates for synthesis of their phosphatidyl-analogue phosphatidyl-propanediol.

However, in the case of 1,3-propanediol the cardiolipin analogue di-phosphatidyl-1,3-

propanediol (di-p-1,3-prOH), which contains a 1,3-propanediol headgroup instead of a

glycerol, was produced as well. This demonstrates that not only phosphatidyl-glycerol (PG),

but also phosphatidyl-1,3-propanediol (p-1,3-prOH) can serve as an acceptor in the ClsA

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CHAPTER 3

mediated phosphatidyl-transfer.

To further test the promiscuity of the enzyme, methylated (2,2-dimethyl-1,3-propanediol)

and phenylated (2-phenyl-1,3-propanediol) derivatives of 1,3-propanediol were tested as

substrates (Fig.4B). Although this resulted in the production of both phosphatidyl-2,2-

dimethyl-1,3-propanediol (p-2,2-Me-1,3-prOH) and phosphatidyl-2-phenyl-1,3-propanediol

(p-2-Phe-1,3-prOH), the latter was produced at only low quantities. Moreover, in presence of

2,2-dimethyl-1,3-propanediol, production of PG and PA was reduced, and nearly completely

absent in the presence of 2-phenyl-1,3-propanediol, which indicates steric hindrance at

the second carbon impairs the overall hydrolytic enzyme activity. A similar effect of these

two substrates is observed during the synthesis of cardiolipin, indicating that the steric

hindrance inhibits the overall activity of the enzyme (fig S3A).

Like the tri-carbon alcohols, also di- and tetra-carbon alcohols could be used by

ClsA to produce PG analogues, which is illustrated by the ethanediol-, 1,3-butanediol-

and 1,4-butanediol-dependent synthesis of phosphatidyl-1,2-ethanediol (p-1,2-etOH),

phosphatidyl-1,3-butanediol (p-1,3-buOH) and phosphatidyl-1,4-butanediol (p-1,4-buOH),

respectively (Fig.4A). However, only in the presence of the tetra-carbon alcohols the

cardiolipin analogues di-phosphatidyl-1,3-butanediol-cardiolipin (di-p-1,3-buOH) and

1,4-butanediol-cardiolipin (di-p-1,4-buOH) could be observed. As only trace amounts of

di-p-1,3-buOH (2% of di-p-1,4-buOH) could be detected, ClsA seems to function better

with the substrate p-1,4-buOH, suggesting that a second primary alcohol is preferred as

phosphatidyl-acceptor. The promiscuity of ClsA to a diversity of alcohols is best exemplified

by its ability to incorporate mannitol 24, which allows for the formation of both phosphatidyl-

mannitol (p-mannitol) and di-phosphatidyl-mannitol (di-p-mannitol) (Fig.4A). This sugar

comprises 6 carbons, all attached to an alcohol moiety, illustrating that ClsA is capable of

incorporating a wide variety of alcohols as polar headgroup, resulting in the synthesis of a

diverse group of phospholipids.

Besides the glycerol related alcohols, some polar headgroups of lipids commonly

found in nature were tested as substrates as well (Fig.4C). Serine, ethanolamine, choline

and inositol were tested as alcohol donors in the CL hydrolysis reaction. Although PS,

PE, PC and PI could be observed by LC-MS analysis, they are only produced in minute

amounts, hence hardly any CL was consumed. Notably, in the presence of ethanolamine

the common hydrolysis of CL into PG and PA was reduced, indicating an inhibiting effect of

ethanolamine. A similar observation was made by using 3-amino-propanol, suggesting that

also this compound impairs the ClsA hydrolytic activity (Fig.S3B). Vice versa, the synthesis

of CL in the presence of either ethanolamine or 3-aminopropanol reduces CL synthesis as

well, illustrating that the overall activity of ClsA is impaired (Fig.S3C).

