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

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

**Pre5ent addre55: Chr15t1an Han5en•5 La60rat0r1um, 869e A11~ 10-12, 2970 H6r5h01m, Denmark tDepartment 0 f 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. 0 ther 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 - 1 h - 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 N A D P H - - ~ N A D H NADP + ~ NAD +

NAD+~Xy11t01 N A D H ~

5u9ar uptake

NADPH NADP +

1 6 Ph05ph091uc0nate

61uc05e

1 6 1 u c 0 5 e

- 6 1 u c 0 5 e 6P

C02

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

~ 5 P ~ . . . . .. 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

N A D + N A D H

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 0101 X 27 Anaer061c 0.07 0.85 20

a9 9 -1 c0n5umed 5u9ar X, Xy105e; 6, 91uc05e NA, N0t app11ca61e 6C0nt1nu0u5 cu1t1vat10n

and 91uc05e are tran5p0rted v1a the 5ame fac111tated d1ffu- 510n 5y5tem, wherea5 5tarvat10n re5u1t5 1n the 1nduct10n 0f tw0 5ymp0rt5, 0ne f0r xy105e and the 0ther f0r 91uc05e (7a- 61e 2). 7he tran5p0rt 5y5tem5 0f P. tann0ph11u5 and 0ther xy105e-ferment1n9 yea5t5 have n0t yet 6een 1nve5t19ated.

L0w- and h19h-aff1n1ty 5y5tem5 w1th Km va1ue5 0f 2.26 and 0.08 mM, re5pect1ve1y, were rep0rted f0r P. 5t1p1t15 C85 7126, 22 wherea5 f0r P. 5t1p1t15 NRRL Y-7124 the c0rre- 5p0nd1n9 va1ue5 were 380 and 0.9 mM 23 (7a61e 2). 7he5e d1fference5 1n k1net1c c0n5tant5 may 6e re1ated t0 5tra1n var1at10n5 and d1fference51n 9r0wth c0nd1t10n5. Neverthe- 1e55,1t 15 apparent that the tran5p0rt 5y5tem5 0fP. 5t1p1t15 are m0re eff1c1ent than th05e 0f C. 5hehatae, and that 91uc05e 15 tran5p0rted m0re eff1c1ent1y than xy105e 1n 60th yea5t5. 1n c0ntra5t, C. ut1115, wh1ch 9r0w5 0n, 6ut fa115 t0 ferment, xY105e, p055e55e5 10w-aff1n1ty (Krn va1ue 0f 67.6 mM) and h19h-aff1n1ty (Km va1ue 0f 1.9 mM) xY105e tran5p0rt 51m11ar t0P. 5//p/t/5. 80th 5y5tem5 appeared t0 6e pr0t0n 5ymp0rt5. 26

A1th0u9h 5. cerev151ae 15 una61e t0 9r0w 0n xy105e, 27,28 the 0r9an15m 15 a61e t0 ut1112e xY105e under certa1n c0nd1- t10n5. 29,3° Xy105e tran5p0rt 1n 5. cerev151ae 0ccur5 6y fac111-

tated tran5p0rt. 31 7w0 tran5p0rt 5y5tem5 f0r 91uc05e, c0n- 515t1n9 0f a 91uc05e-repre55161e, h19h-aff1n1ty 5y5tem and a c0n5t1tut1ve, 10w-aff1n1ty 5y5tem, have 6een de5cr16ed. 32-36 Xy105e 15 apparent1y taken up 6y the5e 5y5tem5, 6ut the aff1n1ty 0f the5e 5y5tem5 15 appr0x1mate1y 200-f01d 10wer than th05e f0r 91uc05e. 37-39 Furtherm0re, 5tarvat10n 1ead5 t01nact1vat10n 0fxy105e tran5p0rt, 6ut th15 can 6e prevented 6y exp05ure t0 xy105e and r1605e. 36

A num6er 0f 5tud1e5 u51n9 ]4C-xy105e, 13C-nuc1ear ma9- net1c re50nance (NMR) 5pectr05c0py, 0r the 5pec1f1c xy105e uptake rate 22,4°,41 1nd1cate that the rate 0f tran5p0rt may 11m1t the ut1112at10n 0f xy105e 1n aer061c and 0xY9en-11m1ted ce115 0fP. 5t1p1t15 and C. 5hehatae 9r0w1n9 0n xy105e. Under anaer061c c0nd1t10n5, tran5p0rt d0e5 n0t appear t0 6e 11m- 1t1n9 1n P. 5t/p/t/5 6ut rather the 1n1t1a1 tw0 5tep5 0f xy105e meta60115m. 40 1n 5. cerev151ae under aer061c c0nd1t10n5, xy- 105e tran5p0rt 15 apparent1y n0t 11m1t1n9 6ut rather the 1n- trace11u1ar meta6011c 5tep5. 36 0xY9en 15 apparent1y nece5- 5ary f0r the uptake 0f certa1n d15acchar1de51n many yea5t5, a1th0u9h 1t 15 n0t re4u1red f0r 91uc05e tran5p0rt. 42,43 1n a recent 5tudy 0n 0xY9en re4u1rement5 f0r xy105e uptake, 44

En2yme M1cr06.7echn01., 1994, v01. 16, N0vem6er 935

Rev1ew5

7a61e 2 7ran5p0rt 5y5tem51n xy105e-ferment1n9 yea5t 5tra1n5

5tra1n 5u9ar Ce11 5tatu5 Krn (mM) 7ype 0f tran5p0rt Ref.

P1ch1a 5t1p1t15 NRRL Y-7124 X n5 380 a Act1ve tran5p0rt 23

0.96 C85 7126 X n5 2.26 a Pr0t0n 5ymp0rt 22

0.086 Pr0t0n 5ymp0rt 22 C85 7126 X 5 1.89 a Pr0t0n 5ymp0rt 22

0.066 Pr0t0n 5ymp0rt 22 C85 7126 6 n5 0.73 Pr0t0n 5ymp0rt 22 C85 7126 6 5 0.26 Pr0t0n 5ymp0rt 22 C85 5773 6 5 0.02 Pr0t0n 5ymp0rt 24

Cand1da 5hehatae C85 2779 X n5 125 Fac111tated d1ffu510n 25 C85 2779 X 5 1 Pr0t0n 5ymp0rt 25 C85 2779 X 5 125 Fac111tated d1ffu510n 25

C85 2779 6 n5 2 Fac111tated d1ffu510n 25 C85 2779 6 5 0.12 Pr0t0n 5ymp0rt 25

n5, N0n5tarved ce115; 5, 5tarved ce115 aL0w aff1n1ty 6H19h aff1n1ty X, Xy105e; 6, 91uc05e

the rat10 6etween uptake under aer061c ver5u5 anaer061c c0nd1t10n5 wa5 h19her f0r aer061ca11y cu1tured ce115 than f0r anaer061ca11y cu1tured ce115,1nd1cat1n9 that 0xY9en 1nduce5 0r act1vate5 a tran5p0rt 5y5tem.

C0nver510n 0f xy105e t0 xy1u105e-5-ph05phate

Yea5t5 u5e a tw0-5tep 0x1d0-reduct10n react10n t0 c0nvert xy105e t0 xy1u105e. 7h1515 f0110wed 6y a ph05ph0ry1at10n t0 xy1u105e 5-ph05phate. 7he f1r5t en2yme 1n the pathway 15 an NAD(P)H-dependent xy105e reducta5e (XR) (a1d05e re- ducta5e EC 1.1.1.21 0f the a1d0/ket0 reducta5e fam11y) wh1ch c0nvert5 xy105e t0 xy11t01. Xy11t01 15 e1ther excreted fr0m the ce11 0r 0x1d12ed t0 xy1u105e 6y an NAD +- dependent xy11t01 dehydr09ena5e (XDH; EC 1.1.1.9) (F19- ur, 1 5 45 47 e ). , - 7he 5u65e4uent ph05ph0ry1at10n 15 cata1y2ed 6y xY1u10k1na5e (EC 2.7.1.17). 5,27,46

7he re4u1rement f0r 0xy9en dur1n9 xy105e fermentat10n 15 fre4uent1y a5cr16ed t0 the apparent red0x 1m6a1ance wh1ch deve10p5 dur1n9 anaer061c c0nd1t10n5 due t0 the d1f- ference 1n c0fact0r re4u1rement5 0f the f1r5t tw0 en2yme5. 48,49 7he red0x 1m6a1ance hyp0the515 wa5 6a5ed 0n re5u1t5 fr0m a 5tudy 0fC. ut1115, wh1ch ha5 a xy105e reduc- ta5e and a xy11t01 dehydr09ena5e re4u1r1n9 NADPH and NAD +, re5pect1ve1y. 7h15 re5u1t5 1n an 0verpr0duct10n 0f NADP + and NADH, re5pect1ve1y. Wherea5 NADP + can 6e reduced 6y cyc11n9 v1a fruct05e 6-ph05phate, C. ut1115 cann0t re0x1d12e NADH 1n the a65ence 0f 0xy9en. 7h15 1m6a1ance 15 further ref1ected 6y excret10n 0f the 1nterme- d1ate xy11t01. 1f an externa1 e1ect0n accept0r (acet01n) wa5 added, xy11t01 pr0duct10n wa5 prevented and anaer061c eth-

48 an01 pr0duct10n 0ccurred. N0 tran5hydr09ena5e (NADH + NADP + <--*NAD + + NADPH) act1v1ty ha5 50 far 6een detected 1n yea5t5, and apparent1y the 1m6a1ance cann0t 6e a11ev1ated 6y th15 r0ute• 0 1n c0ntra5t t0 C. ut1115, P. 5t1p1t15, 44,49 C. 5hehatae, 41 and P. tann0ph11u549 a11 have

XR act1v1ty w1th dua1 c0fact0r 5pec1f1c1ty, wh1ch may a11ev1- ate the red0x 1m6a1ance and perm1t anaer061c ethan01 pr0- duct10n w1th0ut xy11t01 f0rmat10n. 1n P. 5t1p1t15 C85 5773, h0wever, 1n v1tr0 k1net1c mea5urement5 0f pur1f1ed XR 1n- d1cate that XR prefer5 NADPH a5 a c0fact0r. 51

