Recent Progress in Bioconversion of Lignocellulosics

184	N.W.Y. Ho et al.

Fig. 12. Fermentation of glucose and xylose present in the crude hydrolysate of corn fiber by genetically engineered Saccharomyces yeast strain 1400(pLNH32). The corn fiber hydrolysate was provided by Cargill, Inc. The major sugars present in the hydrolysate are glucose, Xylose, and-arabinose. Symbols: solid square glucose; solid circle xylose; solid triangle ethanol; open circle arabinose; open triangle xylitol and arabitol; open square glycerol

Successful Design and Development of Genetically Engineered Saccharomyces Yeasts

185

Fig. 13. Fermentation of glucose and xylose present in the crude hydrolysate of corn stover by genetically engineered Saccharomyces yeast strain 1400 (pLNH32). The hydrolysate was provided by Swan Biomass Company. Symbols: solid square glucose; solid circle xylose; solid triangle ethanol; open circle arabinose; open triangle xylitol and arabitol; open square glycerol

186	N.W.Y. Ho et al.

Fig. 14. Fermentation of glucose and xylose present in the crude hydrolysates of wheat straw by genetically engineered Saccharomyces yeast strain 1400 (pLNH32). The hydrolysate was provided by St. Lawrence Technologies Inc., Canada. Symbols: solid square glucose; solid circle xylose (90%) and small amount of galactose (10%); solid triangle ethanol; open triangle xylitol; open square glycerol

Successful Design and Development of Genetically Engineered Saccharomyces Yeasts

187

Fig. 15. Fermentation of glucose and xylose present in the crude hydrolysates of softwood by genetically engineered Saccharomyces yeast strain 1400 (pLNH32). The hydrolysate was provided by the Chemical Division of Tembec, Inc., Canada. Symbols: solid square glucose; solid circle mannose, xylose, and galactose; solid triangle ethanol; open triangle xylitol and arabitol; open square glycerol

188	N.W.Y. Ho et al.

8

Development of Genetically Engineered Saccharomyces Yeasts Able to Coferment Glucose, Xylose, and L-Arabinose

Although the major fermentable sugars from cellulosic biomass are glucose and xylose, some of them, such as corn fiber, also contain substantial amounts of L-arabinose. It is known that Saccharomyces yeasts are also unable to ferment L-arabinose to ethanol and to use the sugar for growth. Naturally we should try to make our engineered Saccharomyces yeasts also able to ferment L-arabinose to ethanol and also to use the sugar for growth, if possible.

It is known that most bacteria including E. coli can effectively utilize L-arabi- nose for aerobic growth and also can ferment it to ethanol. Bacteria depend on protein products of the araBAD operon, which contains three genes, araB (encoding L-ribulokinase), araA (encoding L-arabinose isomerase) and araD (encoding L-ribulose-5-phosphate 4-epimerase) to convert L-arabinose directly to D-xylulose 5-phosphate through L-ribulose and L-ribulose-5-phosphate [28]). The genes encoding the E. coli L-arabinose metabolizing enzymes have been cloned and sequenced [29].

Although some of the yeasts and fungi have been reported to be able to utilize L-arabinose for growth and a few of them can also ferment the pentose sugar to ethanol [30, 31] , the yeast and fungi enzymes involved in the metabolizing L-arabinose to xylitol have not yet been isolated and the genes encoding these enzymes have not been cloned and sequenced. Thus, the logical approach for making our recombinant Saccharomyces yeast also able to ferment L-arabi- nose is to clone and express the bacterial genes in the recombinant yeast. In the following, we briefly describe our results in the cloning and expression of the three E. coli genes, araA, araB, and araD in S. cerevisiae.

Since the three E. coli genes will be synthesized by PCR techniques and since it is known that mutations may be more frequently introduced into the DNA synthesized by PCR techniques, in order to insure that the cloned genes are functionally expressed prior to being introduced into the yeast cells we used the following strategies.

First, we obtained three E. coli mutant strains and each strain contained a mutation in one of the three genes encoding the synthesis of the three enzymes required for metabolism of L-Arabinose in E. coli. As a results, these three mutant strains could only produce white colonies but not red colonies on MacConkey agar plates [32]. If a functional gene had been cloned into each of these mutants and could complement the specific mutation of the mutant strains, the recombinant strains should then produce red colonies on the MacConkey plates.

Second, we selected or made several yeast glycolytical promoters including pyruvate kinase (Pyk) promoter, phosphoglucose isomerase (PGI) promoter, and phosphoglycerol kinase (PGK) promoter that can actively express genes in both E. coli and Saccharomyces yeasts and also use these promoters to express the E. coli genes both in yeast and E. coli.