77

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RT (min)

0 5 10 15 20 250

2.0x106

4.0x106

6.0x106

Lipi

d sp

ecie

s(I

on c

ount

)741.54 m/z

p-1-prOH

0 5 10 15 20 250

1x104

2x104

Lipi

d sp

ecie

s(I

on c

ount

)

741.54 m/z

p-2-prOH

0 5 10 15 20 250

5.0x106

1.0x107

1.5x107

2.0x107

Lipi

d sp

ecie

s(I

on c

ount

)

757.53 m/z

p-1,2-prOH

757.53 m/z7

0 5 10 15 20 250

1x10 7

2x10

Lipi

d sp

ecie

s(I

on c

ount

)

1440.02 m/z

p-1,3-prOHdi-p-1,3-prOH

0 5 10 15 20 250

5.0x106

1.0x107

1.5x107

2.0x107

Lipi

d sp

ecie

s(I

on c

ount

)

743.52 m/z

p-1,2-etOH

0

2.0x10 6

4.0x10 6

6.0x10 6

8.0x10 6

0

1x10 4

2x10 4

3x10 4

4x10 4

0 5 10 15 20 25

p-1,

3-bu

OH

(Ion

cou

nt)

di-p

-1,3

-buO

H(I

on c

ount

)

p-1,3-buOH

1454.04 m/z

771.55 m/z

di-p-1,3-buOH

0 5 10 15 20 250

1x10 7

2x10 7

0

2.0x105

4.0x105

6.0x105

p-1,

4-bu

OH

(Ion

cou

nt)

di-p

-1,4

-buO

H(I

on c

ount

)771.55 m/z

1454.04 m/zp-1,4-buOHdi-p-1,4-buOH

0 5 10 15 20 250

5.0x10 6

1.0x10 7

1.5x10 7

Lipi

d sp

ecie

s(I

on c

ount

)

863.56 m/z

1546.05 m/z

p-mannitol

di-p-mannitol

R-group

Control ClsA ClsA +1,3-prOH

ClsA +glycerol

ClsA +2,2-Me-1,3-prOH

ClsA +2-Phe-

1,3-prOH

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Lipi

d sp

ecie

s(n

orm

aliz

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n co

unt)

CLPGPAp-1,3-prOH

di-p-1,3-prOHp-2,2-Me-1,3-prOHp-2-Phe-1,3-prOH

A

B C

RT (min)

Control ClsA ClsA +choline

ClsA +serine

ClsA +inositol

ClsA +ethanol-amine

0.0

0.1

0.2

0.3

0.4

0.5 CLPGPAPC

PSPIPE

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

B

A

C

Figure 4. in vitro ClsA activity in the presence of various alcohols. (a) Glycerol-like alcohols: 1-propanol,

2-propanol, 1,2-propanediol, 1,3-propanediol, 1,2-ethanediol, 1,3-butanediol, 1,4-butanediol and

mannitol. (b) 1,3-propanediol and its derivatives: glycerol (1,2,3-propanetriol), 2,2-dimethyl-1,3-

propanediol and 2-phenyl-1,3-propanediol. (c) Common polar lipid headgroup alcohols: choline,

serine, inositol, ethanolamine. Lipid species were analyzed by LC-MS, if noted normalized for the

internal standard and plotted.

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ClsA ACTIVITY WITH THE ARCHAEAL ARCHAETIDYLGLYCEROL

In archaea, phospholipids consist of highly-methylated isoprenoid chains that are ether-

linked to a glycerol-1-phosphate backbone, whereas in bacteria and eukaryotes, fatty

acid chains are ester bonded to the enantiomeric backbone glycerol-3-phosphate 25.

Thus, the bacterial and archaeal phospholipids are chiral distinct. To further examine

the substrate promiscuity, ClsA (Ec_ClsA) was incubated with archaetidylglycerol (AG),

the archaeal equivalent of PG. This resulted in the formation of an archaeal cardiolipin

(aCL) equivalent and the byproduct archaeatidic acid (AA) (fig 5A). Subsequently, a mixture

of PG and AG was applied. Although in this case ClsA did not synthesize aCL, two other

distinct cardiolipin species were produced. (Fig.5B). Besides bacterial cardiolipin (bCL), an

additional cardiolipin species containing both isoprenoid lipid-tails and fatty acid lipid-tails

was observed, thereby forming an archaeal-bacterial hybrid cardiolipin (hCL). Interestingly,

not only aCL, but also AA was absent, indicating no hydrolysis of AG, thereby suggesting

that ClsA prefers PG over AG as phosphatidyldonor in CL synthesis. Nevertheless, ClsA

appears highly promiscuous as it also accepts the archaeal AG as a substrate.