5evera19r0up5 have 5tud1ed the effect 0f 0xY9en 0n the red0x 1m6a1ance 6y varY1n9 the 0xY9enat10n when xy105e 0r 91uc05e 15 u5ed a5 car60n 50urce. M05t xy105e-ferment1n9 yea5t5 pr0duce 50me xy11t011n add1t10n t0 ethan01 (7a61e 1 ). Xy11t01 pr0duct10n 1ncrea5e5 a5 0xy9en decrea5e5, 8 and xy- 11t01 y1e1d5 a5 h19h a5 0.85 per 9ram c0n5umed xy105e have 6een 065erved 1n P. tann0ph11u5 1f68 0101. 2° 1n c0ntra5t, 50me 5tra1n5 0f P. 5t1p1t15 pr0duce ne9119161e am0unt5 0f xy11t01 under 0xy9en-11m1ted c0nd1t10n5, and the 6e5t etha- n01 y1e1d5 are f0und under the5e c0nd1t10n5 (7a61e 1). 0x- y9en may thu5 n0t 0n1y he1p a11ev1ate the red0x 1m6a1ance 1n d1fferent 5tra1n5, 6ut may a150 5erve 0ther purp05e5. Further 5upp0rt f0r th15 1nterpretat10n 15 der1ved fr0m the fact that 1n 5tud1e5 0f P. 5t1p1t15 5tra1n5 under 0xy9en-11m1ted c0nd1t10n5, 0xy9en wa5 f0und t0 6e nece55ary f0r eff1c1ent ethan01 pr0duct10n fr0m 91uc05e, wh1ch d0e5 n0t 1nv01ve XR 0r XDH. 8,52

7he 10wer xy11t01 accumu1at10n 6y P. 5t1p1t15 than P. tann0ph11u5 may a150 6e re1ated t0 an a1ternat1ve re5p1ra- t0ry pathway recent1y d15c0vered w1th 1nh161t0r 5tud1e51n P. 5t1p1t15. 53 7he re5p1rat0ry pathway5 pre5ent 1n P. 5t1p1t15 ev1dent1y re11eve the red0x 1m6a1ance that 1ead5 t0 xy11t01 accumu1at10n. Furtherm0re, a 5ec0nd P. 5t1p1t15 C85 6054 9ene, c0rre5p0nd1n9 t0 an XDH, ha5 6een c10ned. 1t rema1n5 t0 6e e1uc1dated h0w th15 5ec0nd XDH en2yme 15 1nv01ved 1n the xy11t01 meta60115m 0f P. 5t1p1t15. 54

7he 1n v1tr0 act1v1t1e5 0f XR and XDH have 6een mea- 5ured 1n ce115 at d1fferent 0xY9en 1eve15. 1n P. 5t1p1t15 C85 7126 and C85 605444,55 (F19ure 2) and C. 5hehatae A7CC 22984, 41 the rat10 0f NADH- t0 NADPH-11nked 1n v1tr0 act1v1t1e5 0f XR wa5 5h0wn t0 6e c0n5tant 1n re1at10n t0 the 0xY9en 5upp1y dur1n9 fermentat10n (7a61e 3). 7he rat10 1n

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

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

3.0

t 6 c 2.5

~ 2.0

~ 1.5

0.0 1 1 1 1 1 1 1 1 1

>411 411 182 51 31 11 8 2 <1 <1 0

07R (mm011Ah -H

A

0.6

0.5 •=

~ 0.4 -6 E 9 0.3

0 2

0.0

• NADPH

>411 411 182 51 31 13 8 2 <1 <1 0

07R (rnm011-1h "1)

8

F19ure 2 5pec1f1c act1v1ty 0f (A) xy105e reducta5e mea5ured w1th NADPH (1) and NADH (1~) and 0f (8) xy11t01 dehydr09ena5e mea- 5ured w1th NAD + 1n P1ch1a 5t1p1t15 C85 6054 cu1t1vated at d1fferent 0xy9en tran5fer rate544

C. 5hehatae A7CC 22984 41 wa5 marked1y 10wer than that 1n C. 5hehatae C85 2779. 55 1n c0ntra5t, the rat10 1n P. tan- n0ph11u5 NRRL Y-2460 1ncrea5ed w1th decrea51n9 aerat10n, 56 wh1ch c0nf1rm5 the re5u1t5 06ta1ned 1n a 5tudy 57 where d1fferent 0xy9en-11m1t1n9 c0nd1t10n5 were u5ed. 1n the 5tra1n5 0f P. 5t1p1t15 and C. 5hehatae 1nve5t19ated, the 1eve1 0f the 0xY9en 5upp1y dur1n9 fermentat10n had appar- ent1y 11tt1e effect 0n the 1n v1tr0 act1v1ty 0f XDH (F19ure 2). 41,44,55 50 far, the XDH act1v1ty 1n P. tann0ph11u5 ha5 n0t 6een 5tud1ed 1n re1at10n t0 0xY9en.

7he accumu1at10n and excret10n 0f xy11t01 6y P. 5t1p1t15 under 50me c0nd1t10n5 ha5 6een exp1a1ned 1n part 6y the unfav0ra61e therm0dynam1c e4u1116r1um c0n5tant5, wh1ch have 6een e5t1mated t0 6e 6 × 109 1 m01 - 1 f0r the c0nver- 510n 0fxy105e t0 xy11t0158 and 7 x 10 -11 m01 1-1 f0r the c0nver510n 0f xy11t01 t0 xy1u105e 59 at phy5101091ca1 pH va1- ue5. H0wever, 1t 15 n0t fru1tfu1 0n1y t0 100k at a therm0dy- nam1c e4u1116r1um c0n5tant f0r 0ne react10n 1n a meta6011c pathway, 51nce the c0ncentrat10n5 0f the reactant5 are 1n- f1uenced 6y preced1n9 and 5u65e4uent react10n5. Further- m0re, xy11t01 accumu1at10n decrea5e5 1n the pre5ence 0f 0xy9en 0r 0ther e1ectr0n accept0r5, there6y 1nd1cat1n9 that the c0ncentrat10n5 0f NAD + and NADH a150 1nf1uence the e4u1116r1a 0f the react10n5.

5urpr151n91Y, xy11t01 15 e1ther n0t fermented 0r 0n1y p00r1y fermented 6y the xy105e-ferment1n9 yea5t5, wherea5

1 218 60 9r0wth •5 n0t affected t0 the 5ame de9ree. , , 1nP. 5t1p1t15 C85 6054, the 1n v1tr0 act1v1t1e5 0f XR and pyruvate decar- 60xy1a5e 1n xY11t01-9r0wn ce115 were 20- and 10-f01d 1e55, re5pect1ve1y, than 1n xY105e-9r0wn ce115 under 0xy9en- 11m1ted c0nd1t10n5, wh11e the 1n v1tr0 act1v1ty 0f XDH re- ma1ned unchan9ed. 7 Why xy11t01 15 n0t a551m11ated under

7a61e 3 EffeCt 0f 0XY9en 0n Xy105e redUCta5e (XR) and Xy11t01 dehydr09ena5e (XDH) act1V1ty 1n Xy105e-ferment1n9 yea5t 5tra1n5

XR a

5tra1n Aerat10n NADPH NADH Rat10 NADH/NADPH XDH a NAD • Ref.

P1ch1a 5t1p1t15 C85 6054 Aer061c 1.15 0.73 0.63 0.40 7 C85 6054 0xY9en 11m1ted 1.71 1.10 0.62 0.44 44 C85 6054 Anaer061c 0.89 0.55 0.62 0.33 44

C85 7126 Aer061c 0.44 0.24 0.55 0.77 55 C85 7126 0xY9en 11m1ted 0.57 0.34 0.60 0.72 55 C85 7126 Anaer061c 0.30 0.18 0.60 0.65 55

Cand1da 5hehatae C85 2779 Aer061c 0.58 0.37 0.64 0.74 55 C85 2779 0xY9en 11m1ted 0.54 0.28 0.52 1.27 55 C85 2779 Anaer061c 0.51 0.28 0.55 1.01 55

A7CC 22984 Aer061c 0.28 0.07 0.27 0.15 40 A7CC 22984 0xY9en 11m1ted 0.31 0.08 0.28 0.17 40 A7CC 22984 Anaer061c 0.33 0.10 0.31 0.24 40

Pachy501en tann0ph11u5 NRRL Y-2460 Aer061c 0.01 0.002 0.14 nm 56 NRRL Y-2460 0xY9en 11m1ted 0.22 0.17 0.76 nm 56 N RRL Y-2460 Anaer061c 0.05 0.06 1.14 n m 56

a5pec1f1c en2yme act1v1t1e5 are expre55ed a5 mm01 9 - 1 m1n - nm, N0t mea5ured

En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er 937

Rev1ew5

anaer061c c0nd1t10n5 when the 1n0cu1a have 6een 9r0wn 0n xy105e 15 uncerta1n. 0ne p05516111ty c0u1d 6e that NADH f0rmed 1n the c0nver510n 0f xy11t01 t0 x~,1u105e cann0t 6e re0x1d12ed under anaer061c c0nd1t10n5. 48,49

