Functional

analysis

of

genes

involved

in

the

biosynthesis

of

isoprene

in

Bacillus

subtilis

In

comparison

to

other

bacteria

Bacillus

subtilis

emits

the

volatile

compound

isoprene

in

high

concentrations

Isoprene

is

the

smallest

representative

of

the

natural

product

group

of

terpenoids

A

search

in

the

genome

of

B.

subtilis

resulted

in

a

set

of

genes

with

yet

unknown

function

but

putatively

involved

in

the

methylerythritol

phosphate

-LRB-

MEP

-RRB-

pathway

to

isoprene

Further

identification

of

these

genes

would

give

the

possibility

to

engineer

B.

subtilis

as

a

host

cell

for

the

production

of

terpenoids

like

the

valuable

plant-produced

drugs

artemisinin

and

paclitaxel

Conditional

knock-out

strains

of

putative

genes

were

analyzed

for

the

amount

of

isoprene

emitted

Differences

in

isoprene

emission

were

used

to

identify

the

function

of

the

enzymes

and

of

the

corresponding

selected

genes

in

the

MEP

pathway

We

give

proof

on

a

biochemical

level

that

several

of

these

selected

genes

from

this

species

are

involved

in

isoprene

biosynthesis

This

opens

the

possibilities

to

investigate

the

physiological

function

of

isoprene

emission

and

to

increase

the

endogenous

flux

to

the

terpenoid

precursors

isopentenyl

diphosphate

and

dimethylallyl

diphosphate

for

the

heterologous

production

of

more

complex

terpenoids

in

B.

subtilis

Introduction

Isoprene

-LRB-

I

-RRB-

or

actually

isopentenyl

diphosphate

-LRB-

IDP

II

-RRB-

is

the

general

precursor

of

all

terpenoids

that

represent

a

very

diverse

class

of

natural

products

Two

evolutionary

distinct

routes

occur

in

nature

for

the

biosynthesis

of

IDP

The

spread

of

the

two

pathways

is

well

investigated

for

organisms

with

a

sequenced

genome

-LRB-

Lange

et

al.

Boucher

and

Doolittle

Rohdich

et

al.

-RRB-

In

eukaryotes

and

archaea

IDP

and

its

isomer

dimethylallyl

diphosphate

-LRB-

DMADP

III

-RRB-

are

formed

via

the

mevalonate

pathway

-LRB-

reviewed

by

Kuzuyama

-RRB-

This

pathway

is

well

studied

and

for

many

organisms

the

enzymes

are

characterized

and

the

encoding

genes

identified

More

recently

another

pathway

to

IDP

was

discovered

in

some

eubacteria

and

in

plastids

of

higher

plants

that

proceeds

via

the

intermediate

methylerythritol

phosphate

-LRB-

MEP

-RRB-

-LRB-

Rohmer

et

al.

Rohmer

-RRB-

Most

Gram-negative

bacteria

including

Escherichia

coli

use

the

MEP

pathway

and

for

E.

coli

this

is

now

well

documented

For

Gram-positive

bacteria

the

situation

is

less

clear

Gram-positive

cocci

have

been

reported

to

only

possess

genes

for

the

mevalonate

pathway

-LRB-

Wilding

et

al.

-RRB-

In

Streptomyces

aeriouvifer

both

pathways

are

found

-LRB-

Seto

et

al.

-RRB-

In

Bacillus

subtilis

being

regarded

as

the

prototype

for

Gram-positives

only

homologues

of

the

genes

for

the

MEP

pathway

are

present

-LRB-

Wagner

et

al.

-RRB-

For

E.

coli

the

complete

MEP

pathway

has

been

elucidated

and

the

genes

involved

have

been

identified

and

the

corresponding

enzymes

described

-LRB-

reviewed

by

Eisenreich

et

al.

-RRB-

The

pathway

exists

of

seven

subsequent

enzymatic

steps

-LRB-

Fig.

-RRB-

The

first

reaction

of

the

MEP

pathway

is

catalyzed

by

a

transketolase

-LRB-

DXS

-RRB-

and

concerns

the

condensation

of

pyruvate

-LRB-

IV

-RRB-

and

-LRB-

V

-RRB-

to

-LRB-

VI

-RRB-

-LRB-

Sprenger

et

al.

Lois

et

al.