aCL homologs are found in some Archaea, most notably in certain halophiles and

methanogens, organisms that belong to the clade of euarchaeota. To identify a possible

cardiolipin synthesizing enzyme in archaea, we performed a BLAST homology search

with ClsA which revealed 2 main clusters of archaeal homologs with phospholipase D

like activity (Fig.S4A). One cluster consists of halophilic archaea and the other concerns

methanogenic archaea. The putative cardiolipin synthase from Methanospirilum hungatei

was selected for further analysis. Although this enzyme only shows 27% sequence identity

with the E. coli ClsA, its average hydropathy-profile also predicts two hydrophobic regions

at the N-terminus, thereby showing a high structural similarity between these two proteins.

M. hungatei is a strict anaerobe, growing at mesophilic temperatures with a pH around

7. Additionally, the salt requirement of M. hungatei is more comparable to E. coli. The M.

hungatei ClsA homologue (Mh_Cls) was cloned into an overexpression vector and, after

expression in E. coli, purified (Fig.S4B) and tested in vitro. The Mh_Cls more efficiently

converted AG into aCL as compared to ClsA, thereby confirming its functionality as an

archaeal cardiolipin synthesizing enzyme (Fig.5C). Moreover, by feeding Mh_Cls with both

PG and AG not only archaeal CL (aCL), but also the hybrid cardiolipin (hCL) and bacterial

cardiolipin (bCL) could be synthesized, indicating a high degree of promiscuity for this

archaeal enzyme as well (Fig.5D).

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Control Ec_ClsA0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

PG AG PA AA bCL aCL hCL

Control Ec_ClsA0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Lipi

d sp

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unt)

AG AA aCL

Control Mh_Cls0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

AG AA aCL

Control Mh_Cls0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

PG AG PA AA bCL aCL hCL

Lipi

d sp

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Lipi

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s(n

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Lipi

d sp

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s(n

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unt)

A B

C DD

BA

C

Figure 5. Activity of the bacterial Ec_ClsA in the presence of (a) AG (b) together with PG, compared to

the activity of the archaeal Mh_Cls in the presence of (c) AG (d) together with PG. Lipid species were

analyzed by LC-MS, normalized for the internal standard and plotted.

DISCUSSION

Here, we report on the in vitro activity of ClsA, the primary cardiolipin synthesizing enzyme

in E. coli. By reconstituting this enzyme into liposomes, we were able to study this enzyme in

the absence of its native membrane and in a controlled environment. In vitro activity assays

in the presence of PG and/or CL revealed there is a dynamic equilibrium between these lipid

species, which is, among others, depending on the amount of glycerol. The hydrolysis of

cardiolipin does not only yield PG, but also results in the synthesis of the by-product PA. As

PA cannot be reutilized by ClsA, we developed a recycling system that converts PA back into

PG via an enzymatic cascade consisting of the three proteins CdsA, PgsA and PgpA. This

recycling system will allow for sustainable phospholipid biosynthesis, without accumulation

of the by-product PA. Because of the recycling additional energy is consumed, as CTP and

G3P are lost in a futile cycle. This enzymatic cascade was further extended with the enzymes

FadD, PlsB and PlsC, thereby allowing the synthesis of cardiolipin from simple fatty acid

and glycerol building blocks. Together with PssA and PsD 7, this enables a complete in vitro

mimic of phospholipid biosynthesis in E. coli that may serve as a phospholipid-membrane

expanding system in a synthetic cell.