A1th0u9h 5. cerev151ae can ne1ther 9r0w 0n xy105e n0r pr0duce 519n1f1cant am0unt5 0f ethan01 fr0m xy105e, a 510w d1551m11at10n may 0ccur, re5u1t1n9 1n xy11t01 accumu1at10n when 0ther m0n05acchar1de5, 5uch a5 r1605e 0r 9a1act05e, are c0meta60112ed w1th xy105e.11,29,30 1n v1tr0 act1v1t1e5 0f the5e en2yme5, re5p0n5161e f0r the c0nver510n 0f xy105e t0 xy1u105e, are tw0 0rder 0f ma9n1tude5 1e55 than 1n the xy105e-ferment1n9 yea5t5, 36,61 and they are m05t 11ke1y n0n- 5pec1f1c a1d05e reducta5e5 and 5u9ar a1c0h01 dehydr09ena- 5e5. 7he c0fact0r 5pec1f1c1ty 0f the en2yme act1v1t1e5 1n 5. cerev151ae are re5tr1cted t0 NADPH and NAD +, re5pec- t1ve1y. A 1ar9e num6er 0f 5. cerev151ae 5tra1n5 can pr0duce ethan01 fr0m xy1u105e, an 150mer1c f0rm 0f xy105e. 11,62-64 A c0mpar150n 0f xy1u105e ver5u5 91uc05e meta60115m 1n the pre5ence 0f xy105e 1nd1cated xy11t01 t0 6e pr0duced fr0m 60th xy105e and xy1u105e. 11

1n add1t10n t0 the 1n1t1a1 5tep5 0f Xy105e meta60115m, the re5U1t5 0f m01eCU1ar and 9enet1C 5tUd1e5 1nd1Cate that the th1rd 5tep 1n the xy105e meta60115m fr0m xy1U105e t0 xy1U105e 5-ph05phate Cata1y2ed 6y xy1U10k1na5e may a150 6e 11m1t1n9.65, 66

7he pent05e ph05phate pathway Xy1u105e 5-ph05phate 15 further meta60112ed v1a the pen- t05e ph05phate pathway (PPP) (F19ure 1). 1n 0vera11 meta6- 0115m, the PPP pr0v1de5 the 6105ynthet1c re4u1rement5 0f the ce11 f0r NADPH, r1605e 5-ph05phate, and erythr05e 4-ph05phate. A11 the xy105e meta60112ed 6y a ce11 mu5t 6e r0uted 6y the PPP, 1n c0ntra5t t0 91uc05e where 0.9-10% 0f th15 5u65trate ha5 6een e5t1mated t0 6e meta60112ed v1a the PPP, a1th0u9h th15 can vary. 6,67 When 5tra1n5 0fP. tann0- ph11u5 were mutated and then 5e1ected f0r h19her PPP en- 2yme act1v1t1e5, enhanced 5pec1f1c ethan01 pr0duct1v1t1e5 were apparent c0mpared w1th c0ntr01 5tra1n5, 46,65 1nd1cat- 1n9 the 1mp0rtance 0f an ade4uate1y funct10n1n9 PPP f0r eff1c1ent xy105e meta60115m.

1n 5. cerev151ae, 9reater accumu1at10n 0f 5ed0heptu105e 7-ph05phate wa5 f0und 1n xy1u105e-ferment1n9 5tra1n5 than 1n 91uc05e-ferment1n9 5tra1n5, even th0u9h the 5pec1f1c ac- t1v1ty 0f tran5a1d01a5e wa5 the 5ame. 7he5e f1nd1n95 were 1nterpreted a5 a re5u1t 0f c0mpet1t10n 6etween the PPP and 91yc01y515 f0r 91Ycera1dehyde 3-ph05phate 68 and/0r a p00r1y expre55ed PPP. 7h15 may a150 exp1a1n why the rate 0f xy1u- 105e meta60115m 15 10-f01d 10wer than that 0f 91uc05e. 11 51m11ar c0nc1u510n5 were drawn fr0m 065ervat10n5 0n xy105e a551m11at10n 6y a rec0m61nant 5tra1n 0f 5. cerev151ae c0nta1n1n9 9ene5 expre551n9 XR and XDH. 39 5ed0heptu105e 7-ph05- phate and 6-ph05ph091uc0nate 1eve15 were 519n1f1cant1y h19her dur1n9 xy105e meta60115m c0mpared 60th w1th ce115 cu1t1vated 0n 91uc05e and w1th ce115 0f P. 5t1p1t15 cu1t1vated 0n xy105e. 61ycera1dehyde 3-ph05phate 1eve15 1n 5. cerev151ae cu1t1vated 0n xy105e were 1n519n1f1cant. 7he auth0r5 c0nc1uded that the tran5a1d01a5e/tran5ket01a5e react10n5 were 1n5uff1c1ent.

Fruct05e 6-ph05phate can 6e meta60112ed e1ther 6y cy- c11n9 v1a the 0x1dat1ve p0rt10n 0f the PPP where car60n d10x1de 15 pr0duced, 0r v1a 91yc01y515. 7he the0ret1ca1 y1e1d

0f ethan01 fr0m xy105e depend5 0n whether a p0rt10n 0f fruct05e 6-ph05phate 15 cyc1ed v1a the 0x1dat1ve PPP. 1f a11 fruct05e 6-ph05phate 15 meta60112ed 6y 91yc01y515, the y1e1d w1116e 0.51 9 9-1, wherea5 cyc11n9 0f fruct05e 6-ph05phate may decrea5e the y1e1d t0 a5 10w a5 0.31 9 9-1.1f a ph05- ph0ket01a5e wa5 pre5ent, h0wever, the y1e1d c0u1d 1ncrea5e t0 0.61 9 9-1.69 511n1n9er et a1. 70 mea5ured car60n d10x1de pr0duct10n dur1n9 ethan01 f0rmat10n fr0m xy105e at d1f- ferent 9r0wth rate5 1n P. 5t1p1t15 C85 5773 and 065erved unexpected1y h19h 1eve15, pre5uma61y der1ved fr0m the PPP. When, 0n the 0ther hand, 91uc05e- and xy105e-9r0w1n9 P. 5t1p1t15 C85 6054 were c0mpared under aer061c c0nd1- t10n5 where n0 ethan01 wa5 f0rmed, car60n d10x1de pr0duc- t10n and 5u65trate c0n5umpt10n were c0mpara61e and 1n- dependent 0f the car60n 50urce. 7

F1na1 fermentat10n 5tep5

7he meta6011te5 (fruct05e 6-ph05phate and 91ycera1dehyde 3-ph05phate) fr0m the PPP are further meta60112ed 6y 91Y- c01y515 t0 pyruvate (F19ure 1). Pyruvate c0n5t1tute5 a junc- t10n at wh1ch the ce115 may f0110w a fermentat1ve pathway and/0r an 0x1dat1ve pathway. 1n the f0rmer pathway, pyru- vate decar60xy1a5e (PDC) and a1c0h01 dehydr09ena5e (ADH) c0nvert pyruvate t0 ethan01, and the NADH f0rmed 1n the 0x1dat10n 0f 91ycera1dehyde 3-ph05phate 15 re0x1d12ed. 7he 91yc01yt1c en2yme ph05ph0fruct0k1na5e 15 act1vated 6y fruct05e 2,6-615ph05phate,71 wh05e 5ynthe51515 cata1y2ed 6y tw0 6-ph05ph0fruct0-2 k1na5e 150en2yme5. 7he 1eve1 0f fruct05e 2,6-615ph05phate wa5 5h0wn t0 de- pend 0n a 91yc01yt1c meta6011te up5tream 0f the ph05ph0- 91uc05e 150mera5e react10n. 72 F0r c0mp1ete 1nduct10n 0f pyruvate k1na5e, the 5ynthe515 0f 91uc05e 6-ph05phate and fruct05e 6-ph05phate 15 needed, wherea5 the 1nduct10n 0f PDC ma1n1y depend5 0n a 519na1 1n the 10wer part 0f the 91yc01y515. 7he 1ntrace11u1ar meta6011te c0ncentrat10n 0f fruct05e-1,6 615ph05phate 1n 5. cerev151ae ce115 meta601121n9 xy1u105e 15 0ne 0rder 0f ma9n1tude 10wer than 1n ceU5 me- ta601121n9 91uc05e. 11 7he 10w 1eve1 0f fruct05e 1,6- 615ph05phate may 1nd1cate that the meta6011te c0ncentra- t10n51n the upper part 0f the 91yc01y515 are n0t h19h en0u9h t0 fu11y act1vate pyruvate k1na5e. 7h15 cau5e5 PDC n0t t0 6e act1vated, wh1ch 1n turn 1ead5 t0 a 10wer ethan01 pr0- duct10n rate 0n xy1u105e c0mpared t0 91uc05e. Further 5tud1e5 are needed t0 determ1ne t0 what extent 91yc01y515 11m1t5 the xy1u105e fermentat10n.

5tud1e5 have 6een carr1ed 0ut t0 1nve5t19ate PDC and ADH 1n re1at10n t0 xy105e meta60115m. 7he 1n v1tr0 PDC 1eve15 1n P. 5t1p1t15 C85 6054 cu1ture5 9r0wn at var10u5 0xy- 9en 1eve15 0n e1ther 91uc05e 0r xy105e fa11ed t0 5h0w d1fference5. 44,52 7he 1n v1tr0 PDC act1v1ty 1ncrea5ed w1th 1ncrea51n9 ethan01 pr0duct10n. Dur1n9 xy11t01 a551m11at10n, h0wever, the 1n v1tr0 PDC act1v1ty wa5 10-f01d 10wer than w1th 91uc05e- and xy105e-a551m11at1n9 ce115. 7

ADH ha5 6een exten51ve1y 5tud1ed 1n 5. cerev151ae, where 1t 0ccur5 a5 f0ur 1502yme5, ADH 1, 73 ADH 11, 7M ADH 111, 75 and ADH 1V. 76 ADH 1 and ADH 111 are re5p0n5161e f0r ethan01 pr0duct10n, wherea5 ADH 11 15 re5p0n5161e f0r ethan01 0x1dat10n. 7he funct10n 0f ADH 1V rema1n5 un- kn0wn. 1n C. 5hehatae A7CC 22984, the 1n v1tr0 act1v1ty 0f ADH 1ncrea5e5 w1th decrea51n9 0xy9en 5upp1y dur1n9

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

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

fermentat10n. 77,78 Apart fr0m the fact that the 1n v1tr01eve10f ADH 1510werwhen xy105e 15 u5ed than when 91uc05e 15 u5ed, 79 there w0u1d 5eem t0 6e n0 11m1tat10n 0f the c0nver510n 0f xy105e t0 ethan011n the f1na1 fermentat10n 5tep5.