-RRB-

is

a

branching

intermediate

in

the

biosynthesis

of

isoprenoids

thiamine

-LRB-

vitamin

-RRB-

-LRB-

White

David

et

al.

-RRB-

and

pyridoxol

-LRB-

vitamin

-RRB-

respectively

-LRB-

Hill

et

al.

-RRB-

The

second

step

is

catalyzed

by

the

IspC

protein

initiating

a

rearrangement

in

the

carbon

skeleton

followed

by

a

reduction

in

the

keto

function

of

-LRB-

VI

-RRB-

and

finally

delivering

-LRB-

Takahashi

et

al.

-RRB-

Five

other

subsequently

acting

enzymes

are

needed

for

the

synthesis

of

IDP

including

a

phosphocytidyl

transferase

-LRB-

IspD

-RRB-

a

kinase

-LRB-

IspE

-RRB-

a

cyclophosphate

synthase

-LRB-

IspF

-RRB-

and

two

reductases

-LRB-

IspG

IspH

-RRB-

-LRB-

Fig.

-RRB-

Fig.

phosphate

pathway

of

isoprenoids

-LSB-

Dxs

-LRB-

-RRB-

deoxy-d-xylulose

synthase

IspC

-LRB-

-RRB-

synthase

IspD

-LRB-

-RRB-

4-diphosphocytidyl-2C-methyl-d-erythritol

synthase

IspE

-LRB-

-RRB-

kinase

IspF

-LRB-

-RRB-

synthase

IspG

-LRB-

-RRB-

IspH

-LRB-

-RRB-

reductase

Idi

-LRB-

-RRB-

isopentyl

diphosphate

isomerase

IspA

-LRB-

-RRB-

farnesyl

diphosphate

synthase

-RSB-

The

putative

genes

are

mentioned

in

italics

for

every

biosynthetic

step

including

the

essential

gene

YpgA

which

is

indicated

with

an

Numbering

of

compounds

and

biosynthetic

steps

refers

to

the

text

Bacteria

use

the

precursors

IDP

and

DMADP

for

the

synthesis

of

several

compounds

including

the

side

chains

of

ubiquinone

or

menaquinone

Next

to

that

several

bacterial

species

use

these

precursors

to

synthesize

isoprene

that

is

emitted

to

their

environment

-LRB-

Kuzma

et

al.

-RRB-

B.

subtilis

emits

isoprene

in

high

levels

compared

to

other

bacterial

species

as

has

been

described

for

B.

subtilis

B.

subtilis

and

B.

subtilis

-LRB-

Kuzma

et

al.

-RRB-

Using

a

bioreactor

system

the

emission

of

isoprene

by

B.

subtilis

was

found

to

occur

in

three

phases

during

the

growth

curve

-LRB-

Wagner

et

al.

-RRB-

The

phases

correspond

with

respectively

glucose

metabolism

and

secretion

of

acetoin

catabolism

of

acetoin

and

the

early

stage

of

sporulation

The

general

laboratory

strain

B.

subtilis

showed

another

pattern

of

isoprene

emission

lacking

phases

and

-LRB-

Fall

and

Copley

-RRB-

The

function

of

the

emitted

isoprene

has

been

postulated

as

being

a

signal

molecule

in

the

natural

environment

of

the

microorganism

Another

possible

explanation

for

the

emitted

isoprene

is

the

efflux

as

an

overflow

metabolite

in

the

bacterial

pathway

to

isoprenoid

structures

-LRB-

Fall

and

Copley

-RRB-

The

uptake

of

isoprene

by

microorganisms

present

in

soil

samples

has

been

described

as

a

sink

of

atmospheric

isoprene

-LRB-

Cleveland

and

Yavitt

-RRB-

However

there

is

no

full

evidence

supporting

one

of

the

hypotheses

mentioned

Isoprene

originates

from

DMADP

The

conversion

of

DMADP

in

isoprene

is

known

to

be

an

enzymatic

process

in

the

poplar

tree

from

which

a

gene

has

been

isolated

and

characterized

-LRB-

Miller

et

al.

Schnitzler

et

al.

-RRB-

Attempts

to

prove

the

enzymatic

conversion

to

isoprene

in

B.

subtilis

suggested

the

involvement

of

an

enzymatic

step

Enzymatic

activity

has

been

partially

purified

but

the

enzyme

turned

out

to

be

very

labile

-LRB-

Sivy

et

al.

