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Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris

Inokuma, Kentaro Miyamoto, Shunya Morinaga, Kohei Kobayashi, Yuma Kumokita, Ryota Bamba, Takahiro Ito, Yoichiro Kondo, Akihiko Hasunuma, Tomohisa 神戸大学

2023.04

概要

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.

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Table 1 Characteristics of yeast strains and plasmids used in this study

Yeast strains and

Relevant genotype

Source

CBS7435

Wild type

ATCC

Pp-UbiC

CBS7435/pIPrUbiC [GAPP–PrUbiC–AOX1T, G418R]

This study

Pp-BG-

CBS7435/pIBG-PpGMSed1 [GAPP–MFα(L42S)SP–A. aculeatus

This study

GMSed1

BGL1–SED1A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG5 [GAPP–MFα(L42S)SP–A. aculeatus

GMG5

BGL1–GCW5A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG12 [GAPP–MFα(L42S)SP–A. aculeatus

GMG12

BGL1–GCW12A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG14 [GAPP–MFα(L42S)SP–A. aculeatus

GMG14

BGL1–GCW14A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG19 [GAPP–MFα(L42S)SP–A. aculeatus

GMG19

BGL1–GCW19A -AOX1T, G418R]

Pp-BG-

CBS7435/pIBG-PpGMG21 [GAPP–MFα(L42S)SP–A. aculeatus

GMG21

BGL1–GCW21A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG28 [GAPP–MFα(L42S)SP–A. aculeatus

GMG28

BGL1–GCW28A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG30 [GAPP–MFα(L42S)SP–A. aculeatus

GMG30

BGL1–GCW30A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG34 [GAPP–MFα(L42S)SP–A. aculeatus

GMG34

BGL1–GCW34A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG42 [GAPP–MFα(L42S)SP–A. aculeatus

GMG42

BGL1–GCW42A -AOX1T, G418R]

Pp-BG-

CBS7435/pIBG-PpGMG45 [GAPP–MFα(L42S)SP–A. aculeatus

GMG45

BGL1–GCW45A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG49 [GAPP–MFα(L42S)SP–A. aculeatus

GMG49

BGL1–GCW49A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG51 [GAPP–MFα(L42S)SP–A. aculeatus

GMG51

BGL1–GCW51A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpGMG61 [GAPP–MFα(L42S)SP–A. aculeatus

GMG61

BGL1–GCW61A -AOX1T, G418 ]

Pp-BG-

CBS7435/pIBG-PpSSG61 [SPI1P–SPI1SP–A. aculeatus BGL1–

SSG61

GCW61A -AOX1T, G418R]

Pp-EG-

CBS7435/pIBG-PpGMSed1 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMSed1

SED1A -AOX1T, G418 ]

plasmids

P. pastoris

This study

Pp-EG-

CBS7435/pIBG-PpGMG5 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG5

GCW5A -AOX1T, G418R]

Pp-EG-

CBS7435/pIBG-PpGMG12 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG12

GCW12A -AOX1T, G418 ]

Pp-EG-

CBS7435/pIBG-PpGMG14 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG14

GCW14A -AOX1T, G418 ]

Pp-EG-

CBS7435/pIBG-PpGMG19 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG19

GCW19A -AOX1T, G418 ]

Pp-EG-

CBS7435/pIBG-PpGMG21 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG21

GCW21A -AOX1T, G418R]

Pp-EG-

CBS7435/pIBG-PpGMG28 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG28

GCW28A -AOX1T, G418R]

Pp-EG-

CBS7435/pIBG-PpGMG30 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG30

GCW30A -AOX1T, G418 ]

Pp-EG-

CBS7435/pIBG-PpGMG34 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG34

GCW34A -AOX1T, G418 ]

Pp-EG-

CBS7435/pIBG-PpGMG42 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG42

GCW42A -AOX1T, G418 ]

Pp-EG-

CBS7435/pIBG-PpGMG45 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG45

GCW45A -AOX1T, G418R]

Pp-EG-

CBS7435/pIBG-PpGMG49 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG49

GCW49A -AOX1T, G418R]

Pp-EG-

CBS7435/pIBG-PpGMG51 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG51

GCW51A -AOX1T, G418 ]

Pp-EG-

CBS7435/pIBG-PpGMG61 [GAPP–MFα(L42S)SP–T. reesei EGII–

GMG61

GCW61A -AOX1T, G418 ]

Pp-EG-

CBS7435/pIBG-PpSSG34 [SPI1P–SPI1SP–T. reesei EGII–GCW34A

SSG34

-AOX1T, G418 ]

Pp-BEC

CBS7435/ pIBG-PpSSG61 [SPI1P–SPI1SP–A. aculeatus BGL1–

This study

GCW61A -AOX1T, G418R], pIH-EG-PpSSG34 [SPI1P–SPI1SP–T.

reesei EGII–GCW34A -AOX1T, HygR], pIZ-CBH-PpSSG34 [SPI1P–

SPI1SP–T. emersonii CBH1–GCW34A -AOX1T, ZeoR]

Pp-BEC-

CBS7435/ pIBG-PpSSG61 [SPI1P–SPI1SP–A. aculeatus BGL1–

UbiC

GCW61A -AOX1T, G418 ], pIH-EG-PpSSG34 [SPI1P–SPI1SP–T.

This study

reesei EGII–GCW34A -AOX1T, HygR], pIZ-CBH-PpSSG34 [SPI1P–

SPI1SP–T. emersonii CBH1–GCW34A -AOX1T, ZeoR], pIN-PrUbiC

[GAPP–PrUbiC–AOX1T, NATR]

Plasmids

pPGP_L42S_

G418R GAPP–MFα(L42S)SP-scFv–AOX1T

(Ito et al. 2022)

scFv

pPGPH_DO

HygR GAPP–MjDOD–AOX1T

(Ito et al. 2020)

ZeoR GAPP–EGFP–AOX1T

(Kumokita et al.

pPGPZEGFP

pPNS-NHCH

2022)

NAT GAPP–EcNMCH–AOX1T

(Kumokita et al.