Third, we fused the E. coli araA structural gene to the Pyk promoter and resulted in the construction of the recombinant gene Pyk-araA . Similarly, we also constructed PGI-araB and PGK-araD. We also fused each of these recombinant

Successful Design and Development of Genetically Engineered Saccharomyces Yeasts

189

genes with a yeast 3¢ noncoding sequence. According to our early work, the 3¢ noncoding sequence of the cloned yeast xylulokinase gene (XKt) contains effective genetic signals for polyadenylation, mRNA termination, and so on [26, 33]. Thus the intact 3¢ noncoding sequence XKt was fused to the 3¢ coding sequences of each of the three recombinant ara genes, resulting in the construction of

Pyk-araA-XKt , PGI-araB-XKt ,and PGK-araD-XKt . Each of these recombinant ara genes was cloned into a yeast-E. coli . shuttle plasmid and each of the resulting

plasmids pYaraA, pYaraB, and pYaraC were used to transform at least two Saccharomyces yeast strains. Analysis of the yeast transformants containing each of these plasmids showed that all three E. coli genes were expressed and active enzymes encoded by these genes were produced in the yeast transformants during growth and fermentation. Furthermore, back transformation of the E. coli L-arabinose mutants by plasmids isolated from the yeast transformants produced E. coli transformants that complemented the respective mutations and produced red colonies on MacConkey plates. Analysis of the specific activity showed that the specific activity of L-ribulokinase and L-ribulose-5-phos- phate-4-epimerase reached at least 70% of the activity of the wild type E. coli., but that the specific activity of L-arabinose isomerase was only 10% of that of the wild type E. coli.

The three recombinant genes, Pyk-araA-XKt , PGI-araB-XKt , and PGK-araD- XKt , were then cloned on a single yeast-E. coli plasmid, resulting in the construction of pYaraDBA plasmid. Saccharomyces yeasts containing this plasmid still could produce all three active E. coli L-arabinose metabolizing enzymes and the plasmids isolated from the yeast transformants could still complement the arabinose mutation of the three E. coli L-arabinose mutants. The yeast transformants containing pYaraDBA were then used to ferment L-arabinose and unfortunately very little ethanol was accumulated during the course of fermentation. Currently we are considering to modify the araA gene to make it more actively expressed in yeast. At the same time, we are also trying to clone the L-arabinose metabolizing genes from yeasts and/or fungi to make the Saccharomyces yeasts able to ferment L-arabinose.

9

Further Improvement of Glucose-Xylose-Fermenting Saccharomyces Yeasts for Effective, Economical Conversion of Sugars from Cellulosic Biomass to Biofuel Ethanol.

Even though our genetically engineered Saccharomyces yeasts are as ideal as can be for cofermenting glucose and xylose, there are at least three types of improvements that could make our engineered Saccharomyces yeasts more effective and economical for the conversion of cellulosic biomass sugars to ethanol. One is to make ethanol fermentation faster, particularly xylose fermentation; the second is to reduce the formation of byproducts; and the third is to make the genetically engineered yeasts also able to produce various high-value byproducts.

Some of the ongoing processes to improve our yeasts in these directions are briefly discussed below:

190	N.W.Y. Ho et al.

1Our genetically engineered Saccharomyces yeasts rely on the enzymes encoded by the cloned P. stipitis XR and XD genes to convert xylose to xylulose. However, the cloned XR produces a xylose reductase that can use either NADH or NADPH as the cofactor but prefers NADPH. On the contrary, the cloned XD gene produces a xylitol dehydrogenase specifically requires only NAD as its cofactor. The general consensus is that if the cofactor requirement for the XR enzyme can be modified to using NADH only, the resulting yeast might be able to ferment xylose more effectively by completely regenerating the necessary cofactors within its own xylose fermentation circuit. For testing this hypothesis, we carried out site-specific mutagenesis on the XR gene. One mutated gene XR(M43) produced a xylose reductase with improved affinity towards NADH cofactor (the ratio of the NADH activity to the NADPH activity is 0.9 for the mutant enzyme while the ratio of the NADH activity to the NADPH activity is 0.63 for the wild-type enzyme). 1400 Yeast transformants containing the plasmid p(LNH33)-M43, which is identical with pLNH33 except that the XR gene has been replaced by the XR-M43, did ferment xylose slightly faster than the same yeast strain transformed with pLNH33, but not substantially. However, much less xylitol was produced by using the yeast containing the mutant XR than the one containing the regu-

lar XR [34].

2 Recombinant Saccharomyces yeast still transports glucose much more preferentially than it transports xylose. Improvement of xylose transport would substantially improve our genetically engineered Saccharomyces yeasts in cofermenting glucose and xylose. We have already carried out substantial studies on improving yeast transport of xylose. For example, we have cloned a low affinity glucose transport gene from another yeast species and also some high affinity glucose transport genes from S. cerevisiae. The effect of these genes on our engineered yeasts in cofermenting glucose and xylose will be analyzed.