-RRB-

Until

now

the

encoding

gene

could

not

be

identified

A

search

for

a

homologue

protein

of

the

isoprene

synthase

from

poplar

tree

in

the

genome

of

the

B.

subtilis

did

not

yield

a

candidate

Based

on

homology

with

known

genes

mostly

from

E.

coli

candidate

genes

for

the

other

enzymatic

steps

in

the

MEP

pathway

in

B.

subtilis

have

been

suggested

Identification

of

the

genes

involved

in

this

pathway

should

shed

some

light

on

the

function

of

isoprene

synthesis

in

Bacillus

physiology

Due

to

the

essentiality

of

the

products

downstream

of

the

isoprenoid

biosynthesis

like

menaquinone

it

is

not

remarkable

that

the

candidate

genes

except

for

the

isomerase

have

been

shown

to

be

essential

genes

for

the

survival

of

the

bacterial

cell

-LRB-

Kobayashi

et

al.

-RRB-

In

the

construction

of

the

BFAN

collection

these

knock-out

strains

were

not

viable

and

therefore

conditional

mutants

have

been

made

using

a

The

expression

in

these

strains

can

be

regulated

by

the

isopropyl-beta-d-thiogalactopyranoside

-LRB-

IPTG

-RRB-

inducible

Pspac

promoter

-LRB-

Yansura

and

Henner

Vagner

et

al.

-RRB-

Using

this

mutant

system

expression

levels

can

be

regulated

to

a

minimum

for

survival

by

decreasing

the

amounts

of

IPTG

In

that

way

the

function

of

the

genes

can

be

investigated

Identification

of

the

involvement

of

certain

genes

in

the

isoprenoid

biosynthetic

pathway

may

create

possibilities

to

redirect

the

metabolic

flux

toward

IDP

The

possibilities

of

pathway

engineering

have

shown

their

strength

already

in

other

microorganisms

like

E.

coli

and

Saccharomyces

cerevisiae

The

metabolic

flux

was

dependent

mainly

on

the

deoxyxylulose

synthase

-LRB-

DXS

-RRB-

the

isopentenyl

diphosphate

isomerase

-LRB-

Idi

-RRB-

and

the

prenyltransferase

delivering

farnesyl

diphosphate

-LRB-

IspA

-RRB-

-LRB-

Harker

and

Bramley

Kim

and

Keasling

Kajiwara

et

al.

Martin

et

al.

-RRB-

By

up-regulation

of

the

MEP

pathway

B.

subtilis

can

be

developed

as

an

interesting

host

organism

for

the

production

of

complex

terpenoid

compounds

such

as

the

valuable

drugs

artemisinin

and

paclitaxel

In

the

present

study

we

describe

the

functional

analysis

of

genes

in

B.

subtilis

putatively

encoding

enzymes

involved

in

the

biosynthesis

of

the

terpenoid

precursor

isopentenyl

diphosphate

We

used

quantitative

isoprene

emission

of

different

well

controllable

knock-out

Bacillus

strains

in

their

environment

to

determine

the

function

of

the

tested

genes

Materials

and

methods

Bacterial

strains

and

media

Bacterial

strains

used

are

listed

in

Table

B.

subtilis

strains

and

were

obtained

from

the

American

Type

Culture

Collection

-LRB-

ATCC

-RRB-

-LRB-

Rockville

USA

-RRB-

B.

subtilis

mutant

strains

were

obtained

through

a

chromosomal

integration

of

derived

plasmids

-LRB-

Vagner

et

al.

-RRB-

Experiments

were

performed

using

Luria-Bertani

-LRB-

LB

-RRB-

medium

containing

bacto-tryptone

yeast

extract

and

NaCl

-LRB-

all

purchased

from

Duchefa

Zwijndrecht

the

Netherlands

-RRB-

If

required

medium

was

supplemented

with

erythromycin

-LRB-

μg/ml

Duchefa

the

Netherlands

-RRB-

or

the

specified

concentration

of

isopropyl-β-d-thiogalactopyranoside

-LRB-

IPTG

Duchefa

the

Netherlands

-RRB-

For

growing

on

plates

the

medium

was

solidified

with

agar

Table

genes

for

the

MEP

pathway

in

the

genome

of

B.

subtilis

and

the

percentage

of

identities

with

known

proteins

from

E.