Re5p1rat0ry pathway 1n the pre5ence 0f 0xy9en, pyruvate 15 0x1d12ed thr0u9h the tr1car60xy11c ac1d (7CA) cyc1e and the re5p1rat0ry cha1n (0x1dat1ve ph05ph0ry1at10n) (F19ure 1). 0x1dat1ve ph05ph0- ry1at10n 15 carr1ed 0ut 6y re5p1rat0ry a55em611e5 10cated 1n the 1nner m1t0ch0ndr1a1 mem6rane 1n eukary0te5.

7here 15 an a6501ute re4u1rement 0f 0xY9en f0r 9r0wth 0n xy105e 1n P. 5t1p1t15 5tra1n5 NRRL Y-7124 and C85 6054, a5 wa5 5h0wn 60th 1n c0nt1nu0u5 cu1ture where the 51~ec1f1c 0xY9en uptake wa5 pr0p0rt10na1 t0 the 9r0wth rate 7,79 and 6y the 1na6111ty 0f the5e yea5t5 t0 9r0w under anaer061c c0nd1t10n5. 7,8° Anaer061c 9r0wth 0n xy105e 15 n0t p055161e f0r P. 5t1p1t15 C85 6054, 7 C. 5hehatae C51R-Y492, ]5 0r P. tann0ph11~ 5tra1n5 NRRL Y-2463 and NRRL Y-2460.]5,8° 0n the 0ther hand, very p00r anaer061c 9r0wth wa5 06- 5erved f0r P. 5t1p1t15 C85 7126 and C. 5hehatae C85 2779, 55 perhap5 due t0 the d1ff1cu1ty 1n ma1nta1n1n9 true anaer061c c0nd1t10n5. 81 1n a 5tudy where 9reat care wa5 taken t0 en- 5ure anaer061c c0nd1t10n5, 510w anaer061c 9r0wth 0n 91u- c05e 1n c0ntra5t t0 xy105e wa5 f0und f0r P. 5t1p1t15 C85 6054.7 5. cerev151ae 9r0w5 we11 0n 91uc05e anaer061ca11y 1f the me- d1um 155upp1emented w1th er905ter01 and un5aturated fatty

82 83 aC1d5. • 5Upp1ementat10n W1th yea5t eXtraCt C0nta1n1n9 the5e C0mp0nent5, h0WeVer, d0e5 n0t perm1t anaer061C 9r0Wth 0n Xy105e 6y the Xy105e-ferment1n9 yea5t5. 7~15~8° 51m- 11ar1y, anaer061c 9r0wth d0e5 n0t 0ccur w1th 5. cerev151ae 0n xy1u105e. 84 51nce the pre5ence 0f 0xY9en enhance5 ethan01 pr0duct10n, 1t 15 m05t 11ke1y that ethan01 pr0duct10n 15 e1ther

70 7 9r0wth re1ated 60th 0n xy105e and 0n 91uc05e 0r depen- dent 0n funct10na1 m1t0ch0ndr1a. 85 7he re1at10n5h1p 6e- tween an 1ntact m1t0ch0ndr1a1 funct10n 85 and the recent1y d15c0vered a1ternat1ve re5p1rat0ry pathway 1n P. 5t1p1t1553 5t111 rema1n5 t0 6e 1nve5t19ated.

7he need f0r an un1mpa1red m1t0ch0ndr1a1 funct10n f0r 9r0wth 0n xy105e and 0pt1ma1xy105e fermentat10n ha5 6een pr0p05ed 6a5ed 0n the f1nd1n95 0f an exten51ve 5tudy where re5p1rat0ry 1nh161t0r5, p0ta551um cyan1de, ant1myc1n, 50- d1um a21de, and r0ten0ne were u5ed dur1n9 xy105e a551m1- 1at10n w1th P. 5t1p1t15 C85 7126, P. tann0ph11u5 NRRL Y-2460, and a150 dur1n9 91uc05e a551m11at10n w1th 5. cerev1- 51ae NRRL Y-132. 85 1n 5. cerev151ae NRRL Y-132, wh1ch can 9r0w w1th0ut m1t0ch0ndr1a1 funct10n, 86 the re5p0n5e wa5 marked1y d1fferent fr0m that 06ta1ned w1th the tw0 xy105e-ferment1n9 5tra1n5 0f P. 5t1p1t15 and C. 5hehatae, wh1ch were 51m11ar t0 each 0ther 1n th15 re5pect. 51nce 50- d1um a21de 87 and p0ta551um cyan1de 88,89 a5 weak ac1d5 can act a5 unc0up1er5 and theref0re m19ht 1nterfere w1th the pr0t0n 5ymp0rt tran5p0rt 0f xy105e 1n P. 5t1p1t15, 22 the d1ffer- ent re5p0n5e 15 n0t e4ua11y c1ear f0r the xy105e-ferment1n9 yea5t5.7he nece551ty 0f un1mpa1red m1t0ch0ndr1a1 funct10n 9a1n5 further 5upp0rt fr0m re5u1t5 06ta1ned 1n a 5tudy 0f a re5p1rat0ry-def1c1ent mutant 0f P. tann0ph11u5 NRRL Y-2460, 9° and the fa11ure t0 06ta1n 5ta61e pet1te mutant51n P. tann0ph11u5 NRRL Y-2463 and Y-24608° and 1n C. 5he- hatae A7CC 22982 and C51R Y492. 91 7he a6111ty 0fyea5t5

t0 9r0w anaer061ca11y wa5 recent1y 11nked t0 the 10cat10n 0f d1hydr00r0tate dehydr09ena5e, an NAD +-dependent en- 2yme 1nv01ved 1n pyr1d1ne 6105ynthe515. 921n 5. cerev151ae the en2yme 15 cyt05011c, wherea5 1n 5. p0m6e, a fermentat1ve, pet1te-ne9at1ve yea5t 1ncapa61e 0f anaer061c 9r0wth, d1hy- dr00r0tate dehydr09ena5e 15 10ca112ed 1n the m1t0ch0n- dr10n, where 1t5 act1v1ty 15 c0up1ed t0 an 1ntact e1ectr0n tran5p0rt cha1n. Whether the 10cat10n and funct10n 0f the d1hydr00r0tate dehydr09ena5e 0f xy105e-ferment1n9 yea5t5 6ear5 any re1at10n5h1p t0 the1r 1na6111ty t0 9r0w anaer061- caUy re4u1re5 further 1nve5t19at10n.

1n P. 5t1p1t15 C85 6054, an0ther d1fference 6etween the aer061c a551m11at10n 0f 91uc05e and pent05e5 (xy105e and xy11t01), when n0 ethan01 15 pr0duced, wa5 f0und 1n the h19her A7P/ADP rat10 06ta1ned when 91uc05e wa5 u5ed. 7 0n the 0ne hand, 91uc05e-a551m11at1n9 ce115 may pr0duce m0re A7P; 0n the 0ther hand, A7P 1n xy105e- 0r xy11t01- a551m11at1n9 ceU5 may 6e u5ed f0r purp05e5 5uch a5 ma1n- ta1n1n9 a funct10n1n9 PPP. N0 anaer061c 9r0wth fr0m pen- t05e5 0ccurred, wherea5 there wa5 a very 510w 9r0wth fr0m 91uc05e 1n the xy105e-ferment1n9 yea5t5. 51m11ar 065erva- t10n5 were made dur1n9 xy1u105e fermentat10n 1n 5. cerev151ae. 15,80,84 7he a65ence 0f anaer061c 9r0wth w1th P. 5t1p1t15 C85 5773 0n xy105e may 6e cau5ed 6y 1nade4uate am0unt5 0f A7P 6e1n9 pr0duced under anaer061c c0nd1- t10n5 t0 a110w 9r0wth. 93

Ethan01 1nh161t10n 1n xy105e-a551m11at1n9 yea5t5 Ethan01 1nh161t10n 0f ce11 9r0wth and fermentat10n var1e5 w1th c0ncentrat10n and temperature (7a61e 4). When xy- 105e 15 a551m11ated at 30°C, ce119r0wth 6yP. 5t1p1t15 C85 7126 and C. 5hehatae C51R-Y492, C51R-Y978, and C51R-Y981 15 1nh161ted at 10wer ethan01 c0ncentrat10n5 (32-34 9 1-1) than are the1r fermentat1ve act1v1t1e5 (42-45 91 - 1).~ A5 the

7a61e 4 C0ncentrat10n 0f ethan011nh161t1n9 9r0wth and fermenta- t10n 6y var10u5 xy105e-ferment1n9 yea5t5 and 5acchar0myce5 cere- v151ae

Ethan011nh161t10n

6r0wth Fermentat10n Cu1t1vat10n 5tra1n (9 1 - 1) (9 1 - 1) temperature, °C Ref.

