Brandt Bianca A, García-Aparicio Maria D P, Görgens Johann F, van Zyl Willem H
Department of Microbiology, Stellenbosch University, Private Bag X1, Stellenbosch, 7602, South Africa.
Department of Process Engineering, Stellenbosch University, Private Bag X1, Stellenbosch, 7602, South Africa.
Biotechnol Biofuels. 2021 Aug 28;14(1):173. doi: 10.1186/s13068-021-02021-w.
The fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable Saccharomyces cerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S. cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential delta-integrations of double-gene cassettes containing one gene conferring broad inhibitor tolerance (ARI1, PAD1 or TAL1) coupled with an inhibitor-specific gene (ADH6, FDH1 or ICT1). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor.
The first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g. TAL1 + FDH1). The TAL1 + ICT1 combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the ICT1 protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes.
The integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some gene combination sequences had a reduced impact on xylose conversion.
将木质纤维素水解产物发酵为乙醇需要强大的能够利用木糖的酿酒酵母菌株，这些菌株要能够在存在微生物抑制胁迫的情况下发挥作用。本研究旨在通过鉴定赋予对多种抑制剂抗性的新基因组合（从而形成累积抑制剂抗性表型），开发对预处理衍生的微生物抑制剂具有更高耐受性的工业酿酒酵母菌株，同时对木糖发酵能力的影响最小。该策略包括对双基因盒进行多次连续的δ整合，双基因盒包含一个赋予广泛抑制剂耐受性的基因（ARI1、PAD1或TAL1）以及一个抑制剂特异性基因（ADH6、FDH1或ICT1）。在生物量生长、乙醇产量和生产率方面，以及在合成抑制剂混合物、甘蔗渣水解产物和硬木亚硫酸盐废液中的解毒能力方面，将转化体的性能与亲本菌株进行了比较。
第一轮和第二轮δ整合转化体在生物量和乙醇产量之间表现出权衡。相对于亲本对照，转化体在2% SC培养基中40%和80% v/v浓度的浓缩亚硫酸盐废液发酵中表现出增强的抑制剂抗性表型。出乎意料的是，与亲本对照相比，转化体的木糖发酵能力降低，但某些基因组合的影响较小（例如TAL1 + FDH1）。TAL1 + ICT1组合对生物量生长和乙醇产量均产生负面影响，这可能与ICT1蛋白由于细胞膜变化而增加转化体对弱酸和温度的敏感性有关。
已证明所选基因的整合可提高对合成或工业水解产物中预处理抑制剂的耐受性，但它们仅限于葡萄糖的发酵。然而，一些基因组合序列对木糖转化的影响较小。