coli

the

essentiality

of

each

gene

the

corresponding

BFAN

mutant

strain

-LRB-

explained

in

detail

in

the

text

with

numbering

of

the

respective

enzymatic

steps

-RRB-

EnzymeE

coliB

subtilisEssential

geneMutant

strainDxs

-LRB-

-RRB-

dxsdxs/yqiE

-LRB-

-RRB-

IyqiEIspC/Dxr

-LRB-

-RRB-

dxryluB

-LRB-

-RRB-

IyluBIspD

-LRB-

-RRB-

ygbPyacM

-LRB-

-RRB-

IyacMIspE

-LRB-

-RRB-

ychByabH

-LRB-

-RRB-

IyabHIspF

-LRB-

-RRB-

ygbByacN

-LRB-

-RRB-

IyacNIspG

-LRB-

-RRB-

gcpEyqfY

-LRB-

-RRB-

IyqfYIspH

-LRB-

-RRB-

lytByqfP

-LRB-

-RRB-

IyqfPIdi

Iidi

Idi

II

-LRB-

-RRB-

ypgA

-LRB-

-RRB-

a

-LRB-

-RRB-

ispAyqiD

-LRB-

-RRB-

IyqiDaHomologue

of

Streptomyces

protein

-LRB-

Kaneda

et

al.

-RRB-

Detection

of

bacterial

isoprene

emission

A

single

colony

of

the

different

B.

subtilis

strains

was

transferred

from

a

plate

to

ml

Luria-Bertani

-LRB-

LB

-RRB-

medium

-LRB-

if

required

supplemented

with

μg/ml

erythromycin

and

μM

IPTG

-RRB-

and

grown

over

night

-LRB-

°C

rpm

-RRB-

Before

the

inoculation

-LRB-

-RRB-

of

fresh

LB

medium

containing

different

specified

concentrations

of

IPTG

cells

were

gently

washed

three

times

with

fresh

LB-medium

without

IPTG

by

resuspending

and

centrifugating

Isoprene

accumulation

was

measured

on-line

by

sampling

every

min

during

a

period

of

nine

hours

ml

of

the

air

above

ml

bacterial

culture

growing

-LRB-

°C

rpm

-RRB-

in

a

ml

Erlenmeyer

air

tight

flask

-LRB-

CBN

the

Netherlands

-RRB-

and

transferring

into

a

gas-chromatography

system

suitable

for

the

sensitive

detection

of

isoprene

-LRB-

Syntech

Spectras

series

Synspec

BV

the

Netherlands

-RRB-

-LRB-

Loreto

and

Delfine

-RRB-

The

air

was

pumped

through

a

Tenax

GA

trap

desorbed

at

°C

and

transferred

to

an

AT

column

under

a

flow

of

ml/min

nitrogen

-LRB-

bars

quality

-RRB-

The

temperature

program

used

was

min

at

°C

followed

by

an

increase

in

temperature

to

°C

at

min

kept

at

this

temperature

until

min

and

than

lowered

to

°C

again

The

isoprene

present

was

detected

by

photo

ionization

at

eV

The

gas

chromatograph

was

calibrated

using

the

dynamic

gas

dilution

principle

with

several

concentrations

of

gaseous

isoprene

using

liquid

isoprene

-LRB-

Sigma

USA

-RRB-

diluted

in

methanol

and

evaporated

with

a

gas

dilutor

-LRB-

MCZ

Umwelttechnik

Germany

-RRB-

During

the

isoprene

detection

the

growth

of

the

bacterial

culture

was

determined

by

measuring

the

optical

density

at

nm

-LRB-

nm

-RRB-

every

hour

Results

Isoprene

emission

in

wild-type

Bacillus

strains

The

emission

of

isoprene

from

B.

subtilis

wild

type

strains

and

was

investigated

For

all

four

strains

isoprene

accumulated

in

the

logarithmic

phase

of

growth

leading

to

a

high

increase

in

isoprene

in

the

flask

The

absolute

maximum

of

wild

type

strains

varied

between

and

Starting

from

the

late

logarithmic

phase

to

the

beginning

of

the

stationary

phase

the

amount

of

isoprene

in

the

flask

decreased

slowly

Figure

shows

the

accumulation

of

isoprene

during

the

growth

of

the

wild

type

strains

and

B.

