新型工业絮凝酵母的构建与乙醇耐性研究
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摘要
絮凝是工业酿酒酵母的重要特征,利用絮凝分离菌体,替代离心分离,可显著降低菌体分离成本,但国内外研究较多的组成型絮凝对生长有抑制作用,如果使用诱导型絮凝菌株,在发酵前期保持游离状态,而发酵结束后开始絮凝沉降,既能保证细胞通过自沉降采收,又可减轻组成型絮凝带来的内扩散效应。此外,选育胁迫耐受性提高的工业酿酒酵母菌株,可以使其在高浓度乙醇等胁迫条件下具有良好的活性,从而提高发酵终点的乙醇浓度,节省下游过程如乙醇精馏和废糟液处理等的能耗。本论文的主要研究目的是构建新型诱导絮凝的工业酿酒酵母,并寻找关键的乙醇耐性相关基因,为选育高效乙醇发酵工业酵母奠定基础。
酵母的絮凝表型可以通过表达絮凝基因来获得。本文克隆了来源于自絮凝酵母SPSCO1的絮凝基因FLO1,并将其整合到工业酿酒酵母菌株Sc4126染色体上,通过SPSCO1中克隆的海藻糖6-磷酸合酶TPS1启动子调控其表达,从而构建了遗传稳定的诱导型絮凝酵母,其絮凝性状可以响应乙醇浓度变化,在发酵前期乙醇浓度较低时絮凝较弱,因此保证酵母细胞能良好生长,在发酵后期乙醇浓度高时絮凝增强,从而实现发酵结束后酵母自沉降采收。所构建的诱导型絮凝酵母的乙醇发酵性能与组成型絮凝酵母相比显著改善,其生长和发酵接近游离酵母宿主。进一步通过模式菌株S288c来源的TPS1启动子对絮凝性状实现精细调控,由于模式酵母S288c的TPSI启动子胁迫响应原件STRE个数比SPSCO1中克隆的TPS1启动子少,所构建的絮凝酵母对乙醇的响应得以弱化,延迟了絮凝的发生并降低了絮凝强度,从而进一步提高了发酵效率,而且在发酵终点酵母可以完全沉降实现菌体与产物的自动分离。
酵母细胞的乙醇耐受性由多基因调控,改造转录因子是提高酵母细胞乙醇耐受性的有效策略。前期研究发现,培养基中添加锌离子可以显著提高酵母细胞的乙醇耐性。因此进一步深入研究了锌离子提高酵母细胞乙醇耐性的机理,从而寻找关键的代谢调控基因进行耐性菌株的构建。利用150g/L葡萄糖的培养基进行乙醇连续发酵,在培养基中添加0.05g/L的硫酸锌后,酵母细胞的乙醇耐受性提高,乙酸和丙酮酸等副产物及胞内活性氧水平下降,絮凝性也下降,但细胞的氮和麦角固醇含量提高。转录组与蛋白组分析共检测到330个转录表达差异基因和71个蛋白表达差异基因,表明锌离子可能通过调节细胞全局基因表达水平对代谢产生影响。转录组分析和蛋白组分析结果不完全一致,说明存在大量转录与翻译水平不一致的情况,推测锌对细胞代谢的调控存在转录和翻译后水平的修饰。受锌离子调控的主要代谢通路包括核糖体合成、糖酵解与乙醇生成、磷脂与麦角固醇合成、ROS响应及相关的氧化还原系统、细胞絮凝、胞内锌离子平衡及转录因子激活或抑制等。可以考虑选取这些途径中的相关酶和差异表达的转录因子作为后续乙醇耐性改造的靶点。还发现了94个功能未知的基因响应锌离子的添加,为进一步探讨锌离子提高酵母菌乙醇耐性的机理,并对关键的调控基因进行代谢工程改造奠定了基础。最后利用人工锌指蛋白技术进行了基因组工程改造,证明人工转录因子技术可以有效选育乙醇耐性提高的工业酿酒酵母。本论文的研究为进一步选育高效乙醇发酵的工业酿酒酵母、提高乙醇发酵效率奠定了基础。
Flocculation of Saccharomyces cerevisiae strains has raised great interest in industry due to its advantage in biomass recovery by sedimentation instead of centrifugation that is required by regular non-flocculating yeast, which consequently saves capital investment on centrifuges and energy consumption on centrifuge operation. However, constitutive flocculation of yeast cells has the disadvantage of slow growth and ethanol fermentation caused by mass transfer limitation. Therefore, inducible flocculation of yeast cells with the flocculation onset near the end of the fermentation is preferred. Moreover, improved ethanol tolerance ensures high yeast viability under high gravity fermentation conditions, and thus is important for fuel ethanol production to improve ethanol titer and save energy consumption for downstream processes such as ethanol distillation and stillage treatment. In this work, a platform for developing inducible flocculating yeast strains was established, and molecular mechanism underlying improved ethanol tolerance by zinc supplementation was investigated by transcriptomic and proteomic analysis, through which key genes involved in stress tolerance were proposed for developing robust industrial yeast strains for efficient ethanol fermentation.
