The accD3 gene for mycolic acid biosynthesis as a target for improving fatty acid production by fatty acid-producing Corynebacterium glutamicum strains

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glutamicum. Appl Microbiol Biotechnol 75:1173-1182.

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High-efficiency extracellular release of free fatty acids from Aspergillus oryzae

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peptidoglycan structures in Corynebacterium glutamicum. Mol Microbiol

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107:312-329. https://doi.org/10.1111/mmi.13883

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Figure legends

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Fig. 1 Lipid metabolism and its proposed regulatory mechanism in C. glutamicum.

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Unlike the majority of bacteria, including E. coli and B. subtilis, coryneform bacteria,

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such as members of the genera Corynebacterium and Mycobacterium, are known to

573

possess type I fatty acid synthase, a multienzyme that performs successive cycles of

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fatty acid synthesis. In C. glutamicum, fatty acids are believed to be synthesized as

575

acyl-CoA (Kawaguchi and Okuda 1977) by two type I fatty acid synthases, FasA and

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FasB, and the CoA derivatives are used for the synthesis of membrane phospholipids

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and the outer layer component mycolic acids. The FasA enzyme produces mainly oleic

578

acid and palmitic acid with a relatively small amount of stearic acid whereas FasB

579

mostly synthesizes palmitic acid (Radmacher et el. 2005). Recently, the FasB enzyme

580

was reported to be involved in biosynthesis of the C8 compound lipoic acid in this

581

organism (Ikeda et al. 2017). The Tes enzyme is assumed to be involved in the cleavage

582

of acyl-CoA to produce free fatty acids, considering the predicted role of the enzyme in

583

fatty acid production in E. coli (Cho and Cronan 1995). The process of free fatty acid

584

excretion remains to be elucidated. Mycolic acids are suggested to be synthesized by

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condensation of α-carboxyl-acyl-CoA and acyl-AMP, which is catalyzed by polyketide

586

synthase Pks13 (Portevin et al. 2004), and the subsequent reduction of the resulting

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keto-ester function to a hydroxyl group by CmrA (Lea-Smith et al. 2007). Whereas

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acyl-AMP is assumed to be produced by acyl-CoA synthetase FadD32 from free fatty

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acid (Portevin et al. 2005), α-carboxyl-acyl-CoAs are believed to be produced by

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carboxylation of acyl-CoAs, which is catalyzed by an enzyme complex consisting of

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two acyl-CoA carboxylase β-subunits, AccD2 and AccD3; a biotinylated α-subunit

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AccBC (represented as BC); and an ε-subunit AccE (represented as E) (Gande et al.

593

2004; Gande et al. 2007). The AccBC and AccE are shared by the acetyl-CoA

25

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carboxylase complex, including the β-subunit AccD1 (Gande et al. 2007). Three genes

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responsible for the β-oxidation of fatty acids are missing from the C. glutamicum

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genome (gray arrows) (Barzantny et al. 2012). Acyl-CoA is thought to inhibit

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acetyl-CoA carboxylase, FasA, and FasB based on knowledge of related bacteria (Erfle

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1973; Morishima and Ikai 1987). The repressor protein FasR, combined with the

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effector acyl-CoA, represses the genes for accD1, fasA, and fasB (Nickel et al. 2010;

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Irzik et al. 2014). Repression and predicted inhibition are indicated by double lines.

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Arrows with solid and dotted lines represent single and multiple enzymatic processes,

602

respectively. Tes, acyl-CoA thioesterase; CmrA, short-chain dehydrogenase Cgl2472;

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FadE, acyl-CoA dehydrogenase; EchA, enoyl-CoA hydratase; FadB, hydroxyacyl-CoA

604

dehydrogenase; FadA, ketoacyl-CoA reductase; MA, mycolic acid; PM, plasma

605

membrane; OL, outer layer.

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Fig. 2 Oleic acid production of strains PAS-15, #43, and PASA-3. These three strains

608

and wild-type strain ATCC 13032 were cultivated on MM agar pieces. After cultivation

609

for 3 days, the agar pieces were transferred onto bioassay plates containing the oleic

610

acid auxotroph OLA-15 as an indicator. The plates were incubated for 1 day at 30ºC.

611

The images show one representative result from three independent experiments.