P1ch1a 5t1p1t15 C85 7126 60.0 C85 5773 64.3 C85 7126 37.5 C85 7126 35.1

Cand1da 5hehatae C85 2779 58.0 C85 2779 30.0 C85 2779 32.3 C51R-Y978 30.5 R-5tra1n 34.1 C51R-Y981 32.1

Pachy501en tann0ph11u5 NRRL Y-2460 44.0

5acchar0myce5 cerev151ae NCYC 479 120

47.1

16-22 95 25 79 30 95 30 a 94

13-17 95 30 95

44.0 30 a 94 38.9 30 a 94 34.1 30 a 94 45.4 30 a 94

30 95

300 23 96

aFed-6atch

E n 2 y m e M1cr06. 7echn01. , 1994, v01 .16 , N 0 v e m 6 e r 9 3 9

Rev1ew5

temperature decrea5e5, ethan01 t01erance 1ncrea5e5 (7a61e 4). 7he max1mum ethan01 c0ncentrat10n 6ef0re t0ta1 1nh1- 61t10n 0f 9r0wth wa5 65 91 - 1 at 25°C 1n P. 5t1p1t15 C85 5 77379 and 58 9 1- ] at 13-17°C 1n C. 5hehatae, 95 wherea5 P. tann0- ph11u5 c0u1d 0n1y t01erate 44 9 1-1 at 30°C 95 (7a61e 4). Ethan01 1nh161t10n 15 m0re pr0n0unced 1n the xy105e- ferment1n9 5tra1n5 than 1n 5. cerev151ae. 1n a 5ake yea5t, 5. cerev151ae NCYC 479, 9r0wth wa5 n0t c0mp1ete1y 4uenched unt11 the ethan01 c0ncentrat10n wa5 a5 h19h a5 120 9 1-1 7he fermentat1ve act1v1ty d1d n0t cea5e unt11 the ethan01 c0ncentrat10n had reached 300 9 1 - 1.96

7he 1nh161t0ry effect 0f ethan01 ha5 6een 5u99e5ted t0 6e due t0 (1) an accumu1at10n 0f aceta1dehyde and acetate, (11) chan9e5 1n the ce11 mem6rane5, and (111) the effect 0f etha- n01 0n ce11u1ar 05m01a11ty.97 Further d15cu5510n w1116e c0n- f1ned t0 the f1r5t tw0 a1ternat1ve5, 51nce the 10w ethan01 pr0duct10n va1ue5 50 far detected w0u1d have ne9119161e effect5 0n 05m01a11ty.

Aceta1dehyde and acetate f0rmat10n

At c0ncentrat10n5 a60ve 0.5 mM, aceta1dehyde 1nh161t5 a11 ce11u1ar funct10n5 6y t0x1c1ty due t0 1t5 react10n w1th the ce11u1ar am1n0 9r0up5. 98 51nce ethan01 15 c0nc0m1tant1y pr0duced and rea551m11ated dur1n9 xy105e fermentat10n, the 1nh161t0ry effect 0f ethan01 may 6e re1ated t0 the effect 0f aceta1dehyde. Ethan01 rea551m11at10n 1ncrea5e5 when the 0xY9enat10n 1eve1 1ncrea5e5 (F19ure 3). 7,99,1°°

Dur1n9 ethan010x1dat10n, aceta1dehyde and NADH are f0rmed, wh1ch may 1ead t0 a red0x 1m6a1ance. 7he m05t c0mm0n r0ute 15 that v1a wh1ch aceta1dehyde dehydr09e- na5e c0nvert5 aceta1dehyde t0 acetate (F19ure 1) and at the 5ame t1me reduce5 NAD +, thu5 amp11fy1n9 the red0x 1m6a1ance. 97 Acetate 15 e1ther accumu1ated, excreted, 0r further 0x1d12ed thr0u9h the 7CA cyc1e. Depend1n9 0n the de9ree 0f red0x 1m6a1ance, aceta1dehyde c0nver510n may 6e prevented. H0wever, when 13C-ethan01 0x1dat10n wa5 5tud1ed 6y 13C-NMR 5pectr05c0py, acetate wa5 the 0n1y pr0duct detected. 99 7he 1na6111ty t0 detect aceta1dehyde wa5 pr06a61y due t0 the 10w 5en51t1v1ty 0f NMR 5pectr05- c0py and the v01at111ty 0f aceta1dehyde. When the acet1c ac1d (pKa = 4.75) accumu1ate51ntrace11u1ar1y, 1t d1550c1ate5 due t0 the neutra1 1ntrace11u1ar pH. 7h15 cau5e5 ac1d1f1ca- t10n 0f the ce11, 51nce the d1550c1ated f0rm 0f acet1c ac1d cann0t d1ffu5e thr0u9h the mem6rane. 7M 1t ha5 6een 5u9- 9e5ted that when the 1ntrace11u1ar pH decrea5e5, the pr0t0n 9rad1ent acr055 the mem6rane cann0t 6e ma1nta1ned, thu5 re5u1t1n9 1n an unc0up11n9 0f the ener9y pr0duct10n and tran5p0rt 5y5tem5, wh1ch are dependent 0n pr0t0n 9rad1ent5.1°2 1n a 5tudy 0f ethan01 rea551m11at10n 1n c0n- t1nu0u5 cu1ture, the A7P/ADP rat10 decrea5ed. 99 7h15 5upp0rt5 the hyp0the515 that the acetate f0rmed cau5e5 the ac1d1f1cat10n 0f the ce115 and unc0up11n9 0f the ener9y pr0duct10n.

Chan9e5 1n the ce11 mem6rane5

1n add1t10n t0 the effect 0f aceta1dehyde and acet1c ac1d, ethan01 may d15tur6 the mem6rane funct10n. Mem6rane5 can 6e c0n51dered a5 p01ar and n0np01ar pha5e5. Ethan0115 0ften a550c1ated w1th the p01ar pha5e and may 1nteract w1th the p01ar 51te5, thu5 repe111n9 water fr0m the mem6rane5.

A

8

50 1(X)

60

50

40

9

~" 30

1(1

0

7tme(h0ur5)

1 150

• 0,5 xy1

A 5 xy1

7 xy1

• 13 xy1

4

1 t~

0 1 t

0 5[~ 1(K1

71mC (h0ur5)

150

• 03 et

• ,1, 5e1

7et

• 13ct

F1¢Jure 3 Xy105e c0n5umpt10n (A) and ethan01 pr0duct10n (8) 1n 6atch fermentat10n w1th Cand1da tr0p1ca115, a5 a funct10n 0f 0xY9en tran5fer rate. (0) 0.5 mM h - 1 ; (A) 5 mM h - 1 ; (~]) 7 mM h - 1; (1) 13 mM h-1 100

7he effect 0n mem6rane5 depend5 1ar9e1Y 0n the med1um c0mp051t10n. 1n a 5tudy 0f xy105e fermentat10n 1n P. tann0- ph11u51F0 1007, the ethan01 y1e1d 1ncrea5ed fr0m 0.2 t0 0.32 9 9 - 1 when the med1um wa5 5upp1emented w1th er905ter01, 11n01e1c ac1d, and 7ween-80, m3 add1t1ve5 wh1ch may 1n- crea5e mem6rane f1u1d1ty.

C0nc1u510n5

1n the 4ue5t f0r 1dea1 xy105e-ferment1n9 yea5t5, 1t 15 appar- ent that a num6er 0f 1nherent phy5101091ca1 re5tr1ct10n5 11m1t the p05516111ty 0f 519n1f1cant1y 1mpr0v1n9 the rate5 0f ethan01 pr0duct10n and the t01erance t0 ethan01 6y env1- r0nmenta10r 9enet1c man1pu1at10n 0fP. 5t1p1t15, C. 5hehatae, and P. tann0ph11u5. 5. cerev151ae, 0n the 0ther hand, exh161t5 a much 9reater ethan01 t01erance and pr0duce5 ethan01 at a much h19her rate under 1e55 demand1n9 0xY9enat10n c0n- d1t10n5 than the xy105e-ferment1n9 yea5t5.7he current re- c0m61nant 5tra1n5 0f 5. cerev151ae, p055e551n9 the a6111ty t0 c0nvert xy105e t0 xY1u105e, ferment xy105e t0 ethan01 p00r1y. 1n 5p1te 0f the5e 11m1tat10n5, 5. cerev151ae rema1n5 the yea5t 0f ch01ce f0r p055161e c0mmerc1a1 xy105e fermentat10n 6e- cau5e 0f 1t5 trad1t10na1 u5e 1n the fermentat10n 1ndu5try, pr0ven a6111ty t0 pr0duce h19h ethan01 c0ncentrat10n5 rap- 1d1y, and the deve10pment 0f m01ecu1ar techn14ue5 that ena61e 9enet1c man1pu1at10n 0f the yea5t.

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

810chem15try and phy510109Y 0f xy105e fermentat10n: 8. Hahn-H~9erda1 et a1.

Ackn0w1ed9ement5 We thank NU7EK (the 5wed15h Nat10na180ard f0r 1ndu5- tr1a1 and 7echn1ca1 Deve10pment), 57U (the 5wed15h Na- t10na1 80ard f0r 7echn1ca1 Deve10pment), 57EV (the Na- t10na1 Ener9y Adm1n15trat10n 0f 5weden), N1 (the N0rd1c Fund f0r 7echn0109y and 1ndu5tr1a1 Deve10pment), NFR (the 5wed15h Natura1 5c1ence Re5earch C0unc11), 55EU (the 5wed15h Ethan01 Deve10pment F0undat10n), and the FRD (the F0undat10n f0r Re5earch Deve10pment 0f 50uth Afr1ca) f0r f1nanc1a15upp0rt. We e5pec1a11y thank Pr0fe550r 6u1d0 2acch1 (Department 0f Chem1ca1 En91neer1n9, Lund) and Pr0fe550r 665ta Petter550n (Department 0f 810chem15try, Lund) f0r va1ua61e d15cu5510n5 c0ncern1n9 en2yme k1net1c5.