subtilis

reached

higher

levels

of

isoprene

but

this

is

probably

not

caused

by

a

higher

production

rate

rather

than

the

amount

of

cells

present

as

reflected

by

the

optical

density

of

these

strains

Corrected

for

the

amount

of

cells

determined

as

the

optical

density

at

nm

B.

subtilis

accumulated

at

maximum

μg

nm

compared

to

μg

nm

for

B.

subtilis

The

other

two

wild

type

strains

tested

and

showed

a

similar

accumulation

of

isoprene

as

the

strains

described

before

Corrected

for

the

amount

of

cells

produced

μg

nm

and

produced

μg

nm

isoprene

at

maximum

accumulation

-LRB-

Table

-RRB-

Fig.

emitted

-LRB-

solid

lines

-RRB-

by

wild

type

strains

B.

subtilis

-LRB-

-RRB-

and

B.

subtilis

-LRB-

-RRB-

during

growth

-LRB-

dashed

lines

-RRB-

Zero

time

-LRB-

t

-RRB-

indicates

the

transition

point

between

the

exponential

and

the

post-exponential

growth

phasesTable

concentration

of

isoprene

accumulated

after

emission

by

the

different

wild

type

Bacillus

subtilis

strains

corrected

for

the

amount

of

cells

-LRB-

nm

-RRB-

Bacillus

subtilis

strainMaximal

isoprene

accumulation

-LRB-

nm

-RRB-

Mutant

strains

To

find

the

optimal

conditions

in

which

expression

of

the

genes

can

be

limited

without

killing

the

cells

each

conditional

mutant

strain

was

subjected

to

IPTG

depletion

by

growing

the

strains

on

agar

plates

containing

different

concentrations

of

IPTG

varying

from

to

mM

Decreasing

the

concentration

of

IPTG

caused

growth

inhibition

after

overnight

incubation

resulting

in

smaller

and

finally

no

colonies

-LRB-

on

plates

without

IPTG

-RRB-

at

°C

Table

shows

the

minimal

concentration

of

IPTG

at

which

still

some

growth

was

observed

for

the

different

conditional

mutant

strains

This

concentration

of

IPTG

was

used

in

our

experiments

and

defined

as

the

condition

causing

limited

induction

Most

mutant

strains

indeed

showed

growth

inhibition

at

lower

concentration

of

IPTG

The

mutant

the

mutant

for

the

putative

gene

encoding

a

homologue

of

the

IspG

protein

-LRB-

step

-RRB-

did

not

show

any

dependency

on

IPTG

on

plates

-LRB-

varying

IPTG

concentrations

from

to

mM

-RRB-

Plates

without

IPTG

incubated

overnight

resulted

in

the

growth

of

normal

colonies

and

the

liquid

cultures

obtained

growth

curves

comparable

with

wild

type

B.

subtilis

independent

of

the

concentration

of

IPTG

For

some

of

the

other

mutant

strains

there

was

some

growth

of

the

bacterial

cultures

without

IPTG

as

well

although

severely

impaired

Table

amount

of

isoprene

accumulated

after

emission

by

the

different

mutant

Bacillus

strains

corrected

for

the

amount

of

cells

-LRB-

nm

-RRB-

Bacillus

strainLimited

expression

-LRB-

μM

IPTG

-RRB-

Maximal

isoprene

accumulation

-LRB-

nm

-RRB-

Relative

decreaseLimited

inductionFull

inductionFull

-LRB-

-RRB-

IyluB

-LRB-

-RRB-

IyacM

-LRB-

-RRB-

IyabH

-LRB-

-RRB-

IyacN

-LRB-

-RRB-

IyqfY

-LRB-

-RRB-

n.d.