612

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Fig. 3 Growth of strains PAS-15 (open circle), PASA-3 (open square), PAS-15ΔaccD3

614

(open triangle), PASA-3/pCaccD3 (solid square), and PAS-15ΔaccD3/pCaccD3 (solid

615

triangle), and wild-type strain ATCC 13032 (solid circle). All strains were cultivated in

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30 mL of LFG1-ASL medium containing 1% glucose in a 300-mL baffled Erlenmeyer

617

flask at 30ºC with rotary shaking at 200 rpm. Values are means of three independent

26

618

cultures, which showed <5% difference from each other. Arrows indicate the time points

619

at which all of the glucose was consumed.

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Fig. 4 Fatty acid production by strains PAS-15, PASA-3, PAS-15ΔaccD3,

622

PASA-3/pCaccD3, and PAS-15ΔaccD3/pCaccD3. Culture supernatants were prepared at

623

the points indicated by the arrows in Fig. 3, and subjected to free fatty acid analysis.

624

The amounts of fatty acids were determined using three independent cultures performed

625

as described in the legend to Fig. 3. The other fatty acids not presented here were not

626

detected or only detected in trace amounts below 0.5 mg/L. Data represent mean value,

627

and the standard deviation from the mean is indicated as error bars.

628

629

Fig. 5 Susceptibilities of strains PAS-15, PASA-3, and PAS-15ΔaccD3 to antibiotics.

630

Exponential cultures of these strains grown in LFG1-ASL medium containing 1%

631

glucose were diluted in the prewarmed same medium containing 0.6% agar to OD 660 of

632

0.03. Five milliliters of each cell diluent were poured onto the 1.5% agar plate

633

containing 17 mL of the same medium in a 90-mm petri dish. After drying, E-test strips

634

of vancomycin (VA) and erythromycin (EM) were applied onto the surface of the plates.

635

The plates for strains PAS-15 and PASA-3 were incubated at 30ºC for 48 h and for

636

strain PAS-15ΔaccD3 for 96 h.

637

638

Fig. 6 Growth of strains PCC-6 and PCCA-3. Strains PCC-6 (circle) and PCCA-3

639

(square) were cultivated in 30 mL of LFG1-ASL media (solid symbols) and MM (open

640

symbols), both containing 1% glucose, in 300-mL baffled Erlenmeyer flasks at 30ºC

641

with rotary shaking at 200 rpm. Values are means of three independent cultures, which

27

642

showed <5% difference from each other. Arrows indicate the time points at which all of

643

the glucose was consumed.

644

645

Fig. 7 Fatty acid production by strains PCC-6 and PCCA-3. Culture supernatants were

646

prepared at the points indicated by the arrows in Fig. 6, and subjected to fatty acid

647

analysis. The amounts of fatty acids were determined using three independent cultures

648

performed as described in the legend to Fig. 6. The other fatty acids not presented here

649

were not detected or only detected in trace amounts below 0.5 mg/L. Data represent

650

mean value, and the standard deviation from the mean is indicated as error bars.

28

Glucose

Acetyl-CoA

BC E

AccD1

FadA

Malonyl-CoA

FasR

FasA

FasB

β-Oxidation

pathway

EchA

FadE

Repression

Inhibition

FadB

Acyl-CoA

Phospholipid

synthesis

BC E

AccD3 AccD2

Tes

α-Carboxylacyl-CoA

Pks13

CmrA

β-keto-MA

CoA

MA

Outer

layer

synthesis

Acyl-AMP

FadD32

Fatty acid

Fatty acid

Fig. 1 Takeno et al.

1 cm

Wild

PAS-15

#43

PASA-3

Fig. 2 Takeno et al.

16

Wild-type

PAS-15

Growth (OD660)

PASA-3

PAS-15ΔaccD3

12

PASA-3/pCaccD3

PAS-15ΔaccD3/pCaccD3

20

40

60

Time (h)

Fig. 3 Takeno et al.

Fatty acid (mg/L)

400

300

Total

Palmitic acid

Oleic acid

Stearic acid

200

100

Fig. 4 Takeno et al.

PAS-15

VA

EM

PASA-3

VA

EM

PAS-15ΔaccD3

VA

EM

Fig. 5 Takeno et al.

12

Growth (OD660)

10

PCC-6, LFG1-ASL

PCCA-3, LFG1-ASL

PCC-6, MM

PCCA-3, MM

10

20

30

40

Time (h)

Fig. 6 Takeno et al.

Total

Fatty acid (mg/L)

400

Palmitic acid

LFG1-ASL

Oleic acid

Stearic acid

MM

300

200

100

Fig. 7 Takeno et al.

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