Besides cardiolipin biosynthesis, ClsA can potentially also generate other phospholipid

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species. The hydrolysis of cardiolipin is not exclusive for the glycerol-dependent production

of two PG molecules, but allows for a wide variety of phosphatidyl-lipids, depending on

the incorporated alcohol. This phenomena has been observed in vivo with mannitol 24 and

other alcoholic substrates 26. In principal, any primary alcohol can serve as a phosphatidyl-

headgroup, provided that it is compatible with the binding pocket of ClsA. This is illustrated

by introducing different side-groups at the second carbon of 1,3-propanediol. Depending on

the specific side-group (size, hydrophobicity, etc.), ClsA incorporates these alcohols with

different efficiency. Interestingly, the length of the carbon-chains seems irrelevant, as ClsA

could incorporate ethanediol, a di-carbon, as well as the hexa-carbon mannitol. Remarkably,

in the presence of extracellular butanol, E. coli was found to produce phosphatidyl-butanol

in a DOPC-dependent manner 26. As this lipid species is not endogenously present in E.

coli, this observation is rather difficult to explain. Moreover, in our in vitro setup, DOPC was

not needed for the production of phosphatidyl-butanol from cardiolipin in the presence of

butanol.

The ability of ClsA to incorporate a wide variety of headgroups, comes with new bio-

catalytic applications of this enzyme, i.e. for the synthesis of natural relevant phospholipid

species. Several alcohols that commonly appear as phospholipid headgroups were tested

with ClsA, which allowed for the synthesis of PE, PC, PS and PI. Although these phospholipid

species could so far only be produced in miniature amounts, the sensitivity of ClsA to such

a wide variety of primary alcohols, could make it an interesting candidate to generate a

membrane with a diverse phospholipid-headgroup composition in synthetic cells. However,

for this purpose further engineering is required.

The versatility of ClsA is not only limited to a wide variety of alcohol-headgroups, as the

enzyme appears to be invariant to the phospholipid-tail, as well as the glycerol-backbone.

This is demonstrated by the ability of ClsA to recognize the archaeal phosphatidylglycerol

(AG), which contains isoprenoid chains that are ether-linked to a G1P backbone, and

convert it into archaeal cardiolipin (aCL). Moreover, when PG and AG were supplied as a

mixed substrate, a novel hybrid-cardiolipin species was produced that contains one pair of

the archaeal-isoprenoid chains and one pair of the bacterial-fatty acid chains. Likewise, an

archaeal cardiolipin synthase, derived from Methanospirillum hungatei (Mh_Cls), showed a

similar promiscuity but was more efficient in the formation of the aCL. In this respect, other

polar head group attaching enzymes from archaeal and/or bacterial sources also appear

invariant to the glycerol core and hydrophobic chains 27. This sheds new light on the ‘lipid

divide’ theory, which states that the lipidome is a crucial and distinct marker separating

the archaeal and bacterial domains of life. The ability of certain enzymes originating from

both domains of life to cross recognize substrates originating from the other domain of life,

further indicates that the later steps in phospholipid biosynthesis, i.e., polar headgroup

attachment and modifications are based on enzymatic reactions that are functionally

equivalent while the enzymes are structurally related.

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MATERIALS AND METHODS

BIOINFORMATIC IDENTIFICATION OF Mh_Cls

Using E. coli K12; MG1655 ClsA (Ec_ClsA: NP_415765.1) as the query sequence, BLAST

homology searches to the domain of Archaea were performed. From these hits a putative

cardiolipin synthase (Mh_Cls: NP_415765.1) from Methanospirilum hungatei JF-1 was

selected.

BACTERIAL STRAINS AND CLONING PROCEDURES

Genomic DNA of Escherichia coli was used as a template for the amplification of genes

encoding for the enzymes ClsA (Ec_ClsA) and ClsA(Δ2-60). An E. coli codon-optimized

synthetic gene of M. hungatei Cls (Mh_Cls) was ordered (GeneArt, Thermo Fisher scientific).

and used as a template for the amplification of Mh_Cls.