There are still quite a few other improvements that can be made to improve xylose fermentation, including choosing more suitable promoters to express the XYL genes, changing some of the promoters of glycolytic genes which might be heavily regulated by the concentration of glucose present in the medium, etc.

Furthermore, with the successful development of our new and convenient method for integrating multiple copies of genes into the yeast chromosome, the engineered yeasts can be programmed to produce different coproducts in addition to ethanol, and there is no limit on what coproducts can be produced by the engineered yeast. Our goal is to make ethanol produced from cellulosic biomass less expensive than gasoline and we are confident that we can accomplish this goal.

Successful Design and Development of Genetically Engineered Saccharomyces Yeasts

191

10

Concluding Remarks Particularly on the Importance of Using Safe and Effective Microorganisms for Biofuel Ethanol Production

The selection of a proper microorganism to carry out large-scale production of bulk products is extremely important. Such a microorganism must be absolutely safe and not have any remote possibility of being hazardous to human health or the environment. In addition,for the production of large volumes of low cost products such as ethanol, it is necessary not only to carefully select the microorganism as described above but also to give serious attention to the conditions that are required for culturing the microorganism. It is ideal that the selected microorganism can be effectively cultured under rather extreme conditions so that no other microorganisms, particularly most of the pathogens, can survive under those conditions. This is because the production of such high-volume low-value products cannot afford and also nearly impossible to be totally aseptic and will be unavoidably to release microorganisms to the environment, both the microorganisms used for the production and the contaminates. Currently, three microorganisms have been genetically engineered which are able to ferment both glucose and xylose to ethanol. These are E. coli [34], Zymomonas [35], and our Saccharomyces yeasts. The efficiencies of the best of each of these three engineered microorganisms in fermenting glucose and xylose present in cellulosic biomass are comparable. However, among the three microorganisms, only the Saccharomyces yeasts have been used for large-scale production of wine and ethanol for hundreds of years without any adverse effects on environment or human health. Furthermore, only Saccharomyces yeasts have been cultured in open vessels for centuries and have not created any ill effects to human health or environment.

In addition to the fact that Saccharomyces yeasts have the proven record to be the absolutely safe microorganisms for large-scale production of industrial products, the engineered yeasts can be substantially further improved for more effective fermentation of celllulosic sugars to ethanol, they have a large enough gnome to allow numerous genes to be inserted into its chromosome to produce various by-products, and our successful development of the new reliable method for integrating multiple copies of gene(s) into the yeast chromosome will further ensure the latter possibility. These extraordinary properties will make our genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts the overwhelming favorite of the ethanol industry for the conversion of cellulosic sugars to ethanol.

Acknowlegements. The authors wish to acknowledge the past support from USDA (the U. S. Department of Agriculture), DOE (the U.S. Department of Energy) and various companies (Amoco, Swan Biomass Co., National Corn Growers Association, Corn Refiners Association, etc.) at different periods from 1982 to 1996 and current support from EPA (the U.S. Environmental Protection Agency) which have in part made it possible for this important work to continue and to reach a successful conclusion. The senior author (N. W. Y. Ho) also wishes to acknowledge the contributions of her many past associates, in particular Dr. P.E. Stevis, Dr. S.A. Rosenfeld, Dr. J.J. Huang, and Dr. S.K. Liu, who

192	N.W.Y. Ho et al.

worked with her on various projects which have contributed to the success of this project one way or the other.

References

1.Jeffries TW (1985) Trends Biotechnol 3:208–212

2.Horecker BL (1963) Pentose Metabolism in Bacteria, New York: John Wiley, New York, p 100

3.Chiang C, Knight SG (1960) Nature 188:79

4.Gong CS, Chen LF, Flickinger MC, Chiang LC, Tsao GT (1981) Appl Environ Microbiol 41:430–436

5.Chiang LC, Hsiao HY, Ueng PP, Chen LF, Tsao GT (1981) Biotech Bioeng SymP 11:263–274

6.Rosenfeld S, Stevis P, Ho NWY (1984) Mol Gen Genetics 194:410–415

7.Ho NWY, Stevis P, Rosenfeld S, Huang JJ,Tsao GT (1983) Biotechnol Bioengineering Symposium 13:245–250

8.Sarthy AV et al. (1987) Appl Environ Microb 53:1996–2000

9.Wilhelm M, Hollenberg CP (1984) The EMBO J 3:2555–2560

10.Amore R, Wilhelm M, Hollenberg CP (1989) Appl Mcirobiol Biotechnol 30:351–357

11.Kötter P, Ciriacy M (1993) Appl Microbiol Biotechnol 38:776–783

12.Walfridsson M, Anderlund M, Bao X, Hahn-Hagerdal B (1997) Appl Microbiol Biotechnol 48:218–224