Reference5 1 5chne1der, H., Wan9, P. Y., Chan, Y. K. and Ma1e52ka, R. C0nver-

510n 0f D-xY105e 1nt0 ethan01 6y the yea5t Pachy501en tann0ph11u5. 810techn01. Lett. 1981, 3, 89-92

2 Jeffr1e5, 7. W. C0nver510n 0f xy105e t0 ethan01 under aer061c c0nd1- t10n5 6y Cand1da tr0p1ca115. 810techn01. Lett. 1981, 3, 213-218

3 511n1n9er, P. J., 80tha5t, R. J., Van Cauwen6er9e, J. E. and Kurt2- man, C. P. C0nver510n 0f D-xy105e t0 ethan016y the yea5tPachy501en tann0ph11u5. 810techn01. 810en9. 1982, 24, 371-384

4 Ch1an9, C. and Kn19ht, 5. 6. Meta60115m 0f D-xY105e 6y m0u1d5. Nature 1960, 188, 79-81

5 Chakrav0rty, M., Ve19a, L. A., 8ac11a, M. and H0recker, 8. L. Pen- t05e meta60115m 1n Cand1da 11.7he d1ph05ph0pyr1d1ne nuc1e0t1de- 5pec1f1c p01y01 dehydr09ena5e 0f Cand1da ut1115. 8101. Chem. 1962, 237, 1014-1020

6 5trathern, J. N., J0ne5, E, W. and 8r0ach, J. R., ed5. 7he M01ecu1ar 810109Y 0f the Yea5t 5acchar0myce5 cerev151ae. Meta60115m and 6ene Expre5510n. C01d 5pr1n9 Har60r La60rat0ry, C01d 5pr1n9 Har60r, NY, 1982, pp, 1-37

7 5k009, K. 7he 1nf1uence 0f0xY9en 1n pent05e fermentat10n 6y yea5t5. Ph.D. 7he515, Un1ver51ty 0f Lund, 5weden, 1992

8 L19the1m, M. E., Pr10r, 8. A. and du Pree2, J. C. 7he 0xY9en re4u1re- ment5 0f yea5t5 f0r the fermentat10n 0f 0-xy105e and D-91uc05e t0 ethan01.App1. M1cr06101. 810techn01. 1988, 28, 63-68

9 6r00tjen, D. R. J.,van der Lan5, R. 6. J. M. and Luy6en, K. C. A. M. Effect5 0fthe aerat10n rate 0n the fermentat10n 0f 91uc05e and xy105e 6y P1ch1a 5t1p1t15 C85 5773. En2yme M1cr06. 7echn01. 1990,12, 20-23

10 La5t1ck, 5. M., M0ha9he9h1, A., 7ucker, M. P. and 6r0hmann, K. 51mu1tane0u5 fermentat10n and 150mer12at10n 0fxy105e t0 ethan01 at h19h xy105e c0ncentrat10n. App1. 810chem. 810techn01. 1990, 24/25, 431-439

11 5enac, 7. and Hahn-H~19erda1, 8.1ntermed1ary meta6011te c0ncen- trat10n5 1n xy1u105e- and 91uc05e-ferment1n9 5acchar0myce5 cerev1- 51ae ce115. App1. Env1r0n. M1cr06101. 1990, 56, 120-126

12 Jeffr1e5, 7. W. and Kurt2man, C. P. 5tra1n 5e1ect10n, tax0n0my, and 9enet1c5 0f xy105e-ferment1n9 yea5t5. En2yme M1cr06. 7echn01. 1994, 16, 922-932

13 Pr10r, 8. A., K111an, 5. 6. and du Pree2, J. C. Fermentat10n 0f D-xy105e 6y the yea5t5 Cand1da 5hehatae and P1ch1a 5t1p1t15. Pr0ce55 810chem. 1989, 24, 21-32

14 511n1n9er, P. J., 801en, P. L. and Kurt2man, C. P. Pachy501en tann0- ph11u5:Pr0pert1e5 and pr0ce55 c0n51derat10n5 f0r ethan01 pr0duct10n fr0m D-xy105e. En2ymeM1cr06. 7echn01. 1987, 9, 5-15

15 du Pree2, J. C,, Pr10r, 8. A. and M0nte1r0, A. M. 7. 7he effect 0f aerat10n 0n xy105e fermentat10n 6y Cand1da 5hehatae and Pachy501en tann0ph11u5. App1. M1cr06101. 810techn01. 1984, 19, 261-266

16 Wat50n, N. E., Pr10r, 8. A., du Pree2, J. C. and Late9an, P. M. 0xY9en re4u1rement5 f0r D-xy105e fermentat10n t0 ethan01 and p01y- 015 6y Pachy501en tann0ph11u5. En2yme M1cr06. 7echn01. 1984, 6, 447-450

17 Lee, H. and 5chne1der, H. Ethan01 pr0duct10n fr0m xy11t01 and 50me 0ther p01y015 6yP1ch1a an90ph0rae. 810techn01. Lett. 1987, 9, 581-584

18 du Pree2, J. C., 805ch, M. and Pr10r, 8. A. 7he fermentat10n 0f

hex05e and pent05e 5u9ar5 6y Cand1da 5hehatae and P1ch1a 5t1p1t15. App1. M1cr06101. 810techn01. 1986, 23, 228-233

19 Yu, 5., Wayman, M. and Parekh, 5. K. Fermentat10n t0 ethan010f pent05e-c0nta1n1n9 5pent 5u1ph1te 114u0r. 810techn01. 810en9. 1987, 29, 1144-1150

20 De6u5, D., Methner, H., 5chu12e, D. and De11we9, H. Fermentat10n 0f xy105e w1th the yea5t Pachy501en tann0ph11u5. Eur. J. App1. M1cr0- 6101. 810techn01. 1983, 17, 287-291

21 Lee, H., 81e1y, P., Latta, R. K., 8ar605a, M. F. 5. and 5chne1der, H. Ut1112at10n 0f xy1an 6y yea5t5 and 1t5 c0nver510n t0 ethan016y P/ch/a 5t1p1t15 5tra1n5.App1. Env1r0n. M1cr06101. 1986, 52, 320-324

22 K111an, 5.6. and van Uden, N. 7ran5p0rt 0fxy105e and 91uc05e 1n the xy105e-ferment1n9 yea5t P1ch1a 5t1p1t15. App1. M1cr06101. 810techn01. 1988, 27, 545-548

23 D0e5, A. L. and 81550n, L. F. Character12at10n 0f xy105e uptake 1n the yea5t5 P1ch1a heed1 and P1ch1a 5t1p1t15. App1. Env1r0n. M1cr06101. 1989, 55, 159-164

24 van Urk, H., P05tma, E., 5cheffer5, W. A. and van D1jken, J. P. 61uc05e tran5p0rt 1n Cra6tree-p051t1ve and Cra6tree-ne9at1ve yea5t5. J. 6en. M1cr06101. 1989, 135, 2399-2406

25 Luca5, C. and van Uden, N. 7ran5p0rt 0f hem1ce11u105e m0n0mer5 1n the xy105e-ferment1n9 yea5t Cand1da 5hehatae. App1. M1cr06101. 810techn01. 1986, 23, 491-495

26 K111an, 5. 6,, Pr10r, 8. A. and du Pree2, J. C. 7he k1net1c5 and re9u1at10n 0f D-xy105e tran5p0rt 1n Cand1da ut1115. W0rdJ. M1cr06101. 810techn0L 1993, 9, 357-360

27 8arnett, J. A.Adv. Car60hydr. Chem. 810chem. (71p50n, R. 5. and H0rt0n, D., ed5.). Academ1c Pre55, New Y0rk, 1976, 32, 125-235

28 8arnett, J. A., Payne, R. W. and Yarr0w, D. Yea5t5:Character15t1c5 and1dent1f1cat10n. Cam6r1d9e Un1ver51ty Pre55, New Y0rk, 1983, p. 56

29 8att, C. A., Carva110, 5., Ea550n, D. D., Jr., Aked0, M. and 51n5key, A. J. D1rect ev1dence f0r a xy105e meta6011c pathway 1n 5acchar0my- ce5 cerev151ae. 810techn01. 810en9. 1986, 28, 549-553

30 van 2y1, C., Pr10r, 8. A., K111an, 5. 6. and K0ck, J. L. F. D-Xy105e ut1112at10n 6y 5acchar0myce5 cerev151ae. J. 6en. M1cr06101. 1989, 135, 2791-2798

31 C1r1110, V. P. 5u9ar tran5p0rt 1n m1cr00r9an15m5.Annu. Rev. M1cr0- 6101. 1961, 15, 197-218

32 81550n, L. F. and Fraenke1, D. 6. Expre5510n 0f k1na5e-dependent 91uc05e uptake 1n 5acchar0myce5 cerev151ae. J. 8acter101. 1984, 159, 1013-1017

33 81550n, L. F. Derepre5510n 0f h19h-aff1n1ty 91uc05e uptake re4u1re5 a funct10na15ecret0ry 5y5tem 1n 5acchar0myce5 cerev151ae. J. 8acter101. 1988, 170, 2654-2658

34 Lan9, J. M. and C1r1110, V. P. 61uc05e tran5p0rt 1n a k1na5e1e55 5acchar0myce5 cerev151ae mutant. J. 8acter10L 1987, 169, 2932-2937

35 Ram05, J., 52kutn1cka, K. and C1r1110, V. P. Re1at10n5h1p 6etween 10w- and h19h-aff1n1ty 91uc05e tran5p0rt 5y5tem5 0f 5acchar0myce5 cerev151ae. J. 8acter10L 1988, 170, 5375-5377

36 van 2y1, C., Pr10r, 8. A., K111an, 5. 6. and 8randt, E. V. R01e 0f D-r1605e a5 a c0meta6011te 1n the 0-xy105e meta60115m 6y 5acchar0- myce5 cerev151ae. App1. Env1r0n. M1cr06101. 1993, 59, 1487-1494

37 5erran0, R. and De1afuente, 6. Re9u1at0ry pr0pert1e5 0f the c0n- 5t1tut1ve hex05e tran5p0rt 1n 5acchar0myce5 cerev151ae. M01. Ce11. 810chem. 1974, 5, 161-171

38 8u5tur1a, A. and La9una5, R. Cata6011te 1nact1vat10n 0f the 91uc05e tran5p0rt 5y5tem 1n 5acchar0myce5 cerev151ae. J. 6en. M1cr06101. 1986, 132, 379-385

39 K6tter, P. and C1r1acy, M. Xy105e fermentat10n 6y 5acchar0myce5 cerev151ae. AppL M1cr06101. 810techn01. 1993, 38, 776-783

40 L19the1m, M. E., Pr10r, 8. A., du Pree2, J. C. and 8randt, V. An 1nve5t19at10n 0f D-(1-13C) xy105e meta60115m 1n P1ch1a 5t1p1t15 under aer061c and anaer061c c0nd1t10n5.App1. M1cr06101. 810techn01. 1988, 28, 293-296

41 A1exander, M. A., Yan9, V. W. and Jeffr1e5, 7. W. Leve15 0f pent05e ph05phate pathway en2yme5 fr0m Cand1da 5hehatae 9r0wn 1n c0n- t1nu0u5 cu1ture.App1. M1cr0610L 810techn0L 1988, 29, 282-288

