隨著科技發展日新月異，石化燃油為生活中不可缺的必需品，但隨而代之的也增加許多汙染。尋求可替代的能源已成當今的重要議題，可再生資源成為一大新興議題。自然界中的木質纖維素為一新興來源，其主要由木質素、纖維素及半纖維素組合。為了從木質纖維素材料中有效得到小分子醣類，需透過稀酸前處理破壞高度結晶性且複雜的結構，但同時也會產生許多具有毒性的副產品。其會劇烈抑制微生物生長，也會對水解酶酵素產生抑制效果，進而影響生質酒精之產率。
本研究以木質纖維素抑制物中常見之呋喃醛類化合物 (furans compound) 作為目標抑制物，透過基因工程之設計，建構出可以耐受更高濃度抑制物的菌株，並測試菌株發酵的能力是否不受抑制物影響。同時，分別探討兩面向，其一，針對在含有抑制物的環境下利用葡萄糖與木糖培養Y.lipolytica探討轉化木糖醇效率；另一方面，針對其發酵作用進行探討。
本研究成功建立目標蛋白HMFH與HMFO在S.cerevisiae 及Y.lipolytica。經由耐受性測試，重組菌株S.cerevisiae 可生長於50 mM HMF；Y.lipolytica可生長於60 mM HMF。接著，蛋白活性測試發現約40小時候，HMF皆能被降解完畢。菌株透過以不同比例的葡萄醣與木糖培養重組Y.lipolytica在含有60 mM HMF環境下，得到木糖與葡萄糖比例 (1:1與1:0.5) 轉化效率最好，可達到約50 %轉化率。最後，以重組菌株S.cerevisiae進行厭氧發酵實驗，於72小時後，其乙醇產率皆可達到80% 以上。
最後，實驗證明該重組酵母菌株可以用於處理抑制物HMF。並且可以以此為模板，對不同木質纖維素水解產物進行後續探討。

Renewable energy source has become an important issue due to the pollution caused by petrochemical fuel. Lignocellulose primarily composed of lignin, cellulose and hemicellulose is a potential source for making renewable energy. In order to effectively release sugar from lignocellulosic materials, it is necessary to pretreat by dilute acid methods. However, many inhibitors are also released during pretreatment. It will inhibit the growth of microorganisms and conversion of ethanol.
In this study, furan compounds were used as target inhibitors. Through genetic engineering, strains that can tolerate higher concentrations of inhibitors were constructed, and tested their ability of the fermentation. Two aspects are discussed separately. Testing the conversion of xylitol by co-metabolism glucose and xylose within inhibitors. Also, the fermentation is performed to verify this experiment.
The study already successfully constructed the target proteins HMFH and HMFO in S. cerevisiae and Y. lipolytica and verified by agarose gel. Through the tolerance test, the recombinant strain S. cerevisiae could tolerant 50 mM HMF and Y. lipolytica can tolerant 60 mM HMF. Next, testing the protein activity showed that HMF could be degraded completely in around 40 hours. Different proportions of glucose and xylose with 60 mM HMF were used to culture the recombinant Y. lipolytica strain. The ratio of xylose to glucose (1:1 and 1:0.5) had the best result, xylitol conversion rate was about 50%. Last, using recombinant strain S. cerevisiae in the anaerobic fermentation to produce ethanol, the ethanol yield could reach more than 80% after 72 hours.
Therefore, the recombinant yeast strain can be used for inhibitors in detoxifying lignocellulosic hydrolysates and may be widely used for the treatment of other inhibitors.
 

1. Chow, J., R.J. Kopp, and P.R. Portney, Energy Resources and Global Development. Science, 2003. 302(5650): p. 1528-1531.
2. Sanderson, K., A field in ferment. Nature, 2006. 444: p. 673.
3. Taherzadeh, M.J. and K. Karimi, Pretreatment of Lignocellulosic Wastes to Improve Ethanol and Biogas Production: A Review. International Journal of Molecular Sciences, 2008. 9(9): p. 1621-1651.
4. Klinke, H., A. Bjerre, and B. Ahring, Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Vol. 66. 2004. 10-26.
5. Larsson, S., et al., The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme and Microbial Technology, 1999. 24(3): p. 151-159.
