Biochemistry and physiology of xylose fermentation by yeasts

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  • 810chem15try and phy510109y 0f xy105e fermentat10n 6y yea5t5 8. Hahn-Hf19erda1,* H. Jepp550n,* K. 5k009** and 8. A. Pr10r t

    *Department 0f App11ed M1cr0610109Y, Chem1ca1 Centre, Lund 1n5t1tute 0f 7echn0109Y, Lund Un1ver51ty, Lund, 5weden

    **Pre5ent addre55: Chr15t1an Han5en5 La60rat0r1um, 869e A11~ 10-12, 2970 H6r5h01m, Denmark tDepartment 0f M1cr0610109Y and 810chem15try, Un1ver51ty 0f the 0ran9e Free 5tate, 810emf0nte1n, 50uth Afr1ca

    7he rate 0f ethan01 pr0duct10n and the ethan01 c0ncentrat10n5 atta1ned 6y the m05t pr0m151n9 xy105e-ferment1n9 yea5t5, P1ch1a 5t1p1t15, Cand1da 5hehatae, and Pachy501en tann0ph11u5, c0mpare p00r1y w1th that 0f c0mmerc1a1 ethan01fermentat10n 6y n0n-xy105e-ferment1n9 5acchar0myce5 cerev151ae u51n991uc05e-6a5ed 5u65trate5. 7he 0xY9en re4u1rement f0r eff1c1ent fermentat10n 6y the xY105e-ferment1n9 yea5t5 and the 1ack 0f 5uch a 9enera1 re4u1rement 6y 5. cerev151ae 1nd1cate5 6a51c underty1n9 d1fference51n the1r phy5101091ca1 re1at10n5 t0 0xy9en. 7he red0x 1m6a1ance 1n the 1n1t1a1 c0nver510n 0f xy105e t0 xy1u105e, 5en51t1v1ty t0 h19h c0ncentrat10n5 0f ethan01, d1fference5 1n the re5p1rat0ry pathway and 5en51t1v1ty t0 m1cr061a1 1nh161t0r5, part1cu1ar1y th05e 116erated dur1n9 pretreatment and hydr01y515 0f 119n0ce11u105e 5u65trate5, have 6een 1denuf1ed a5 maj0r fact0r511m1t1n9 ethan01fermentat10n 6y the xy105e-ferment1n9 yea5t5. Rec0m61nant 5. cerev151ae, c0nta1n1n9 funct10na1 xy105e reducta5e and xy11t01 dehydr09ena5e, 9r0w5 0n, 6ut p00rty ferment5, xy105e. 7he unfav0ra61e k1net1c pr0pert1e5 0f the5e en2yme5 and an 1nade4uate pent05e ph05phate pathway apparent1y 11m1t the a6111ty 0f the rec0m61nant yea5t t0 ferment xy105e.

    Keyw0rd5: Yea5t5; xy105e fermentat10n; ethan01 pr0duct10n; 0xY9en re1at10n5

    1ntr0duct10n

    51nce the d15c0very 0f the a6111ty 0f yea5t5 t0 ferment xy105e 1n 1981,1-3 the pathway f0r the meta60115m 0f xy105e ha5 6een exten51ve1y 1nve5t19ated. Wherea5 6acter1a were 5h0wn t0 150mer12e xy105e d1rect1y t0 xy1u105e a5 ear1y a5 1960, Ch1an9 and Kn19ht 4 f0und that the f11ament0u5 fun- 9u5 Pen1c1111um chry509enum p055e55ed en2yme5 f0r the 1n1- t1a1 5tep5 0f pent05e meta60115m that d1ffered fr0m xy105e 150mer12at10n 1n 6acter1a. Yea5t5 5uch a5 Cand1da ut1115, wh1ch are a61e t0 0x1d12e xy105e and 9r0w 0n the 5u65trate, were f0und t0 1n1t1a11y reduce xy105e t0 xy1u105e v1a xy11t01. 5 7hereafter, xy1u105e 15 ph05ph0ry1ated t0 xy1u105e-5- ph05phate. 7h15 1ntermed1ate 15 then meta60112ed 6y the pent05e ph05phate pathway (F19ure 1). Xy105e-ferment1n9 yea5t5 were f0und t0 f0110w the 0x1d0-reduct1ve r0ute v1a xy11t011n the c0nver510n 0fxy105e t0 xy1u105e. 7he 519n1f1cant