E. coli DH5α (Invitrogen) was used for cloning. Plasmids for the E. coli enzymes FadD,

PlsB, PlsC, CdsA, PgsA and PgpA were as reported 7. All primers and plasmids used in

the present study are listed in Tables 1 and 2. E. coli BL21 (DE3) was used as a protein

overexpression host strain for FadD, PlsB, PlsC, CdsA, PgsA and PgpA and E. coli Lemo21

(DE3) protein as overexpression host strain for ClsA, ClsA(Δ2-60) and Mh_Cls. All strains

were grown under aerobic conditions at 37◦C in LB medium supplemented with the required

antibiotics, kanamycin (50 μg/ml), chloramphenicol (34 μg/ml) and ampicillin (50 μg/ml).

EXPRESSION AND PURIFICATION OF PHOSPHOLIPID SYNTHESIZING ENZYMES

The proteins FadD, PlsB, PlsC, CdsA, PgsA and PgpA were expressed and purified as

described 7. ClsA, ClsA(Δ2-60) and Mh_Cls were overexpressed in E. coli Lemo21 (DE3)

strain and induced with 0.5 mM IPTG. After 2.5 hrs. of induction cytoplasmic and membrane

fractions were separated as described 28. The total membranes fractions were resuspended

in buffer A (50 mM Tris/HCl, pH 8.0, 100 mM KCl and 15% glycerol) after which they could

be stored at -80. For further purification, 0.5 mg/ml of membranes were solubilized in 2%

n-dodecyl-β-D-maltopyranoside (DDM) detergent for 1 hr. at 4◦C. The material was subjected

to a centrifugation (17000xg) step for 15 min at 4◦C to remove insolubilized material and the

supernatant was incubated with Ni-NTA (Ni2+- nitrilotriacetic acid) beads (Sigma) for 2 hrs.

at 4◦C. The Ni-NTA beads were washed 10 times with 40 column volumes (CV) of buffer B (50

mM Tris/HCl, pH 8.0, 100 mM KCl, 15% glycerol and 0.05% DDM) supplemented with 10 mM

imidazole, and the proteins were eluted three times with 0.5 CV of buffer B supplemented

with 300 mM imidazole. Purity of the eluted proteins were assessed on 15% SDS/PAGE

stained with Coomassie Brilliant Blue and the protein concentration was determined

by measuring the absorbance at 280 nm. Extinction coefficients were obtained from the

ProtParam tool from ExPASy by applying the specific amino acid sequence.

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Table 1. Cloning and expression vectors used in this study.

Plasmid Description Reference

pRSF-Duet-1 Expression vector (KanR), T7 promoter Novagen

pET-Duet-1 Expression vector (AmpR), T7 promoter Novagen

pET-28b Expression vector (KanR), T7 promoter Novagen

pACYC-Duet-1 Expression vector (CmR), T7 promoter Novagen

pME001 fadD gene with C-terminus His-tag from E. coli MG1655 cloned into pRSF-Duet-1 vector using the primers PrME001 and PrME002

Exterkateet al.a

pME002 plsB gene with C-terminus His-tag from E. coli MG1655 cloned into pet-28b vector using the primers PrME003 and PrME004

Exterkateet al.a

pME003 plsC gene with C-terminus His-tag from E. coli MG1655 cloned into pet-28b vector using the primers PrME005 and PrME006

Exterkateet al.a

pME004 ClsA (Ec_ClsA) gene with N-terminus His-tag from E. coli MG1655 cloned into pRSF-Duet vector using the primers PrME007 and PrME008.

This study

pME005 ClsA(Δ2-60) gene with N-terminus His-tag from E. coli MG1655 cloned into pRSF-Duet vector using the primers PrME009 and PrME008.

This study

pNDK001 Mh_Cls gene with N-terminus His-tag from M. hungatei JF-1 cloned into pRSF-Duet vector using the primers NDKo027 and NDKo028.

This study

pSJ148 cdsA gene with N-terminus His-tag from E. E. coli MG1655 cloned into pACYC-Duet vector using the primers 103 and 106

Caforioet al.b

pAC015 pgsA gene with C-terminus His-tag from E. coli MG1655 cloned into pRSF-Duet vector using the primers 551 and 552

Caforioet al.b

pAC017 pgpA gene with C-terminus His-tag from E. coli MG1655 cloned into pET-Duet vector using the primers 562 and 563

Caforioet al.b

a 7; b 27

Table 2. Oligonucleotide primers used in this study.

