Research Progress in the Regulation of Isobutanol Synthesis Pathway in Saccharomyces cerevisiae

doi:10.13560/j.cnki.biotech.bull.1985.2022-1350

Abstract

Abstract:

As one of the microorganism commonly used in industrial production, Saccharomyces cerevisiae is of low pH resistance and strong stress resistance. The use of microorganisms to produce bio-based products has become an important development direction of green biomanufacturing. Isobutanol is a kind of branched-chain alcohol, which has a good application prospect in the chemical industry, energy, and other fields. As a biofuel, the synthesized isobutanol from S. cerevisiae is a sustainable, renewable, economical and environment-friendly approach to meet the needs of human development. The construction of an efficient isobutanol synthesizing S. cerevisiae cell factory may enable the green manufacturing of isobutanol as a biofuel. However, the ability of S. cerevisiae to synthesize isobutanol has limited the industrial development of isobutanol, the rational metabolic regulation strategy combined with synthetic biology is one of the effective methods to improve the ability of microbial isobutanol synthesis. This review summarized the latest developments in the regulating strategies of isobutanol biosynthesis in S. cerevisiae, including promoting isobutanol synthesis pathways, blocking competitive pathways, balancing cofactors, relocating synthetic pathways, regulating transcription factors, and improving microbial isobutanol tolerance. These strategies have significantly improved the conversion and yield of isobutanol in engineering yeast, but there are still many deficiencies in the industrial production of isobutanol. In order to promote the industrial production of isobutanol, the current bottlenecks and their solutions for isobutanol production in S. cerevisiae were discussed, aiming to provide valuable information for the green manufacturing of isobutanol.

Key words: Saccharomyces cerevisiae, isobutanol, biofuel, metabolic regulation, synthetic biology

CHENG Ting, YUAN Shuai, ZHANG Xiao-yuan, LIN Liang-cai, LI Xin, ZHANG Cui-ying. Research Progress in the Regulation of Isobutanol Synthesis Pathway in Saccharomyces cerevisiae[J]. Biotechnology Bulletin, 2023, 39(7): 80-90.