    Addre55 repr1nt re4ue5t5 t0 Pr0f. Hahn-H/19erda1 at the Department 0f App11ed M1cr0610109Y, Chem1ca1 Centre, Lund 1n5t1tute 0f 7echn0109y, Lund Un1ver51ty, P. 0.80x 124, 5-221 00 Lund, 5weden 7h15 art1c1e 15 a rev15ed and updated ver510n 0f pa9e5 242-2591n 8. Hahn- H/19erda1, J. Ha1160rn, H. Jepp550n, L. 01550n, K. 5k009, M. Wa1fr1d550n, Pent05e Fermentat10n t0 A1c0h01 1n 810c0nver510n 0f F0re5t and A9r1- cu1tura1P1antRe51due5, Ed. J. N. 5add1er, CA8 1nternat10na1, Wa111n9f0rd, UK, 1993 Rece1ved 13 5eptem6er 1993; accepted 1 Fe6ruary 1994

    d1fference 6etween hex05e and pent05e meta60115m 15 that a11 pent05e5, c0ntrary t0 hex05e5, 6 have t0 6e 5hutt1ed thr0u9h the PPP. 7he 610techn01091ca11y 1mp0rtant yea5t 5acchar0myce5 cerev151ae 15 una61e t0 ferment xy105e 6ut can ferment xy1u105e, and when the xy105e 150mera5e 15 pre5ent 1n the med1um, xy105e 15 c0nverted t0 xy1u105e wh1ch 15 then fermented t0 ethan01.7he ethan01 f0rmat10n rate 1n xy105e- ferment1n9 yea5t5 when xy105e 15 a551m11ated 15 a60ut ha1f that when 91uc05e 15 a551m11ated. 7-9 C0rre5p0nd1n9 re5u1t5 were 06ta1ned dur1n9 the fermentat10n 0f xy1u105e 6y 5ch1205acchar0myce5 p0m6e 1 and 6y 5. cerev151ae. 11 7h15 rev1ew w111 exam1ne the phy5101091ca1 a5pect5 0f xy105e fer- mentat10n 6y P1ch1a 5t1p1t15, Cand1da 5hehatae, and Pachy- 501en tann0ph11u5, a5 the5e three yea5t5 have 6een m05t exten51ve1y 5tud1ed. 0ther yea5t5 w111 6e referred t0 where appr0pr1ate.

    C0mpar150n 0f the xy105e-ferment1n9 a6111ty 0f yea5t5

    7he yea5t5 P. 5t1p1t15 and the c105e1y re1ated C. 5hehatae are the 6e5t xy105e-ferment1n9 yea5t5 de5cr16ed t0 date 12 ( 7a61e 1). 1n 9enera1, 5tra1n5 0f the5e yea5t5 pr0duce ethan01 at max1mum 5pec1f1c rate5 up t0 0.51 9 9 -1h- 1 w1th ethan01 y1e1d5 up t0 0.50 9 9-1.13C0mpared w1th P. tann0ph11u5, the5e yea5t5 ferment xy105e up t0 f1ve t1me5 fa5ter w1th a