Primers Primer sequence 5’ -> 3’Restriction

site

PrME001 TACTAGGAATTCATGAAGAAGGTTTGGCTTAACCG EcoRI

PrME002 AGTCATGCGGCCGCTCAGTGGTGGTGGTGGTGGTGGGCTTTATTGTCCACTTTGC NotI

PrME003 CATCCTGCCCATGGCCGGCTGGCCACGAATTTACTAC NcoI

PrME004 CTTCATGATTCTCGAGCCCTTCGCCCTGCGTCGCAC XhoI

PrME005 CACTACGACCATGG TATATATCTTTCGTCTTATTATTACCG NcoI

PrME006 CATGTCTACTCGAGAACTTTTCCGGCGGCTTC XhoI

PrME007 CGTAGCATATGCACCACCACCACCACCACACAACCGTTTATACGTTGGTGAGTTGGTTGGCCATTCTGGG NdeI

PrME008 ACGTCCTCGAGTTACAGCAACGGACTGAAG XhoI

PrME009 CTAGTCATATGCACCACCACCACCACCACCTCCATTTAGGCAAACGCCG NdeI

NDKo027 ACAGTTCCATGGCCCATCACCATCATCACCACATCCATGATCTGATTCTGGTGATCCACAATTTTC NcoI

NDKo028 ACTTACGAGCTCTTATTATTACAGCAGCGGACTAAACAGACG SacI

103 GCGCCTCGAGTTAGTGATGGTGATGGTGGTGATGATGAAGCGTCCTGAATACCAGTAAC XhoI

106 GCCGCCATGGGCAGCCATCACCATCATCACCACAGCCTGAAGTATCGCCTGATATCTGC NcoI

551 GCGCCATATGCAATTTAATATCCCTACGTTGC NdeI

552 CGCGCTCGAGTCAGTGATGGTGATGGTGGTGATGATGCTGATCAAGCAAATCTGCACGC XhoI

562 CGCGGAATTCATGACCATTTTGCCACGCCATAAAG EcoRI

563 CGGCGCGGCCGCCTAGTGATGGTGATGGTGGTGATGATGCGACAGAATACCCAGCG NotI

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LIPOSOMES PREPARATION

Chloroform stocks of the lipid species DOPG, Cardiolipin (CL), POPG, DOPA, DOPE, DOPC

were purchased from Avanti (Avanti Biochemicals, Birmingham, AL), whereas AG (Fig. S5)

was synthesized by Ruben L.H. Andringa, from the Chemical Biology Department, and will

be detailed elsewhere. For liposomes with a heterogeneous lipid mixture, the desired lipid

chloroform stocks were mixed together in the stated ratio. Next the lipid solution was dried

under a nitrogen gas stream for multiple hrs., after which the dry lipid film was resuspended

in a 300mM KPi, pH 7.0 buffer. For formation of liposomes, a sonication cycle of 15 sec on,

15 sec off was repeated (10-40x) till the solution became transparent.

IN VITRO ASSAYS FOR PHOSPHOLIPID PRODUCTION

All in vitro reactions were performed in 100 μl of assay buffer A containing a final concentration

of 300 mM potassium phosphate pH 7.0, 0.1% DDM and 2 mM DTT, except for the combined

activity of ClsA with the enzymes derived from the earlier described in vitro phospholipid

biosynthesis pathway, which was performed in buffer B 100 mM MES, pH 7.0, 10 mM MgCl2,

100 mM KCl, 2 mM DTT. The activity of ClsA in comparison to ClsA(Δ2-60) was assayed in

buffer A using 1 μM enzyme and 1 mM of DOPG. The glycerol-dependent dynamic equilibrium

of ClsA was assayed in buffer A using 1 μM enzyme and either 1 mM DOPG, 0.5 mM CL, 1 mM

POPG together with 0.5 mM CL, or 1 mM DOPG together with 1 mM POPA.