42 K1uyver, A. J. and Cu5ter5, M. 7. J. 7he 5u1ta6111ty 0f d15acchar1de5 a5 re5p1rat10n and a551m11at10n 5u65trate5 f0r yea5t5 wh1ch d0 n0t fer- ment the5e 5u9ar5. Ant0n1e van Leeuwenh0ek 1940, 6, 121-162

43 51m5, A. P. and 8arnett, J, A. 7he re4u1rement 0f 0xY9en f0r the ut1112at10n 0f ma1t05e, ce1106105e and D-9a1act05e 6y certa1n anaer0- 61ca11y ferment1n9 yea5t5 (K1uyver effect). J. 6en. M1cr06101. 1978, 106, 277-288

En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er 941

Rev1ew5

44 5k009, K. and Hahn-H~19erda1, 8. Effect 0f 0xY9enat10n 0n xy105e fermentat10n 6y P1ch1a 5t1p1t15. App1. Env1r0n. M1cr06101. 1990, 56, 3389-3394

45 5m11ey, K. L. and 801en, P. L. Dem0n5trat10n 0f D-xy105e reducta5e and D-xy11t01 dehydr09ena5e 1n Pachy501en tann0ph11u5. 810techn01. Lett. 1982, 4, 607-610

46 Lachke, A. H. and Jeffr1e5, 7. W. Leve15 0f en2yme5 0f the pent05e ph05phate pathway 1n Pachy501en tann0ph11u5 Y-2460 and 5e1ected mutant5. En2yme M1cr06. 7echn01. 1986, 8, 353-359

47 81ch0, P. A., Runna15, P. L., Cunn1n9ham, J. D. and Lee, H. 1nduc- t10n 0fxy105e reducta5e and xy11t01 dehydr09ena5e act1v1t1e51n Pach- y501en tann0ph11u5 and P1ch1a 5t1p1t15 0n m1xed 5u9ar5.App1. Env1r0n. M1cr06101. 1988, 54, 50-54

48 8ru1nen6er9, P. M., de 80t, P. H. M.,van D1jken, J. P. and 5cheffer5, W. A. 7he r01e 0f red0x 6a1ance5 1n the anaer061c fermentat10n 0f xy105e 6y yea5t5. Eur. J. App1. M1cr06101. 810techn01. 1983,18, 287-292

49 8ru1nen6er9, P. M., de 80t, P. H. M., van D1jken, J. P. and 5cheffer5, W. A. NADH-11nked a1d05e reducta5e: 7he key t0 anaer061c a1c0- h011c fermentat10n 0f xy105e 6y yea5t5. App1. M1cr06101. 810techn01. 1984, 19, 256-260

50 8ru1nen6er9, P. M., J0nker, R., van D1jken, J. P. and 5cheffer5, W. A. Ut1112at10n 0f f0rmate a5 an add1t10na1 ener9y 50urce 6y 91uc05e-11m1ted chem05tat cu1ture5 0f Cand1da ut1115 C85 621 and 5acchar0myce5 cerev151ae C85 8066. Arch. M1cr06101. 1985, 142, 302-306

51 Verduyn, C., van K1eef, R., Frank J2n, J., 5chreuder, H., van D1jken, J. P. and 5cheffer5, W. A. Pr0pert1e5 0f the NAD(P)H-dependent xy105e reducta5e fr0m the xy105e-ferment1n9 yea5t P/ch/a 5t1p1t15. 810- chem. J. 1985, 226, 669-677

52 5k009, K., Jepp550n, H. and Hahn-H/19erda1, 8 .7he effect 0f 0xy- 9enat10n 0n 91uc05e fermentat10n w1th P1ch1a 5t1p1t15. App1. 810chem. 810techn0L 1992, 34/35, 369-375

53 Jepp550n, H., A1exander, N. J. and Hahn-H~19erda1, 8. Cyan1de 1n5en51t1ve re5p1rat10n 1n the xy105e ferment1n9 yea5t P1ch1a 5t1p1f15 C85 6054. 810re50urce 7echn01. (1n pre55)

54 Per550n, 8., Ha1160rn, J., Wa1fr1d550n, M., Hahn-H~19erda1, 8., Ker- ~1nen, 5., Pentt11~1, M. and J(~rnva11, H. Dua1 re1at10n5h1p5 0f xy11t01 and a1c0h01 dehydr09ena5e51n fam111e5 0f tw0 pr0te1n type5. FE85 Lett. 1993, 324, 9-14

55 du Pree2, J. C., van Dr1e55e1, 8. and Pr10r, 8. A. Effect 0f aer0610515 0n fermentat10n and key en2yme 1eve15 dur1n9 9r0wth 0f P1ch1a 5t1p1t15, Cand1da 5hehatae and Cand1da tenu15 0n D-xy105e. Arch. M1cr0610L 1989, 152, 143-147

56 van Cauwen6er9e, J. E., 801en, P. L, McCracken, D. A. and 80tha5t, R. J. Effect 0f 9r0wth c0nd1t10n5 0n c0fact0r-11nked xy105e reducta5e act1v1ty 1n Pachy501en tann0ph11u5. En2yme M1cr06. 7ech- n01. 1989, 11, 662-667

57 Verduyn, C., Frank J2n, J., van D1jken, J. P. and 5cheffer5, W. A. Mu1t1p1e f0rm5 0f xy105e reducta5e 1n Pachy501en tann0ph11u5 C85 4044. FEM5 M1cr06101. Lett. 1985, 30, 313-317

58 R1221, M., Edemann, P., 8u1-7hanh, N.-A. and De11we9, H. Xy105e fermentat10n 6y yea5t5.4. Pur1f1cat10n and k1net1c 5tud1e5 0f xy105e reducta5e fr0m P1ch1a 5t1p1t15. App1. M1cr0610L 810techn01. 1988, 29, 148-154

59 R1221, M., Harwart, K., 8u1-7hanh, N.-A. and De11we9, H. A k1net1c 5tudy 0f the NAD +-xy11t01-dehydr09ena5e fr0m the yea5t P1ch1a 5t1p1t15. J. Ferment. 810en9. 1989, 67, 25-30

60 Ne1r1nck, L 6., 75a1, C. 5., La6e11e, J. L. and 5chne1der, H. Xy11t01 a5 a car60n 50urce f0r 9r0wth and ethan01 pr0duct10n 6yPachy501en tann0ph11u5. Can J. M1cr06101. 1985, 31, 451-455

61 L1nd6n, 7., Peetre, J. and Hahn-Hf19erda1, 8.1501at10n and charac- ter12at10n 0f acet1c ac1d-t01erant 9a1act05e-ferment1n9 5tra1n5 0f 5acchar0rnyce5 cerev151ae fr0m a 5pent 5u1f1te 114u0r fermentat10n p1ant. App1. Env1r0n. M1cr0610L 1992, 58, 1661-1669

62 Wan9, P. Y., 5h0p515, C. and 5chne1der, H. Fermentat10n 0f a pen- t05e 6y yea5t5. 810chem. 810phy5. Re5. C0mmun. 1980, 94, 248-254

63 Ch1an9, L-C., 60n9, C.-5., Chen, L.-F. and 75a0, 6 .7 . D-Xy1u105e fermentat10n t0 ethan016y 5acchar0myce5 cerev151ae. App1. Env1r0n. M1cr06101. 1981, 42, 284-289

64 Uen9, P. P., Hunter, C. A., 60n9, C.-5. and 75a0, 6 .7 . D-Xy1u105e fermentat10n 1n yea5t5.810techn01. Len. 1981, 3, 315-320

65 Jeffr1e5, 7. W. Mutant5 0fPachy501en tann0ph11u5 5h0w1n9 enhanced rate5 0f 9r0wth and ethan01 f0rmat10n fr0m D-xy105e. En2yme M1- cr06. 7echn01. 1984, 6, 254-258

66 H0, N. W. Y. and Chan9, 5.-F. C10n1n9 0f yea5t xy1u10k1na5e 9ene 6y c0mp1ementat10n 0f E. c011 and yea5t mutat10n5. En2yme M1cr06. 7echn01. 1989, 11, 417-421

67 6anced0, J. M. and La9una5, R. C0ntr16ut10n 0f the pent05e- ph05phate pathway t0 91uc05e meta60115m 1n 5acchar0myce5 cerev1- 51ae: A cr1t1ca1 ana1y515 0n the u5e 0f 1a6e11ed 91uc05e. P1ant 5c1. Lett. 1973, 1, 193-200

68 C1r1acy, M. and P0rep, H. C0nver510n 0f pent05e5 t0 ethan01 6y 8aker5• yea5t. 1n: 810m01ecu1ar En91neer1n91n Eur0pean C0mmun1ty (Ma9n1en, E., ed.). Mart1nu5 N1jh0ff, D0rdrecht, 7he Nether1and5, 1986, pp. 677-679

69 Evan5, C. 7. and Rat1ed9e, C. 1nduct10n 0f xy1u105e-5-ph05phate ph05ph0ket01a5e 1n a var1ety 0f yea5t5 9r0wn 0n D-xy105e: 7he key t0 eff1c1ent xy105e meta60115rn. Arch. M1cr06101. 1984, 139, 48-52

70 511n1n9er, P. J., 8ran5trat0r, L. E., L0m0nt, J. M., D1en, 8.5., 0k05, M. R., Lad15ch, M. R. and 80tha5t, R. J. 5t01ch10metry and k1net1c5 0f xy105e fermentat10n 6y P1ch1a 5t1p1t15. Ann. NY Acad. 5c1. 1990, 589, 25-40

71 Av19ad, 6. 5t1mu1at10n 0f yea5t ph05ph0fruct0k1na5e act1v1ty 6y fruct05e 2,6-615ph05phate. 810chem. 810phy5. He5. C0mmun. 1981, 102, 985-991

72 801e5, E., He1n15ch, J. and 21mmerman, F. K. D1fferent 519na15 c0n- tr01 the act1vat10n 0f 91yc01y5151n the yea5t 5acchar0myce5 cerev151ae. Yea5t 1993, 9, 761-770