    13 15 h19her ethan01y1e1d. - Neverthe1e55, the 5pec1f1c ethan01

    1994 8utterw0rth-He1nernann En2yme M1Cr06.7eChn01., 1994, V01. 16, N0Vem6er 933

  • Rev1ew5

    Xy105e

    Xy105e NADPH- -~NADH NADP + ~ NAD +

    NAD+~Xy11t01 NADH~

    5u9ar uptake

    NADPH NADP +

    1 6 Ph05ph091uc0nate

    61uc05e

    1 61uc05e

    - 61uc05e 6P

    C02

    Xy1u105e ~ DP+ ~-- -~7- - - - - - FruCt05e 6P R16u105e ~,

    ~5P ~. . . . .. f~" 1" 1 FruCt05e 1,6-d1ph05phate Xy1u105e 5P R1605e 5P / /

    / / / / ,2~ / /

    5ed0heptu105e 7P 1,2~.-~ -~-1r--~/ /

    ~- Erythr05e 41:)/// Fruct0.5e 6P / /

    /

    Fruct05e 6P --~ 61ycera1dehyde 3P - - -~ Pyruvate

    NAD + NADH

    Re5p1rat0ry cha1n NADH NAD +

    61ycera1dehyde 3P

    ~ NAD NADH

    C02 ~ C02

    Acety1 C0A Aceta1dehyde

    D1hydr0xyacet0ne P

    ~ NADH NAD + 61ycer01 3P

    61ycer01

    Acetate NADH NAD +

    Ethan01

    F19ure 1 Pr0p05ed pathway f0r the meta60115m 0f xy105e and 91uc05e 1n the xy105e-ferment1n9 yea5t5

    pr0duct1v1ty 0f the 6e5t 0f the xy105e-ferment1n9 yea5t515 at 1ea5t f1vef01d 10wer than that 06ta1ned w1th 5. cerev151ae when cu1t1vated 0n 91uc05e. 0xy9en ha5 6een 5h0wn t0 6e nece55ary f0r eff1c1ent ethan01 f0rmat10n fr0m xy105e 6y the xy105e-ferment1n9 yea5t5.1,16 Xy11t01 15 the pr1nc1pa1 p01y01 pr0duced dur1n9 xy105e fermentat10n 6y the5e yea5t5. H0w- ever, C. 5hehatae and P. 5t1p1t15 pr0duce c0n51dera61y 10wer am0unt5 0f xy11t01 than th05e 065erved 1n P. tann0ph11u5 fermentat10n5. 8 Un11ke P1ch1a an90ph0rae, 17 the5e yea5t5 are 1ncapa61e 0f pr0duc1n9 ethan01 fr0m xy11t01.61ycer01, ara61t01, and r161t01 are a150 accumu1ated dur1n9 xy105e fer- mentat10n 6y P. 5t1p1t15, C. 5hehatae, and P. tann0ph11u5, 6ut 1n m05t 1n5tance5 the c0ncentrat10n5 are 10wer than that 0f xy11t01. 8

    Fermentat10n 0f 5u9ar5 0ther than D-xy105e 7he a6111ty 0f xy105e-ferment1n9 yea5t5 t0 ferment 5u9ar5 c0mm0n1y f0und 1n 119n0ce11u1051c 610ma55151mp0rtant 1n the1r p0tent1a1 app11cat10n f0r fue1 pr0duct10n. C. 5hehatae

    18 19 and P. 5t1p1t15 ferment 91uc05e, mann05e, and 9a1act05e. , Ce1106105e 15 fermented 6yP. 5t1p1t15 6ut n0t 6y C. 5hehatae. Ne1ther L-ara61n05e, L-rhamn05e, n0r xy11t01 15 fermented 6y the5e yea5t5. 7M 5tra1n5 0fP. 5t1p1t15 and C. 5hehatae 9r0w 0n the a60ve-ment10ned 5u65trate5 w1th the except10n 0f C.

    5hehatae C85 2779, wh1ch cann0t ut1112e L-rhamn05e. 7he a6111ty 0f 0ther xy105e-ferment1n9 yea5t5 t0 ut1112e 0ther 5u9- ar5 ha5 6een 1e55 exten51ve1y 1nve5t19ated. P. tann0ph11u5 can 9r0w 0n 91uc05e, 9a1act05e, xy105e, and fruct05e 6ut n0t

    20 0n 5ucr05e and 1act05e. 1n add1t10n, P. 5t1p1t15 5tra1n5 C85 5773 and C85 5775 are a61e t0 ferment 1archw00d xy1an d1rect1y t0 ethan01. 21 8eechw00d xy1an 15 1nc0mp1ete1y hy- dr01y2ed, wh1ch 1nd1cate5 that the5e 5tra1n5 d0 n0t pr0duce a c0mp1ete c0mp1ement 0f en2yme5 nece55ary f0r xy1an hydr01y515.