Figures/Tables 3

References 55

[1]	Zörgö E, Chwialkowska K, Gjuvsland AB, et al. Ancient evolutionary trade-offs between yeast ploidy states[J]. PLoS Genet, 2013, 9(3): e1003388.
[2]	田宇, 王义强, 王启业. 异丁醇生物合成的研究进展[J]. 生物技术通报, 2013(5): 40-44.
	Tian Y, Wang YQ, Wang QY. Research progress of isobutanol biosynthesis[J]. Biotechnol Bull, 2013(5): 40-44.
[3]	Lakshmi NM, Binod P, Sindhu R, et al. Microbial engineering for the production of isobutanol: current status and future directions[J]. Bioengineered, 2021, 12(2): 12308-12321. doi: 10.1080/21655979.2021.1978189 pmid: 34927549
[4]	Promdonkoy P, Mhuantong W, Champreda V, et al. Improvement in D-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering[J]. J Ind Microbiol Biotechnol, 2020, 47(6/7): 497-510. doi: 10.1007/s10295-020-02281-9 URL
[5]	Eden A, Van Nedervelde L, Drukker M, et al. Involvement of branched-chain amino acid aminotransferases in the production of fusel alcohols during fermentation in yeast[J]. Appl Microbiol Biotechnol, 2001, 55(3): 296-300. pmid: 11341309
[6]	Chen ECH. The relative contribution of Ehrlich and biosynthetic pathways to the formation of fusel alcohols[J]. J Am Soc Brew Chem, 1978, 36(1): 39-43.
[7]	Kłosowski G, Mikulski D, Macko D, et al. Influence of various yeast strains and selected starchy raw materials on production of higher alcohols during the alcoholic fermentation process[J]. Eur Food Res Technol, 2015, 240(1): 233-242. doi: 10.1007/s00217-014-2323-8 URL
[8]	Larroy C, Fernández MR, González E, et al. Characterization of the Saccharomyces cerevisiae YMR318C(ADH6)gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction[J]. Biochem J, 2002, 361(Pt 1): 163-172. doi: 10.1042/0264-6021:3610163 pmid: 11742541
[9]	李杨, 张爱利, 高玉菡, 等. 酿酒酵母菌中过量表达BAT2和缺失PDC6提高异丁醇产率[J]. 中国科技论文, 2015, 10(12): 1443-1449.
	Li Y, Zhang AL, Gao YH, et al. Overexpression of BAT2 and deletion of PDC6 to increase isobutanol in Saccharomyces cerevisiae[J]. China Sci, 2015, 10(12): 1443-1449.
[10]	Funovics M, Montet X, Reynolds F, et al. Nanoparticles for the optical imaging of tumor selectin[J]. Neoplasia, 2005, 7(10): 904-911. pmid: 16242073
[11]	Lane S, Zhang YF, Yun EJ, et al. Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae[J]. Biotechnol Bioeng, 2020, 117(2): 372-381. doi: 10.1002/bit.v117.2 URL
[12]	Generoso WC, Schadeweg V, Oreb M, et al. Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers[J]. Curr Opin Biotechnol, 2015, 33: 1-7. doi: 10.1016/j.copbio.2014.09.004 URL
[13]	冯鹏鹏, 孙丽静, 肖冬光, 等. 啤酒酵母高级醇的代谢与调控研究进展[J]. 食品研究与开发, 2021, 42(8): 153-159.
	Feng PP, Sun LJ, Xiao DG, et al. Research progress on metabolism and regulation of higher alcohols in beer yeast[J]. Food Res Dev, 2021, 42(8): 153-159.
[14]	Chen X, Nielsen KF, Borodina I, et al. Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism[J]. Biotechnol Biofuels, 2011, 4: 21. doi: 10.1186/1754-6834-4-21 URL
[15]	Kondo T, Tezuka H, Ishii J, et al. Genetic engineering to enhance the Ehrlich pathway and alter carbon flux for increased isobutanol production from glucose by Saccharomyces cerevisiae[J]. J Biotechnol, 2012, 159(1/2): 32-37. doi: 10.1016/j.jbiotec.2012.01.022 URL
[16]	González E, Fernández MR, Marco D, et al. Role of Saccharomyces cerevisiae oxidoreductases Bdh1p and Ara1p in the metabolism of acetoin and 2, 3-butanediol[J]. Appl Environ Microbiol, 2010, 76(3): 670-679. doi: 10.1128/AEM.01521-09 URL
[17]	Li P, Guo XW, Shi TT, et al. Reducing diacetyl production of wine by overexpressing BDH1 and BDH2 in Saccharomyces uvarum[J]. J Ind Microbiol Biotechnol, 2017, 44(11): 1541-1550. doi: 10.1007/s10295-017-1976-2 URL
[18]	White WH, Gunyuzlu PL, Toyn JH. Saccharomyces cerevisiae is capable of de Novo pantothenic acid biosynthesis involving a novel pathway of beta-alanine production from spermine[J]. J Biol Chem, 2001, 276(14): 10794-10800. doi: 10.1074/jbc.M009804200 pmid: 11154694
[19]	Wess J, Brinek M, Boles E. Improving isobutanol production with the yeast Saccharomyces cerevisiae by successively blocking competing metabolic pathways as well as ethanol and glycerol formation[J]. Biotechnol Biofuels, 2019, 12: 173. doi: 10.1186/s13068-019-1486-8
[20]	Ida K, Ishii J, Matsuda F, et al. Eliminating the isoleucine biosynthetic pathway to reduce competitive carbon outflow during isobutanol production by Saccharomyces cerevisiae[J]. Microb Cell Fact, 2015, 14: 62. doi: 10.1186/s12934-015-0240-6 URL
[21]	Bricker DK, Taylor EB, Schell JC, et al. A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans[J]. Science, 2012, 337(6090): 96-100. doi: 10.1126/science.1218099 pmid: 22628558
[22]	Lee KM, Kim SK, Lee YG, et al. Elimination of biosynthetic pathways for l-valine and l-isoleucine in mitochondria enhances isobutanol production in engineered Saccharomyces cerevisiae[J]. Bioresour Technol, 2018, 268: 271-277. doi: 10.1016/j.biortech.2018.07.150 URL
[23]	Holmberg S, Litske Petersen JG. Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae[J]. Curr Genet, 1988, 13(3): 207-217. pmid: 3289762
[24]	Tavoulari S, Thangaratnarajah C, Mavridou V, et al. The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state[J]. EMBO J, 2019, 38(10): e100785.
[25]	Park SH, Kim S, Hahn JS. Improvement of isobutanol production in Saccharomyces cerevisiae by increasing mitochondrial import of pyruvate through mitochondrial pyruvate carrier[J]. Appl Microbiol Biotechnol, 2016, 100(17): 7591-7598. doi: 10.1007/s00253-016-7636-z URL
[26]	高玉菡, 李敬知, 张爱利. 酿酒酵母的甘油和乙醇生物合成对异丁醇产率的影响[J]. 中国科技论文, 2015, 10(18): 2145-2151.
	Gao YH, Li JZ, Zhang AL. The effect of glycerol and ethanol biosynthesis on the yield of isobutanol in Saccharomyces cerevisiae[J]. China Sci, 2015, 10(18): 2145-2151.
[27]	Saint-Prix F, Bönquist L, Dequin S. Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation[J]. Microbiology(Reading), 2004, 150(Pt 7): 2209-2220.