For recycling of PA, buffer B was supplemented with 3 mM liposomes (DOPE,DOPC,DOPA;

ratio 1:1:1), 2 μM CdsA, 4 mM CTP, 2 μM PgsA, 1 μM PgpA and 1 μM ClsA. Conversion of

oleic acid into PG and CL was assayed under the same conditions, except liposomes without

DOPA were used, and the following compounds were additionally added: 2 μM FadD, 50 μM

CoA, 2700 μM oleic acid, 4 mM ATP, 9 mM G3P, 2 μM PlsB, 3 μM PlsC.

The promiscuity toward primary alcohols was assayed in buffer A supplemented with

1 μM ClsA or Mh_Cls, 0.5 mM CL or 1 mM DOPG, and 100 mM alcoholic substrate. The

activity of ClsA (Ec_ClsA) and Mh_Cls with AG/PG was assayed in buffer A with 100 μM of

lipid substrate(s) and 1 μM of enzyme.

All reactions were incubated overnight at 37◦C unless stated differently. Lipids were

extracted two times with 0.3 ml of n-butanol, and evaporated under a stream of nitrogen

gas and resuspended in 50 μl of methanol for LC-MS analysis.

LC–MS ANALYSIS OF LIPIDS

Samples from the in vitro reactions were analyzed using an Accela1250 high-performance

liquid chromatography (HPLC) system coupled with an electrospray ionization mass

spectrometry (ESI–MS) Orbitrap Exactive (Thermo Fisher Scientific). A sample of 5 μl

was injected into an ACQUITY UPLC® CSH™ C18 1.7μm Column, 2.1 x 150 mm (Waters

Chromatography Ireland Ltd) operating at 55◦C with a flow rate of 300 μl/min. Separation of

the compounds was achieved by a changing gradient of Mobile phase A (5 mM ammonium

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formate in water/acetonitrile 40:60, v/v), and mobile phase B (5 mM ammonium formate in

acetonitrile/n-butanol, 10:90, v/v). The column effluent was injected directly into the Exactive

ESI-MS Orbitrap operating in negative ion mode. Voltage parameters of 3 kV (spray), −75 V

(capillary), −190 V (tube lens) and -46 V (Skimmer voltage) were used. Capillary temperature

of 300°C, sheath gas flow 60, and auxiliary gas flow of 5 was maintained during the analysis.

Spectral data constituting total ion counts were analyzed using the Thermo Scientific

XCalibur processing software by applying the Genesis algorithm based automated peak area

detection and integration. The total ion counts of the extracted lipid products: oleic acid (m/z

281.25 [M-H]-), LPA (C18:1) (m/z 435.26 [M-H]-), PA/DOPA (C18:1-18:1) (m/z 699.49 [M-H]-),

CDP-DAG (m/z 1004.54 [M-H]-), PGP (m/z 853.50 [M-H]-), PG/DOPG (C18:1-18:1) (m/z 773.53

[M-H]-), POPA (C18:1-16:0) (m/z 673.48 [M-H]-), POPG (C18:1-16:0) (m/z 747.52 [M-H]-), CL/

DODOCL/bCL (C18:1-18:1-18:1-18:1) (m/z 1456.03 [M-H]-), POPOCL (C16:0-18:1-16:0-18:1)

(m/z 1403.99 [M-H]-), DOPOCL (C18:1-18:1-16:0-18:1) (m/z 1430.01 [M-H]-), DOPE (m/z 742.54

[M-H]-), p-NH2-prOH (m/z 756.55 [M-H]-), DOPC (m/z 830.59 [M-H]-), DOPS (m/z 786.53 [M-H]-

), DOPI (m/z 861.55 [M-H]-), p-2,2-Me-1,3-prOH (m/z 786.56 [M-H]-), p-2-Phe-1,3-prOH (m/z

833.56 [M-H]-), p-1-prOH and p-2-prOH (m/z 741.54 [M-H]-), p-1,2-prOH and p-1,3-prOH (m/z