    7ran5p0rt 0f xy105e and 91uc05e P. 5t1p1t15 NRRL Y-7124 and C85 7126 take up 60th xy105e 22,23 and 91uc05e 22,24 6y mean5 0f a pr0t0n 5ymp0rt 22 (7a61e 2). 1n P. 5t1p1t15 C85 7126, there are tw0 uptake 5y5tem5 f0r xy105e, a h19h- and a 10w-aff1n1ty 5y5tem, wherea5 there 15 0n1y 0ne uptake 5y5tem f0r 91uc05e. 22 7he 10w-aff1n1ty 5y5tem 15 5hared 6y xy105e and 91uc05e, wherea5 the h19h-aff1n1ty 5y5tem f0r xy105e 15 n0nc0mpet1t1ve1y 1nh16- 1ted 6y 91uc05e. 7he 10w-aff1n1ty 5y5tem wa5 5u6ject t0 5u6- 5trate ( > 0.2 mM) 1nh161t10n when 91uc05e 6ut n0t xy105e wa5 the 5u65trate. 1n c0ntra5t t0 P. 5t1p1t15, C. 5hehatae C85 2779 ha5 a fac111tated d1ffu510n 5y5tem 1n add1t10n t0 the pr0t0n 5ymp0rt. 25 1n n0n5tarved ce115 0f C. 5hehatae, xy105e

    934 En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er

  • 810chem15try and phy510109y 0f xy105e fermentat10n: 8. Hahn-H~9erda1 et a1.

    7a61e 1 Effect 0f 0xY9en 0n ethan01 pr0duct10n and xy11t01 f0rmat10n

    Ethan01 Car60n Ethan01 5pec1f1c Xy11t01 810ma55 50urCe y1e1d a pr0duct1v1ty y1e1d a y1e1d a

    5tra1n (9 1-1) Aerat10n (9 9-1) (9 9-1 h-1) (9 9-1) (9 9-1) Ref.

    P1ch1a 5t1p1t15 C85 6054 X 206 Aer061c 0 0 0 0.33 7 C85 6054 X 506 0xY9en 11m1ted 0.46 0.2 0 0.13 44 C85 6054 X 20 Anaer061c 0.35 0.02 0 0 7 C85 6054 6 206 Aer061c 0 0 NA 0.35 52 C85 6054 6 506 0xY9en 11m1ted 0.33 0.38 NA 0.15 52 C85 6054 6 20 Anaer061c 0.50 0.04 NA 0.04 7

    C85 7126 X 40 Aer061c 0.18 0.17 0 0.39 8 C85 7126 X 40 0xY9en 11m1ted 0.47 0.2 0.06 0.05 8 C85 7126 X 40 Anaer061c 0.4 0.02 0 0.03 8

    C85 7126 6 40 Aer061c 0.26 0.17 NA 0.23 8 C85 7126 6 40 0xY9en 11m1ted 0.38 0.28 NA 0.14 8 C85 7126 6 40 Anaer061c 0.33 0.13 NA 0.10 8

    Cand1da 5hehatae C85 2779 X 40 Aer061c 0.22 0.21 0.04 0.33 8 C85 2779 X 40 0xY9en 11m1ted 0.37 0.32 0.13 0.01 8 C85 2779 X 40 Anaer061c 0.41 0.15 0.18 0.01 8

    C85 2779 6 40 Aer061c 0.33 0.35 NA 0.21 8 C85 2779 6 40 0xY9en 11m1ted 0.42 0.51 NA 0.03 8 C85 2779 6 40 Anaer061c 0.44 0.29 NA 0.02 8

    Pachy501en tann0ph11u5 N RR L Y-2460 X 40 Aer061c 0.10 0.04 0.17 0.25 8 NRRL Y-2460 X 40 0xY9en 11m1ted 0.28 0.10 0.30 0.01 8 NRRL Y-2460 X 40 Anaer061c 0.26 0.07 0.30 0.01 8

    NRRL Y-2460 6 40 Aer061c 0.31 0.38 NA 0.14 8 NRRL Y-2460 6 40 0xY9en 11m1ted 0.43 0.49 NA 0.06 8 NRRL Y-2460 6 40 Anaer061c 0.42 0.18 NA 0.04 8

    1f86 0101 X 30 Aer061c 0 0 0 0.40 20 1f86 0101 X 25 0xY9en 11m1ted 0.12 0.61 20 1f86

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