[28]	Milne N, Wahl SA, van Maris AJA, et al. Excessive by-product formation: a key contributor to low isobutanol yields of engineered Saccharomyces cerevisiae strains[J]. Metab Eng Commun, 2016, 3: 39-51. doi: 10.1016/j.meteno.2016.01.002 pmid: 29142820
[29]	Park SH, Kim S, Hahn JS. Metabolic engineering of Saccharomyces cerevisiae for the production of isobutanol and 3-methyl-1-butanol[J]. Appl Microbiol Biotechnol, 2014, 98(21): 9139-9147. doi: 10.1007/s00253-014-6081-0 URL
[30]	李敬知, 冯瑞琪, 张爱利. 过量表达酿酒酵母ZWF1基因对异丁醇产量的影响[J]. 中国科技论文, 2017, 12(6): 607-613.
	Li JZ, Feng RQ, Zhang AL. Effect of ZWF1 gene overexpression in Saccharomyces cerevisiae on the production of isobutanol[J]. China Sci, 2017, 12(6): 607-613.
[31]	Gambacorta FV, Wagner ER, Jacobson TB, et al. Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway[J]. Synth Syst Biotechnol, 2022, 7(2): 738-749. doi: 10.1016/j.synbio.2022.02.007 pmid: 35387233
[32]	Tan SZ, Manchester S, Prather KLJ. Controlling central carbon metabolism for improved pathway yields in Saccharomyces cerevisiae[J]. ACS Synth Biol, 2016, 5(2): 116-124. doi: 10.1021/acssynbio.5b00164 URL
[33]	Park SH, Hahn JS. Development of an efficient cytosolic isobutanol production pathway in Saccharomyces cerevisiae by optimizing copy numbers and expression of the pathway genes based on the toxic effect of α-acetolactate[J]. Sci Rep, 2019, 9(1): 3996. doi: 10.1038/s41598-019-40631-5
[34]	Avalos JL, Fink GR, Stephanopoulos G. Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols[J]. Nat Biotechnol, 2013, 31(4): 335-341. doi: 10.1038/nbt.2509 pmid: 23417095
[35]	Lee WH, Seo SO, Bae YH, et al. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes[J]. Bioprocess Biosyst Eng, 2012, 35(9): 1467-1475. doi: 10.1007/s00449-012-0736-y URL
[36]	Outten CE, Albetel AN. Iron sensing and regulation in Saccharomyces cerevisiae: ironing out the mechanistic details[J]. Curr Opin Microbiol, 2013, 16(6): 662-668. doi: 10.1016/j.mib.2013.07.020 pmid: 23962819
[37]	Camire EJ, Grossman JD, Thole GJ, et al. The yeast Nbp35-Cfd1 cytosolic iron-sulfur cluster scaffold is an ATPase[J]. J Biol Chem, 2015, 290(39): 23793-23802. doi: 10.1074/jbc.M115.667022 pmid: 26195633
[38]	Martínez-Pastor MT, Perea-García A, Puig S. Mechanisms of iron sensing and regulation in the yeast Saccharomyces cerevisiae[J]. World J Microbiol Biotechnol, 2017, 33(4): 75. doi: 10.1007/s11274-017-2215-8 URL
[39]	Hammer SK, Avalos JL. Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis[J]. Metab Eng, 2017, 44: 302-312. doi: S1096-7176(17)30303-8 pmid: 29037781
[40]	Deng C, Wu YK, Lv XQ, et al. Refactoring transcription factors for metabolic engineering[J]. Biotechnol Adv, 2022, 57: 107935. doi: 10.1016/j.biotechadv.2022.107935 URL
[41]	Pang SS, Duggleby RG. Expression, purification, characterization, and reconstitution of the large and small subunits of yeast acetohydroxyacid synthase[J]. Biochemistry, 1999, 38(16): 5222-5231. doi: 10.1021/bi983013m pmid: 10213630
[42]	Lee K, Hahn JS. Interplay of Aro80 and GATA activators in regulation of genes for catabolism of aromatic amino acids in Saccharo-myces cerevisiae[J]. Mol Microbiol, 2013, 88(6): 1120-1134. doi: 10.1111/mmi.2013.88.issue-6 URL
[43]	Wang YP, Liu L, Wang XS, et al. GAT1 gene, the GATA transcription activator, regulates the production of higher alcohol during wheat beer fermentation by Saccharomyces cerevisiae[J]. Bioengineering(Basel), 2021, 8(5): 61.
[44]	Natarajan K, Meyer MR, Jackson BM, et al. Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast[J]. Mol Cell Biol, 2001, 21(13): 4347-4368. doi: 10.1128/MCB.21.13.4347-4368.2001 pmid: 11390663
[45]	Vallejo B, Orozco H, Picazo C, et al. Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking[J]. FEMS Yeast Res, 2017, 17(1): 10.1093/femsyr/fow106 doi: 10.1093/femsyr/fow106
[46]	Eder M, Sanchez I, Brice C, et al. QTL mapping of volatile compound production in Saccharomyces cerevisiae during alcoholic fermentation[J]. BMC Genom, 2018, 19(1): 166. doi: 10.1186/s12864-018-4562-8
[47]	Zhang W, Shao W, Zhang A. Isobutanol tolerance and production of Saccharomyces cerevisiae can be improved by engineering its TATA-binding protein Spt15[J]. Lett Appl Microbiol, 2021, 73(6): 694-707. doi: 10.1111/lam.v73.6 URL
[48]	Su YD, Shao WJ, Zhang AL, et al. Improving isobutanol tolerance and titers through EMS mutagenesis in Saccharomyces cerevisiae[J]. FEMS Yeast Res, 2021, 21(2): foab012.
[49]	温智慧, 李敬知, 冯瑞琪, 等. EMS诱变高异丁醇耐受性酿酒酵母的筛选[J]. 中国酿造, 2018, 37(10): 66-71.
	Wen ZH, Feng RQ, Su YD, et al. Screening of Saccharomyces cerevisiae with high isobutanol tolerance by EMS mutagenesis[J]. China Brew, 2018, 37(10): 66-71.
[50]	邵文举, 鲁尚昆, 张爱利. 酿酒酵母SRP40基因对细胞耐受性影响的研究[J]. 微生物学报, 2022, 62(3): 1150-1165.
	Shao WJ, Lu SK, Zhang AL. Effect of SRP40 gene on cell tolerance of Saccharomyces cerevisiae[J]. Acta Microbiol Sin, 2022, 62(3): 1150-1165.
[51]	Ha GS, Saha S, Basak B, et al. High-throughput integrated pretreatment strategies to convert high-solid loading microalgae into high-concentration biofuels[J]. Bioresour Technol, 2021, 340: 125651. doi: 10.1016/j.biortech.2021.125651 URL
[52]	El-Dalatony MM, Saha S, Govindwar SP, et al. Biological conversion of amino acids to higher alcohols[J]. Trends Biotechnol, 2019, 37(8): 855-869. doi: S0167-7799(19)30023-X pmid: 30871798
[53]	Hammer SK, Avalos JL. Harnessing yeast organelles for metabolic engineering[J]. Nat Chem Biol, 2017, 13(8): 823-832. doi: 10.1038/nchembio.2429 pmid: 28853733
[54]	Das M, Patra P, Ghosh A. Metabolic engineering for enhancing microbial biosynthesis of advanced biofuels[J]. Renew Sustain Energy Rev, 2020, 119: 109562. doi: 10.1016/j.rser.2019.109562 URL
[55]	Boudaoud S, Aouf C, Devillers H, et al. Sourdough yeast-bacteria interactions can change ferulic acid metabolism during fermentation[J]. Food Microbiol, 2021, 98: 103790. doi: 10.1016/j.fm.2021.103790 URL