2022)

pIPrUbiC

G418R GAPP–PrUbiC–AOX1T

This study

pIN-PrUbiC

NAT GAPP–PrUbiC–AOX1T

This study

pIBG-SS

HIS3 SED1P–GLUASP–A. aculeatus BGL1–SED1A–SAG1T

(Inokuma et al. 2014)

pIBG-

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–SED1A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW5A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW12A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW14A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW19A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW21A -AOX1T

This study

pIBG-

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1I–GCW28A -

This study

PpGMG28

AOX1T

pIBG-

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW30A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW34A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW42A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW45A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW49A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW51A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–A. aculeatus BGL1–GCW61A -AOX1T

This study

G418R SPI1P–SPI1SP–A. aculeatus BGL1–GCW61A -AOX1T

This study

PpGMSed1

pIBGPpGMG5

pIBGPpGMG12

pIBGPpGMG14

pIBGPpGMG19

pIBGPpGMG21

PpGMG30

pIBGPpGMG34

pIBGPpGMG42

pIBGPpGMG45

pIBGPpGMG49

pIBGPpGMG51

pIBGPpGMG61

pIBGPpSSG61

pIEG-SS

HIS3 SED1P–GLUASP–T. reesei EGII–SED1A–SAG1T

(Inokuma et al. 2014)

pIEG-

G418R GAPP–MFα(L42S)SP–T. reesei EGII–SED1A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW5A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW12A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW14A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW19A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW21A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW28A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW30A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW34A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW42A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW45A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW49A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW51A -AOX1T

This study

G418R GAPP–MFα(L42S)SP–T. reesei EGII–GCW61A -AOX1T

This study

G418R SPI1P–SPI1SP–T. reesei EGII–GCW34A -AOX1T

This study

HygR SPI1P–SPI1SP–T. reesei EGII–GCW34A -AOX1T

This study

pIU5-CBH1D

URA3 SED1P–GLUASP–T. emersonii CBH1–SED1A–SAG1T

(Liu et al. 2016)

pICBH1-

G418 SPI1P–SPI1SP–T. emersonii CBH1–GCW34A -AOX1T

This study

ZeoR SPI1P–SPI1SP–T. emersonii CBH1–GCW34A -AOX1T,

This study

PpGMSed1

pIEGPpGMG5

pIEGPpGMG12

pIEGPpGMG14

pIEGPpGMG19

pIEGPpGMG21

pIEGPpGMG28

pIEGPpGMG30

pIEGPpGMG34

pIEGPpGMG42

pIEGPpGMG45

pIEGPpGMG49

pIEGPpGMG51

pIEGPpGMG61

pIEGPpSSG34

pIH-EGPpSSG34

PpSSG34

pIZ-CBH1PpSSG34

506

A. aculeatus, Aspergillus aculeatus; T. reesei, Trichoderma reesei; T. emersonii, Talaromyces

507

emersonii; P, promoter; SP, secretion signal peptide sequence; A, anchoring region; T, terminator,

508

PrUbiC, Providencia rustigianii chorismate pyruvate-lyase; scFv, single-chain variable fragment;

509

GLUA, Rhizopus oryzae glucoamylase; MFα, S. cerevisiae alpha-factor; MjDOD, Mirabilis jalapa

510

DOPA deoxygenase; EcNMCH, Eschscholzia californica N-methylcoclaurine hydroxylase

511

512

513

514

Figure 1 Schematic pathway of 4-HBA biosynthesis in P. pastoris. Green and red arrows represent

515

reactions by heterologous enzymes. The dashed arrows indicate multiple enzymatic steps. G6P,

516

glucose-6-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-D-

517

arabinoheptulosonate 7-phosphate.

518

519

520

521

Figure 2 Time course of 4-HBA production in YPD medium by P. pastoris strains.

522

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Figure 3 Comparison of cell-surface activity of (a) BGL and (b) EG. The enzymes were displayed

527

using different GPI-anchoring domains in P. pastoris after cultivation in YPD medium for 48 h. The

528

relative EG activity of each strain is shown as a fold-change in EG activity relative to the average

529

level observed with strain Pp-EG-GMSed1 which uses Sed1p.

530

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Figure 4 The effect of replacing the promoter- and secretion signal sequences on cell-surface activity

535

of (a) BGL and (b) EG. *p < 0.05 for significant differences between two compared groups.

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Figure 5 PASCase activity of the BGL-, EG-, and CBH co-displaying P. pastoris strain (Pp-BEC).

540

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Figure 6 Time course of direct 4-HBA production through SSF of 10 g/L PASC by Pp-UbiC and

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Pp-BEC-UbiC strains.

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...

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Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris

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Harnessing originally robust yeast for rapid lactic acid bioproduction without detoxification and neutralization

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Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source

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参考文献

分野

大学

学位論文種類・取得年

言語

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Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris

概要

この論文で使われている画像

関連論文

Harnessing originally robust yeast for rapid lactic acid bioproduction without detoxification and neutralization

Study on Xylan Degradation System of New Isolated Thermophilic Facultatively Anaerobic Bacterium Paenibacillus sp. Strain DA-C8

Metabolic engineering for 4-aminophenylalanine production from lignocellulosic biomass by recombinant Escherichia coli

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source

Caffeic acid production from glucose using metabolically engineered Escherichia coli

参考文献