-LRB-

-RRB-

ypgA

-LRB-

-RRB-

-LRB-

-RRB-

The

relative

decrease

is

calculated

as

the

maximal

amount

of

isoprene

at

full

induction

devided

by

the

maximal

amount

of

isoprene

at

limited

induction

-LRB-

dashes

indicate

that

measurement

did

not

apply

here

explained

in

detail

in

the

text

with

numbering

of

the

respective

enzymatic

steps

-RRB-

n.d.

not

determined

For

all

conditional

knock

out

strains

isoprene

emission

was

measured

at

the

concentration

of

IPTG

with

limited

expression

in

comparison

to

mM

-LRB-

full

induction

-RRB-

and

no

IPTG

-LRB-

full

repression

-RRB-

Figure

represents

the

data

for

strain

the

conditional

mutant

strain

for

the

first

step

in

the

biosynthetic

pathway

Full

induction

of

the

dxs

gene

resulted

in

a

normal

growth

curve

of

the

bacterial

culture

and

in

an

isoprene

accumulation

profile

comparable

to

the

profile

of

the

wild

type

stain

B.

subtilis

Depletion

of

IPTG

inhibited

cell

growth

The

maximal

concentration

of

isoprene

accumulated

in

the

flask

and

corrected

for

the

amount

of

cells

was

fold

lower

for

the

culture

supplemented

with

μM

IPTG

-LRB-

μg

nm

-RRB-

than

for

the

culture

with

mM

IPTG

-LRB-

μg

nm

-RRB-

Fig.

emitted

-LRB-

solid

lines

-RRB-

by

the

B.

subtilis

mutant

during

growth

-LRB-

dashed

lines

-RRB-

in

the

presence

of

mM

IPTG

-LRB-

-RRB-

μM

IPTG

-LRB-

-RRB-

and

no

IPTG

-LRB-

-RRB-

Zero

time

-LRB-

t

-RRB-

indicates

the

transition

point

between

the

exponential

and

the

post-exponential

growth

phases

Table

shows

the

maximum

concentration

of

isoprene

accumulated

in

the

flask

calculated

on

the

amount

of

cells

present

at

that

time

point

for

all

tested

mutant

strains

Mutant

strains

for

putative

genes

yluB

yacM

yabH

yacN

and

yqfP

encoding

enzymes

for

respectively

steps

and

showed

significant

differences

in

isoprene

emission

varying

from

a

fold

decrease

at

limited

expression

levels

in

comparison

to

full

induction

The

knock-out

strain

of

ypgA

putatively

encoding

an

isopentenyl

diphosphate

isomerase

-LRB-

step

-RRB-

is

the

only

knock-out

of

a

nonessential

gene

The

mutant

strain

showed

a

normal

growth

curve

and

did

not

show

a

significant

difference

in

the

emission

of

isoprene

in

comparison

to

the

wild

type

strain

B.

subtilis

We

also

tested

the

function

of

yqiD

encoding

a

homologue

for

farnesyl

diphosphate

synthase

-LRB-

FDP

-RRB-

synthase

IspA

-LRB-

step

-RRB-

of

E.

coli

Depletion

of

IPTG

to

μM

did

not

result

in

a

significant

change

in

the

isoprene

emission

by

the

mutant

strain

while

it

did

show

to

be

dependent

on

IPTG

for

growth

Discussion

To

identify

genes

involved

in

the

biosynthesis

of

the

highly

volatile

compound

isoprene

an

efficient

online

detection

system

for

this

terpenoid

-LRB-

precursor

-RRB-

in

the

air

above

bacterial

cultures

was

developed

Since

the

B.

subtilis

strains

and

were

known

to

emit

isoprene

-LRB-

Kuzma

et

al.

-RRB-

we

used

these

strains

to

set

up

the

detection

system

for

B.

subtilis

for

which

the

genome

has

been

sequenced

-LRB-

Kunst

et

al.

-RRB-

and

functional

knock

outs

have

been

made

All

four

strains

emitted

isoprene

in

our

experiment

The

differences

in

the

amounts

of

isoprene

emitted

corresponded

with

the

levels

as

described

before

where

B.

subtilis

and

B.

subtilis

emitted

higher

amounts

than

B.

subtilis

-LRB-

Kuzma

et

al.

-RRB-

Since

B.

subtilis

showed

emission

of

isoprene

the

use

of

the

experimental

set

up

to

investigate

the

isoprene

synthesis

by

the

mutant

strains

was

justified

From

the

profile

of

isoprene

emission

with

a

maximum

at

the

transition

of

the

growth

curve

from

the

logarithmic

phase

to

the

stationary

phase

it

was

concluded

that

from

this

time

point

on

maximum

isoprene

concentrations

had

been

reached

and

that

amounts

sampled

from

the

culture

every

min

exceeded

the

production

of

new

isoprene

We

could

not

detect

isoprene

production

in

three

phases

using

standard

incubation

in

a

shaking

flask

as

has

been

described

for

B.

subtilis

grown

in

a

bioreactor

The

conditional

knock

out

strain

of

the

yqiE

encoding

DXS

the

transketolase

responsible

for

the

first

step

in

the

biosynthesis

of

isoprenoids

appeared

to

be

highly

dependent

on

IPTG

The

dramatic

decrease

in

isoprene

production

at

low

concentration

of

IPTG

supported

the

role

of

this

gene

in

the

biosynthetic

pathway

of

isoprene

yqiE

has

been

identified

as

dxs

encoding

in

B.

subtilis

before

-LRB-

Harker

and

Bramley

Hecht

et

al.