13.Tantirungkij M, Nakashima N, Seki T, Yoshida TJ (1993) Ferment Bioeng 75:83–88

14.Ho NWY, Chen ZD, Brainard A (1993) Genetically engineered yeasts capable of effective fermentation of xylose to ethanol. P 738, Proceedings of Tenth International Symposium on Alcohol Fuels, Colorado Springs, CO, USA. Nov. 7–10

15.Ho NWY, Chen Z, Brainard AP (1998) Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose. Appl Environ Microbiol 64: 1852–1859

16.Takuma S, Nakashima N, Tantirungkij M, Kinoshita S, Okada H, Seki T, Yoshida T (1991) Appl Biochem Biotechnol 28/29:327–340

17.Kotter P, Amore R, Hollenberg CP, Ciriacy M (1990) Curr Genet 18:493–500

18.Ho NWY, Chang SF (1989) Enzyme Microb Technol 11:417–421

19.Ho NWY, Tsao GT Recombinant yeasts for effective fermentation of glucose and xylose. U.S. patent #5,789, 210, issued on 4 August, 1998

20.Burke RL, Tekamp-Olson P, Najarian R (1983) J Biol Chem 258:2193–2201

21.Bennetzen JL, Hall BD (1982) J Biol Chem 257:3018–3025

22.Ammerer G (1983) Methods Enzymol 101:192–201

23.Stratagene, La Jolla, CA

24.Moniruzzaman MB, Dien S, Skory CD, Chen ZD, Hespell RB, Ho NWY, Dale BE, Bothast RJ (1997) World J Microbiol Biotechnol 13:341–346

25.Toon ST, Philippidis GP, Ho NWY, Chen ZD, Brainard A, Lumpkin RE, Riley C (1997) J Appl Biochem Biotechnol 63–65:243

26.Deng XX, Ho NWY (1990) Appl Biochem. Biotechnol 24/25:193–199

27.Ho NWY, Brainard A, Chen Z D (1997) Stable xylose-fermenting Saccharomyces yeasts for the conversion of cellulosic biomass to ethanol developed by using a new method for gene integration, presented at the AIChE annual meeting, Los Angeles, CA, Nov. 16–21

28.Lee N (1978) Molecular aspect for ara regulation. In: Miller J, Reznicoff W (eds) The Operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 389–409

29.Lee N, Gielow W, Martin R, Hamilton E, Fowler A (1986) Gene 47:231–244

30.McMillan JD, Boynton BL (1994) Appl Biochem Biotechnol 45/46:569–584

31.Dien BS, Kurtzman CP, Saha BC, Bothast RJ (1996) Appl Biochem Biotechnol 57/58:233–242

32.Miller JH (1972) Experiments in molecular genetics. Cold Spring Laboratory, 48

33.Chen, Zhengdao, Ho NWY(1993) Appl Biochem Biotechnol 39/40:135–147

34.Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF (1987) Appl Environ Microbiol 53:2420–2425

35.Zhang M, Eddy C, Deanda K, Finkelstein M, Picataggio S (1995) Science 267:240–243

Research Perspectives for Bioconversion of Scrap Paper

H.M. Mühlemann · H.R. Bungay

Howard. P. Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180—3590, USA, e-mail: [e-mail]

Avenues for effective research using cellulose as a substrate for bioconversion that can lead to commercialization are discussed.

Keywords. Bioconversion, Paper, Acetate salts, Calcium and magnesium acetate, Anaerobic digestion Bioreactor design, Bioprocessing of paper

1	Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	193
2	Possible Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	195
3	Laboratory Reactor Design . . . . . . . . . . . . . . . . . . . . . . . .	195
3.1	Head Gases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	197
3.2	Feeding Methods for Cellulose . . . . . . . . . . . . . . . . . . . . . .	197
3.3	Systems for Removal of Fermentation Broth . . . . . . . . . . . . . . .	199
3.4	Measurement of Oxidation/Reduction Potential . . . . . . . . . . . .	199
3.5	Other Instrumentation and Control . . . . . . . . . . . . . . . . . . .	200
3.6	Dilution Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	200
4	Microbial Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	203
5	Other Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	203
6	Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	203
7	Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . .	204
8	Proposed Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . .	205

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

1 Introduction

The carbon cycle in nature uses the energy supplied by sunlight for plants to take atmospheric CO2 to organic fixed carbon that through various chemical or biological steps goes back to atmospheric CO2. Sugars produced by plants are

Advances in Biochemical Engineering /

Biotechnology, Vol. 65

Managing Editor: Th. Scheper

© Springer-Verlag Berlin Heidelberg 1999