6. Palmqvist, E. and B. Hahn-Hägerdal, Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresource Technology, 2000. 74(1): p. 25-33.
7. Jing, X., X. Zhang, and J. Bao, Inhibition performance of lignocellulose degradation products on industrial cellulase enzymes during cellulose hydrolysis. Applied biochemistry and biotechnology, 2009. 159(3): p. 696-707.
8. Wierckx, N., et al., Microbial degradation of furanic compounds: biochemistry, genetics, and impact. Applied Microbiology and Biotechnology, 2011. 92(6): p. 1095-1105.
9. Palmqvist, E. and B. Hahn-Hägerdal, Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresource Technology, 2000. 74(1): p. 17-24.
10. Abril, D. and A. Abril, Ethanol from lignocellulosic biomass. Vol. 36. 2009. 163-176.
11. Koopman, F., et al., Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14. Proceedings of the National Academy of Sciences of the United States of America, 2010. 107(11): p. 4919-4924.
12. Dijkman, W. and M. Fraaije, Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688. Vol. 80. 2013.
13. Koopman, F., et al., Efficient whole-cell biotransformation of 5-(hydroxymethyl)furfural into FDCA, 2,5-furandicarboxylic acid. Bioresource Technology, 2010. 101(16): p. 6291-6296.
14. Ulbricht, R.J., S.J. Northup, and J.A. Thomas, A review of 5-hydroxymethylfurfural (HMF) in parenteral solutions. Toxicological Sciences, 1984. 4(5): p. 843-853.
15. Bardet, M., D.R. Robert, and K. Lundquist, On the reactions and degradation of the lignin during steam hydrolysis of aspen wood. Svensk papperstidning, 1985. 88(6): p. 61-67.
16. Wilson, J.J., L. Deschatelets, and N.K. Nishikawa, Comparative fermentability of enzymatic and acid hydrolysates of steam-pretreated aspenwood hemicellulose by Pichia stipitis CBS 5776. Applied microbiology and biotechnology, 1989. 31(5-6): p. 592-596.
17. Van Zyl, C., B.A. Prior, and J.C. Du Preez, Production of ethanol from sugar cane bagasse hemicellulose hydrolyzate byPichia stipitis. Applied Biochemistry and Biotechnology, 1988. 17(1-3): p. 357-369.
18. Palmqvist, E., et al., Main and interaction effects of acetic acid, furfural, and p‐hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnology and bioengineering, 1999. 63(1): p. 46-55.
19. Larsson, S., et al., Comparison of different methods for the detoxification of lignocellulose hydrolyzates of spruce. Applied biochemistry and biotechnology, 1999. 77(1-3): p. 91-103.
20. Pienkos, P.T. and M. Zhang, Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates. Cellulose, 2009. 16(4): p. 743-762.
21. Parawira, W. and M. Tekere, Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production. Critical reviews in biotechnology, 2011. 31(1): p. 20-31.
22. Cantarella, M., et al., Comparison of different detoxification methods for steam-exploded poplar wood as a substrate for the bioproduction of ethanol in SHF and SSF. Process Biochemistry, 2004. 39(11): p. 1533-1542.
23. Jönsson, L.J., B. Alriksson, and N.-O. Nilvebrant, Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnology for biofuels, 2013. 6(1): p. 16.
24. Feron, V.J., et al., Aldehydes: occurrence, carcinogenic potential, mechanism of action and risk assessment. Mutation Research/Genetic Toxicology, 1991. 259(3): p. 363-385.
25. Jesus, Z., M. Alfredo, and I.L. O., Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli. Biotechnology and Bioengineering, 1999. 65(1): p. 24-33.
26. Almeida, J., et al., Metabolic effects of furaldehydes and impacts on biotechnological processes. Vol. 82. 2009. 625-38.
27. Allen, S.A., et al., Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae. Biotechnology for Biofuels, 2010. 3(1): p. 2.
28. Zhang, J., et al., Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation. Biotechnology for Biofuels, 2010. 3(1): p. 26.
29. Hao, X.-C., et al., Comparative proteomic analysis of a new adaptive Pichia Stipitis strain to furfural, a lignocellulosic inhibitory compound. Biotechnology for Biofuels, 2013. 6(1): p. 34.