73 Lut5t0rf, U. and Me9net, R. Mu1t1p1e f0rm5 0f a1c0h01 dehydr09e- na5e 1n 5acchar0myce5 cerev151ae. Arch. 810chern. 810phy5. 1968,126, 933-944

74 C1r1acy, M. 6enet1c5 0f a1c0h01 dehydr09ena5e 1n 5acchar0myce5 cerev151ae. 1: 1501at10n and 9enet1c ana1y515 0f adh mutant5. Mutat. Re5. 1975, 29, 315-326

75 P119r1m, D. and Y0un9, E. 7. Pr1mary 5tructure re4u1rement5 f0r c0rrect 50rt1n9 0f the yea5t m1t0ch0ndr1a1 pr0te1n ADH 111 t0 the yea5t m1t0ch0ndr1a1 matr1x 5pace. M01ec. Ce11. 8101. 1987, 7, 294-304

76 Pa4u1n, C. E. and W1111am50n, V. M. 7y 1n5ert10n5 acc0unt f0r m05t 0f the 5p0ntane0u5 ant1myc1n A re515tance mutat10n5 dur1n9 9r0wth at 15°C 0f 5acchar0myce5 cerev151ae 1ack1n9 ADH 1. M01ec. Ce11. 8101. 1986, 6, 70-79

77 A1exander, M. A., Chapman, 7. W. and Jeffr1e5, 7. W. Xy105e me- ta60115m 6y Cand1da 5hehatae 1n c0nt1nu0u5 cu1ture. App1. M1cr06101. 810techn0L 1988, 28, 478-486

78 Pr10r, 8. A., A1exander, M. A., Yan9, V. and Jeffr1e5, 7. W. 7he r01e 0f a1c0h01 dehydr09ena5e 1n the fermentat10n 0f 0-xy105e 6y Can- d1da 5hehatae A7CC 22984. 810techn0L Lett. 1988, 10, 37-42

79 511n1n9er, P. J., 8ran5trat0r, L. E., 80tha5t, R. J., 0k05, M. R. and Lad15ch, M. R. 6r0wth, death, and 0xY9en uptake k1net1c5 0f P1ch1a 5t1p1t15 0n xy105e. 810techn01. 810en9. 1991, 37, 973-980

80 Ne1r1nck, L. 6., Ma1e52ka, R. and 5chne1der, H. 7he re4u1rement 0f 0xy9en f0r 1nc0rp0rat10n 0f car60n fr0m D-xy105e and D-91uc05e 6y Pachy501en tann0ph11u5. Arch. 810chem. 810phy5. 1984, 228, 13-21

81 V155er, W., 5cheffer5, W. A., 8aten6ur9-van der Ve9te, W. H. and van D1jken, J. P. 0xY9en re4u1rement5 0f yea5t5. App1. Env1r0n. M1cr0610L 1990, 56, 3785-3792

82 Andrea5en, A. A. and 5t1er, 7. J. 8. Anaer061c nutr1t10n 0f 5accha- r0rnyce5 cerev151ae. 1. Er905ter01 re4u1rement f0r 9r0wth 1n a def1ned med1um. J. Ce11. C0mp. Phy510L 1953, 41, 23-26

83 Andrea5en, A. A. and 5t1er, 7. J. 8. Anaer061c nutr1t10n 0f 5accha- r0myce5 cerev151ae. 11. Un5aturated fatty ac1d re4u1rement f0r 9r0wth 1n a def1ned med1um. J. Ce11. C0mp. Phy5101. 1954, 43, 271-281

84 Ma1e52ka, R. and 5chne1der, H. 1nv01vement 0f 0xy9en and m1t0- ch0ndr1a1 funct10n 1n the meta60115m 0f 0-xY1u105e 6y 5acchar0my- ce5 cerev151ae. Arch. 810chem. 810phy5. 1984, 228, 22-30

85 L19the1m, M. E., Pr10r, 8. A. and du Pree2, J. C. 7he effect 0f re5p1rat0ry 1nh161t0r5 0n the fermentat1ve a6111ty 0f P1ch1a 5t1p1t15, Pachy501en tann0ph11u5 and 5acchar0myce5 cerev151ae under var1- 0u5 c0nd1t10n5 0faer0610515.AppL M1cr0610L 810techn0L 1988, 29, 67-71

86 A1exander, N. J. and Detr0y, R. W. A1c0h01 pr0duct10n 6y m1t0- ch0ndr1a1 mutant5 and w11d type 5acchar0myce5 cerev151ae. 810tech- n01. Lett. 1983, 5, 165-168

87 R0th5te1n, A. and 8erke, H. End09en0u5 a1c0h011c fermentat10n 1n yea5t 1nduced 6y 2,4-d1n1tr0phen01. Arch. 810chem. 810phy5. 1952, 36, 195-201

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

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

88 8ar0n0f5ky, J. J., 5chreur5, W. J. A. and Ka5hket, E. R. Unc0up11n9 6y acet1c ac1d 11m1t5 9r0wth 0f and acet09ene515 6y C/05tr/d/um ther- m0acet1at~ AppL Env1r0~ M1cr0610L 1984, 48,1134-1139

89 P05tma, E., Verduyn, C., 5cheffer5, W. A. and van D1jken, J. P. En2ymat1c ana1y515 0f the Cra6tree effect 1n 91uc05e-11m1ted chem05tat cu1ture5 0f 5acchar0myce5 cerev151ae. App1. Env1r0n. M1- cr06101. 1989, 55, 468-477

90 A1exander, N. J. Character12at10n 0f a re5p1rat0ry-def1c1ent mutant 0f Pachy501en tann0ph11u5. Curr. 6enet. 1990, 17, 493-497

91 Jefff1e5, 7. W. Un5ta61e pet1te and 9rande var1ant5 0f Cand1da 5hehatae. 810techn01. Lett. 1984, 6, 777-782

92 Na9y, M., Lacr0ute, F. and 7h0ma5, D. D1ver9ent ev01ut10n 0f pyr1m1d1ne 6105ynthe515 6etween anaer061c and aer061c yea5t5. Pr0c. Nat1. Acad. 5c1. U5A 1992, 89, 8966-8970

93 R1221, M., K1e1n, C., 5chu12e, C., 8u1-7hanh, N.-A. and De11we9, H. Xy105e fermentat10n 6y yea5t5. 5. U5e 0f A7P 6a1ance5 f0r m0de11n9 0xY9en-11m1ted 9r0wth and fermentat10n 0f yea5t P/- ch1a 5t1p1t15 w1th xy105e a5 car60n 50urce. 810techn01. 810en9. 1989, 34, 509-514

94 du Pree2, J. C., van Dr1e55e1, 8. and Pr10r, 8. A. Ethan01 t01erance 0f P1ch1a 5t1p1t15 and Cand1da 5hehatae 5tra1n51n fed-6atch cu1ture5 at c0ntr011ed 10w d15501ved 0xY9en 1eve15. App1. M1cr06101. 810tech- n01. 1989, 30, 53-58

95 du Pree2, J. C., 805ch, M. and Pr10r, 8. A. 7emperature pr0f11e5 0f 9r0wth and ethan01 t01erance 0f the xy105e-ferment1n9 yea5t5 Can- d1da 5hehatae and P1ch1a 5t1p1t15. App1. M1cr06101. 810techn01. 1987, 25, 521-525

96 8r0wn, 5. W., 011ver, 5.6., Harr150n, D. E. F. and R19he1at0, R. C. Ethan011nh161t10n 0f yea5t 9r0wth and fermentat10n: D1fference5 1n the ma9n1tude and c0mp1ex1ty 0f the effect. Eur. J. App1. M1cr0- 6101. 810techn01. 1981, 11, 151-155

97 J0ne5, R. P. 8101091ca1 pr1nc1p1e5 f0r the effect5 0f ethan0L En2yme M1cr06. 7echn01. 1989, 11, 130-153

98 L1e6ertha1, 5., 01df1e1d, M. and 5han1ey, 8. C. Effect 0f ethan01 and aceta1dehyde 0n mem6rane-60und en2yme51n rat 6ra1n.Adv. Exp. Med. 8101. 1979, 132, 797-805

99 5k009, K., Hahn-H/19erda1, 8., De9n, H., Jac065en, J. P. and Ja- c065en, H. 5. Ethan01 rea551m11at10n and ethan01 t01erance 1n P/- ch1a 5t1p1t15 C85 6054 a55tud1ed 6y 13C nuc1ear ma9net1c re50nance 5pectr05c0py.App1. Env1r0n. M1cr06101. 1992, 58, 2552-2558

100 L0hme1er-V09e1, E., 5k009, K., V09e1, H. and Hahn-H/19erda1, 8. 31p Nuc1ear ma9net1c re50nance 5tudy 0f the effect 0f a21de 0n xy105e fermentat10n 6y Cand1da tr0p1ca115. App1. Env1r0n. M1cr06101. 1989, 55, 1974-1980

101 Pampu1ha, M. E. and L0ure1r0-D1a5, M. C. C0m61ned effect 0f acet1c ac1d, pH and ethan010n 1ntrace11u1ar pH 0f ferment1n9 yea5t. App1. M1cr06101. 810techn01. 1989, 31, 547-550

102 Herrer0, A. A., 60me2, R. F., 5nedec0r, 8., 701man, C. J. and R06ert5, M. F. 6r0wth 1nh161t10n 0f C105tr1d1um therm0ce11um 6y car60xy11c ac1d5: A mechan15m 6a5ed 0n unc0up11n9 6y weak ac1d5. App1. M1cr06101. 810techn01. 1985, 22, 53-62

103 Wat50n, K. Un5aturated fatty ac1d 6ut n0t er905ter0115 e55ent1a1 f0r h19h ethan01 pr0duct10n 1n 5acchar0myce5. 810techn01. Lett. 1982, 4, 397-402

En2yme M1cr06. 7echn01., 1994, v01. 16, N0vem6er 943