-RRB-

However

the

confirming

results

for

this

mutant

strain

proved

that

the

experimental

set

up

of

our

work

was

valid

to

investigate

the

function

of

the

other

putative

genes

Apparently

the

isoprene

emission

is

severely

decreased

when

genes

in

the

pathway

are

expressed

at

lower

level

than

in

the

wild

type

strain

Growth

at

the

end

of

the

incubation

after

several

hours

as

shown

for

many

of

the

liquid

cultures

under

IPTG

depletion

or

even

without

suppletion

of

IPTG

may

be

explained

by

the

occurrence

of

reversions

in

the

mutant

strain

-LRB-

Zanen

et

al.

-RRB-

Another

explanation

could

be

the

presence

of

a

not

fully

repressed

promoter

in

the

complementation

strain

It

has

been

reported

that

in

the

used

vector

the

Pspac

promoter

can

give

some

low

expression

even

in

the

absence

of

IPTG

-LRB-

Vagner

et

al.

Petit

et

al.

-RRB-

For

genes

showing

low

levels

of

expression

in

the

wild

type

strain

it

is

easy

to

get

a

pseudo

wild

type

level

of

expression

in

the

mutant

strain

This

may

be

in

particular

the

case

for

highly

efficient

enzymes

in

the

biosynthetic

pathway

For

the

putative

genes

yluB

yacM

yabH

yacN

and

yqfP

encoding

homologues

of

the

enzymatic

steps

and

the

isoprene

accumulation

of

the

conditional

knock

outs

by

depletion

of

IPTG

clearly

proved

the

involvement

of

these

genes

in

the

biosynthetic

pathway

isoprene

B.

subtilis

-LRB-

step

-RRB-

did

not

depend

on

IPTG

and

therefore

nothing

can

be

concluded

from

the

data

obtained

with

this

mutant

strain

other

than

that

this

enzyme

may

be

highly

efficient

leading

to

complementation

already

obtained

at

the

low

expression

levels

caused

by

leaking

activity

of

the

Pspac

promoter

-LRB-

Zanen

et

al.

-RRB-

The

nonessential

gene

ypgA

encodes

a

homologue

protein

for

an

isopentenyl

diphosphate

isomerase

-LRB-

Idi

-RRB-

-LRB-

step

-RRB-

The

B.

subtilis

gene

ypgA

encodes

for

a

so-called

isopentenyl

diphosphate

isomerase

type

II

protein

-LRB-

Takagi

et

al.

-RRB-

while

the

genome

of

E.

coli

harbours

a

gene

for

a

different

Idi

type

I

enzyme

-LRB-

Hahn

et

al.

-RRB-

Interestingly

all

archaea

contain

the

Idi

type

II

and

all

eukaryotes

contain

Idi

type

I

In

genomes

of

eubacteria

both

are

found

but

there

are

also

genomes

without

any

of

the

described

genes

-LRB-

Boucher

and

Doolittle

-RRB-

In

contrast

to

all

other

genes

investigated

in

this

study

the

gene

ypgA

encoding

for

the

IdiII

was

shown

to

be

a

nonessential

gene

in

B.

subtilis

-LRB-

Takagi

et

al.

-RRB-

We

also

observed

that

the

knock-out

mutant

of

the

bacterial

strain

is

viable

and

produces

isoprene

The

idiI

gene

of

E.

coli

has

also

been

shown

to

be

nonessential

Deletion

mutants

of

this

gene

were

viable

on

minimal

medium

-LRB-

Hahn

et

al.