Strain background	Pathway localization	Cofactor-balance	Overexpressing isobutanol synthesis pathway	Block isobutanol competing pathway	Transcription factor regulation	Improved isobutanol tolerance	Isobutanol titer /（mg·L^-1）	Isobutanol yield/ （mg·g^-1 carbon source）	Carbon source/Medium properties/Fermentation conditions	Reference
YPH499			√	√			143.0	6.6	Glucose/defined/microaerbic	[15]
CEN.PK2-1C			√	√	√		376.9	*	Glucose/defined/aerobic	[29]
YPH499		√	√	√			224.0	12.0	Glucose/defined/aerobic	[20]
CEN.PK2-1C	√			√			330.9	*	Glucose/defined/aerobic	[25]
CEN.PK2-1C		√	√	√	√		1 245.0	12.5	Glucose/defined/aerobic	[37]
D452-2	√	√		√			662.0	6.7	Glucose/defined/microaerbic	[22]
CEN.PK113-7D	√			√			2 090.0	59.6	Glucose/defined/aerobic	[1]
CEN.PK2-1C	√			√	√		263.2	*	Glucose/defined/aerobic	[33]
CEN.PK113-5D	√	√					190.0	*	Glucose/defined/anaerobic	[31]
D452-2	√		√				151.0	*	Glucose/complex/microaerbic	[35]
W303-1A			√			√	404.0	10.1	Glucose/complex/microaerbic	[48]
W303-1A			√		√	√	556.0	*	Glucose/complex/microaerbic	[47]
SR8	√		√				2 600.0	*	xylose/defined/aerobic	[11]
BY4742	√		√			√	92.9	*	xylose/defined/aerobic	[4]