30. Modig, T., G. Lidén, and M.J. Taherzadeh, Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochemical Journal, 2002. 363(Pt 3): p. 769-776.
31. Mills, T.Y., N.R. Sandoval, and R.T. Gill, Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnology for Biofuels, 2009. 2(1): p. 26.
32. Almeida João, R.M., et al., Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. Journal of Chemical Technology & Biotechnology, 2007. 82(4): p. 340-349.
33. Dijkman, W.P. and M.W. Fraaije, Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688. ASM journal, 2014. 80: p. 1082-1090.
34. Walker, G.M., Yeast Physiology and Biotechnology. 1998: Wiley.
35. A. Johnson, E., Biotechnology of non-Saccharomyces yeasts - The ascomycetes. Vol. 97. 2012.
36. Nicaud, J.-M., Yarrowia lipolytica. Yeast, 2012. 29(10): p. 409-418.
37. A Johnson, E., Biotechnology of non-Saccharomyces yeasts – the Basidiomycetes. Vol. 97. 2013.
38. Zinjarde, S., et al., Yarrowia lipolytica and pollutants: Interactions and applications. Biotechnology Advances, 2014. 32(5): p. 920-933.
39. Rodriguez, G.M., et al., Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway. Biotechnology for biofuels, 2016. 9(1): p. 149.
40. Türker, M., Yeast Biotechnology: Diversity and Applications. 2014.
41. Aminoff, C., E. Vanninen, and T. Doty, Xylitol--occurrence, manufacture and properties. Oral health, 1978. 68(4): p. 28-29.
42. Verdi, R. and L. Hood, Advantages of alternative sweetener blends. Food technology, 1993.
43. Mäkinen, K.K., Gastrointestinal disturbances associated with the consumption of sugar alcohols with special consideration of Xylitol: scientific review and instructions for dentists and other health-care professionals. International journal of dentistry, 2016. 2016.
44. reddy shetty, P., S. Ravella, and P.J. Hobbs, Current trends in biotechnological production of xylitol and future prospects. Vol. 3. 2009. 8-36.
45. Hyvönen, L., P. Koivistoinen, and F. Voirol, Food technological evaluation of xylitol, in Advances in food research. 1982, Elsevier. p. 373-403.
46. Prakasham, R., R.S. Rao, and P.J. Hobbs, Current trends in biotechnological production of xylitol and future prospects. Curr Trends Biotechnol Pharm, 2009. 3(1): p. 8-36.
47. Rao, R.S., et al., Xylitol production by Candida sp.: parameter optimization using Taguchi approach. Process Biochemistry, 2004. 39(8): p. 951-956.
48. Rao, R.S., et al., Xylitol production from corn fiber and sugarcane bagasse hydrolysates by Candida tropicalis. Bioresource Technology, 2006. 97(15): p. 1974-1978.
49. Zhu, Q. and E.N. Jackson, Metabolic engineering of Yarrowia lipolytica for industrial applications. Current Opinion in Biotechnology, 2015. 36: p. 65-72.
50. Wang, M., M. Wu, and H. Huo, Life-cycle energy and greenhouse gas emission impacts of different corn ethanol plant types. Environmental Research Letters, 2007. 2(2): p. 024001.
51. Wyman, C.E., Biomass ethanol: technical progress, opportunities, and commercial challenges. Annual Review of Energy and the Environment, 1999. 24(1): p. 189-226.
52. Dolejšová, M., The GutHub: Designing Edible Interactions and Fermented Friendships.
53. Taherzadeh, M., et al., Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae. Applied microbiology and biotechnology, 2000. 53(6): p. 701-708.
54. Sitepu, I., et al., Carbon source utilization and inhibitor tolerance of 45 oleaginous yeast species. Journal of industrial microbiology & biotechnology, 2014. 41(7): p. 1061-1070.
55. Li, Y.-C., et al., Inhibitor tolerance of a recombinant flocculating industrial Saccharomyces cerevisiae strain during glucose and xylose co-fermentation. Brazilian Journal of Microbiology, 2017. 48(4): p. 791-800.