-RRB-

Our

findings

that

the

isoprene

emission

is

not

influenced

by

the

knock

out

of

the

ypgA

gene

can

be

explained

by

the

enzymatic

mechanism

of

the

IspH

protein

step

before

the

isomerase

IDP

and

DMADP

can

be

synthesized

independently

by

the

catalytic

action

of

IspH

A

hypothetical

mechanism

for

this

reaction

has

been

described

-LRB-

Eisenreich

et

al.

-RRB-

This

supports

the

nonessential

character

of

the

gene

The

presence

of

isoprene

proves

that

the

isomer

DMADP

is

still

synthesized

in

the

mutant

and

that

the

cells

do

not

depend

on

the

isomerase

for

the

production

of

DMADP

from

IDP

only

The

isomerase

functions

in

the

balance

of

IDP

and

DMADP

as

a

salvage

protein

-LRB-

Eisenreich

et

al.

-RRB-

The

first

enzymatic

biosynthetic

step

downstream

the

formation

of

the

precursors

IDP

and

DMADP

is

catalyzed

by

IspA

farnesyl

diphosphate

synthase

-LRB-

step

-RRB-

Therefore

the

putative

gene

is

not

involved

in

the

biosynthesis

of

isoprene

itself

The

observation

that

isoprene

is

still

emitted

while

the

expression

of

the

gene

was

depleted

is

easily

explained

by

its

function

in

the

pathway

downstream

of

IDP

biosynthesis

Although

some

accumulation

of

the

precursors

IDP

and

DMADP

could

be

expected

by

blocking

yqiD

no

significant

differences

in

the

flux

toward

the

isoprene

emission

were

detected

Apparently

the

efflux

of

isoprene

is

not

influenced

by

the

changes

in

FDP

synthesis

downstream

in

the

isoprenoid

pathway

This

can

be

regarded

as

a

contradiction

to

the

hypothesis

that

isoprene

emission

functions

as

an

efflux

of

an

overflow

metabolite

In

this

study

the

involvement

of

several

B.

subtilis

genes

in

the

MEP

pathway

to

isoprene

was

established

by

studying

isoprene

emission

of

mutants

Five

genes

essential

for

viability

yluB

yacM

yabH

yacN

and

yqfP

were

shown

to

be

essential

for

the

isoprene

production

as

well

Where

these

genes

were

at

first

instance

depicted

based

on

homology

of

the

encoding

proteins

the

results

of

this

study

proved

the

functional

involvement

of

the

genes

in

the

biosynthesis

of

isoprenoids

on

a

biochemical

level

A

knockout

of

the

sixth

candidate

gene

yqfY

did

not

yield

a

reduction

in

isoprene

emission

nor

a

growth

retardation

which

might

be

explained

by

a

not

fully

repressed

Pspac

promoter

present

in

vector

Knock-outs

of

the

two

other

genes

investigated

in

this

study

showing

no

growth

retardation

as

well

as

no

reduction

in

isoprene

emission

behaved

in

accordance

with

expectation

Although

it

remains

unknown

why

isoprene

is

emitted

by

bacteria

the

knowledge

about

the

pathway

to

isoprene

can

be

used

for

further

investigations

towards

a

better

understanding

of

the

metabolic

flux

to

IDP

and

its

physiological

function

Next

to

that

the

IDP

pool

can

be

optimized

by

metabolic

pathway

engineering

creating

a

B.

subtilis

strain

as

an

efficient

Gram-positive

host

for

the

heterologous

production

of

terpenoids

like

the

valuable

plant-derived

pharmaceuticals

artemisinin

and

paclitaxel

The

potential

of

using

heterologous

production

organisms

for

the

supply

of

terpenoids

is

already

under

investigation

for

E.

coli

and

for

several

yeast

strains

-LRB-

Martin

et

al.

Lindahl

et

al.

Ro

et

al.

Dejong

et

al.

-RRB-

For

the

purpose

of

metabolic

pathway

engineering

it

is

interesting

to

search

for

a

gene

encoding

isoprene

synthase

in

B.

subtilis

as

well

Evidence

for

an

enzymatic

bioconversion

and

the

identification

of

this

specific

enzyme

could

give

the

possibility

to

block

the

efflux

of

isoprene

thereby

increasing

the

amount

of

isoprenoid

available

for

the

synthesis

of

terpenoids

O O O O O O O O O B O B I O O O O O B I O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O

bacillus subtilis
isoprene
mep pathway
terpenoid

methylerythritol phosphate pathway
gas chromatography