Strain background	Pathway localization	Cofactor-balance	Overexpressing isobutanol synthesis pathway	Block isobutanol competing pathway	Transcription factor regulation	Improved isobutanol tolerance	Isobutanol titer /（mg·L^-1）	Isobutanol yield/ （mg·g^-1 carbon source）	Carbon source/Medium properties/Fermentation conditions	Reference
YPH499			√	√			143.0	6.6	Glucose/defined/microaerbic	[15]
CEN.PK2-1C			√	√	√		376.9	*	Glucose/defined/aerobic	[29]
YPH499		√	√	√			224.0	12.0	Glucose/defined/aerobic	[20]
CEN.PK2-1C	√			√			330.9	*	Glucose/defined/aerobic	[25]
CEN.PK2-1C		√	√	√	√		1 245.0	12.5	Glucose/defined/aerobic	[37]
D452-2	√	√		√			662.0	6.7	Glucose/defined/microaerbic	[22]
CEN.PK113-7D	√			√			2 090.0	59.6	Glucose/defined/aerobic	[1]
CEN.PK2-1C	√			√	√		263.2	*	Glucose/defined/aerobic	[33]
CEN.PK113-5D	√	√					190.0	*	Glucose/defined/anaerobic	[31]
D452-2	√		√				151.0	*	Glucose/complex/microaerbic	[35]
W303-1A			√			√	404.0	10.1	Glucose/complex/microaerbic	[48]
W303-1A			√		√	√	556.0	*	Glucose/complex/microaerbic	[47]
SR8	√		√				2 600.0	*	xylose/defined/aerobic	[11]
BY4742	√		√			√	92.9	*	xylose/defined/aerobic	[4]