757.53 [M-H]-), di-p-1,3-prOH (m/z 1440.02 [M-H]-), p-1,2-etOH (m/z 743.52 [M-H]-), p-1,3-

buOH and p-1,4-buOH (m/z 771.55 [M-H]-), di-p-1,3-buOH and di-p-1,4-buOH (m/z 1454.04

[M-H]-), p-mannitol (m/z 863.56 [M-H]-), di-p-mannitol (m/z 1546.05 [M-H]-), AG (m/z 805.66

[M-H]-), AA (m/z 731.63 [M-H]-), aCL (m/z 1520.29 [M-H]-), hCL (m/z 1488.15 [M-H]-), were

normalized for either DDM (m/z 509.3 [M-H]-) or didecanoyl-PG (C10:0-10:0) (m/z 553.31 [M-

H]-) and plotted on the y-axis as normalized ion count.

ACKNOWLEDGEMENTS

This work was supported by the NWO Gravity programme BaSyC and the NWO Building

blocks of life programme.

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SUPPLEMENTARY MATERIALS

A ClsA(Δ2-60)ClsAEV

B.1

Figure S1.

A

B.1

88

3

CHAPTER 3

B.2

Figure S1. (a) Western blot analysis of a purified empty vector control, ClsA and ClsA(Δ2-60) using a

his-tag antibody. (b) Proteomic analysis of (b.1) ClsA and (b.2) ClsA(Δ2-60) after in gel trypsin digestion

and subsequent LC-MS analysis.

B.2

89

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Figure S2. In vitro ClsA activity in the presence of DOPG and POPA. Lipid species were analyzed by LC-

MS, normalized for the internal standard and plotted.

Control ClsA ClsA +2,2-Me-1,3-prOH

ClsA +2-Phe-

1,3-prOH

0.0

0.2

0.4

0.6

0.8

1.0

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

PG CL PA p-2,2-Me-1,3-prOH p-2-Phe-1,3-prOH

Control ClsA ClsA +amino-

propanol

ClsA +ethanol-amine

0.0

0.1

0.2

0.3

0.4

0.5

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

CL PG PA p-NH2-prOH PE

Control ClsA0.0

0.2

0.4

0.6

0.8

1.0

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)

PG CL PA p-1-NH2-prOH PE

ClsA +amino-

propanol

ClsA +ethanol-amine

A

B

C


ClsA +2-Phe-

1,3-prOH

0.0

0.2

0.4

0.6

0.8

1.0

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)



propanol

ClsA +ethanol-amine

0.0

0.1

0.2

0.3

0.4

0.5

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


Control ClsA0.0

0.2

0.4

0.6

0.8

1.0

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


ClsA +amino-

propanol

ClsA +ethanol-amine

A

B

C


ClsA +2-Phe-

1,3-prOH

0.0

0.2

0.4

0.6

0.8

1.0

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)



propanol

ClsA +ethanol-amine

0.0

0.1

0.2

0.3

0.4

0.5

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


Control ClsA0.0

0.2

0.4

0.6

0.8

1.0

Lipi

d sp

ecie

s(n

orm

aliz

ed io

n co

unt)


ClsA +amino-

propanol

ClsA +ethanol-amine

A

B

C

BA

C

Figure S3. In vitro ClsA activity in the presence of various alcohols and the substrate PG or CL. (a)

glycerol (1,2,3-propanetriol), 2,2-dimethyl-1,3-propanediol and 2-phenyl-1,3-propanediol. (b and c)

1-amino-3-propanol, ethanolamine. Lipid species were analyzed by LC-MS, if noted normalized for the

internal standard and plotted.

90

3

CHAPTER 3

A

B

Figure S4. (a) Schematic representation of the BLAST homology search on ClsA, revealing the two

main archaeal clusters containing halophilic archaea and methanogenic archaea respectively. (b)

Coomassie stained SDS-PAGE gel of purified Mh_Cls by Ni-NTA chromatography.

OO

O

OHP

OHOHO

OO

OO

OHP

OHOHO

O

also synthesized 14' - sn-1 - sn-3 diastereomer14 - sn-1, sn-1 diastereomer

Figure S5. Schematic representation of the synthesized racemic archaetidylglycerol AG.

91

3


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