The flocculation phenotypes of S. cerevisiae were controlled by the expression of FLO genes, and thus the gene FLO1was cloned from the self-flocculating yeast SPSC01by PCR amplification, which was subsequently integrated into the chromosome of a non-flocculating industrial yeast Sc4126under the control of the trehalose-6-phosphate synthase1(TPS1) promoter from SPSC01, endowing the genetically modified strain an inducible flocculating phenotype in response to ethanol concentration. As a result, the flocculation of the recombinant strain was weak at low ethanol concentration, but was improved with ethanol accumulation. Almost all yeast cells flocculated at the end of the fermentation, which were recovered by sedimentation. Compared to the constitutive flocculation developed with the same host, no mass transfer limitation was observed with the inducible flocculating strain, which significantly improved its growth and ethanol fermentation performance. When TPS1cloned from the model yeast strain S288c with less STREs (stress response element) in the promoter region than that from SPSC01was employed, the response of the transformant's flocculation to ethanol stress was attenuated, retarding the occurrence of yeast flocculation, and in the meantime the flocculation strength was weakened, which further improved its growth and ethanol fermentation.
Ethanol tolerance of yeast cells is regulated by multiple genes, and thus can be improved by engineering global transcriptional factors. In the previous studies, it was found that zinc supplementation improved ethanol tolerance of the self-flocculating yeast SPSC01. Further study was performed in this work to reveal the mechanism underlying this phenomenon, providing potential targets in the key pathways for metabolic engineering. When continuous ethanol fermentation with the medium containing150g/L glucose was performed under0.05g/L zinc sulfate supplementation conditions, improved yeast viability to ethanol-shock treatment was observed, and production of by-products such as acetic acid and pyruvate acid was decreased. It was also found that the intracellular ROS level was down-regulated, and nitrogen and ergosterol contents were both increased. Transcriptomic and proteomic analysis revealed a total of330differentially expressed genes in transcriptional levels and71differentially expressed genes in translational levels, indicating the global effect of Zn2+on the metabolism of yeast cells. The inconsistency of the transcriptional and translational variations provides insights on the abundant post-transcriptional or post-translational regulations of genes affected by Zn2+, which include the synthesis of ribosome proteins, glycolysis, ergosterol synthesis and phospholipids metabolism. On the other hand, ROS response and intracellular redox balance, as well as zinc homeostasis were also identified to be impacted by the zinc supplementation. Activation or repression of several transcriptional factors was proposed as potential targets for engineering yeast cells with improved ethanol tolerance. Moreover,94genes with unknown functions associated with the response of Zn2were also identified, which will be further studied for their roles in ethanol tolerance.
Finally, artificial transcription factor technique employing zinc finger protein was proved to efficiently alter the global network of yeast metabolism, which results in breeding stable industrial yeasts with improved ethanol tolerance, and provides basis for further developing robust industrial yeasts for more efficient ethanol fermentation.
酵母的絮凝表型可以通过表达絮凝基因来获得。本文克隆了来源于自絮凝酵母SPSCO1的絮凝基因FLO1,并将其整合到工业酿酒酵母菌株Sc4126染色体上,通过SPSCO1中克隆的海藻糖6-磷酸合酶TPS1启动子调控其表达,从而构建了遗传稳定的诱导型絮凝酵母,其絮凝性状可以响应乙醇浓度变化,在发酵前期乙醇浓度较低时絮凝较弱,因此保证酵母细胞能良好生长,在发酵后期乙醇浓度高时絮凝增强,从而实现发酵结束后酵母自沉降采收。所构建的诱导型絮凝酵母的乙醇发酵性能与组成型絮凝酵母相比显著改善,其生长和发酵接近游离酵母宿主。进一步通过模式菌株S288c来源的TPS1启动子对絮凝性状实现精细调控,由于模式酵母S288c的TPSI启动子胁迫响应原件STRE个数比SPSCO1中克隆的TPS1启动子少,所构建的絮凝酵母对乙醇的响应得以弱化,延迟了絮凝的发生并降低了絮凝强度,从而进一步提高了发酵效率,而且在发酵终点酵母可以完全沉降实现菌体与产物的自动分离。
酵母细胞的乙醇耐受性由多基因调控,改造转录因子是提高酵母细胞乙醇耐受性的有效策略。前期研究发现,培养基中添加锌离子可以显著提高酵母细胞的乙醇耐性。因此进一步深入研究了锌离子提高酵母细胞乙醇耐性的机理,从而寻找关键的代谢调控基因进行耐性菌株的构建。利用150g/L葡萄糖的培养基进行乙醇连续发酵,在培养基中添加0.05g/L的硫酸锌后,酵母细胞的乙醇耐受性提高,乙酸和丙酮酸等副产物及胞内活性氧水平下降,絮凝性也下降,但细胞的氮和麦角固醇含量提高。转录组与蛋白组分析共检测到330个转录表达差异基因和71个蛋白表达差异基因,表明锌离子可能通过调节细胞全局基因表达水平对代谢产生影响。转录组分析和蛋白组分析结果不完全一致,说明存在大量转录与翻译水平不一致的情况,推测锌对细胞代谢的调控存在转录和翻译后水平的修饰。受锌离子调控的主要代谢通路包括核糖体合成、糖酵解与乙醇生成、磷脂与麦角固醇合成、ROS响应及相关的氧化还原系统、细胞絮凝、胞内锌离子平衡及转录因子激活或抑制等。可以考虑选取这些途径中的相关酶和差异表达的转录因子作为后续乙醇耐性改造的靶点。还发现了94个功能未知的基因响应锌离子的添加,为进一步探讨锌离子提高酵母菌乙醇耐性的机理,并对关键的调控基因进行代谢工程改造奠定了基础。最后利用人工锌指蛋白技术进行了基因组工程改造,证明人工转录因子技术可以有效选育乙醇耐性提高的工业酿酒酵母。本论文的研究为进一步选育高效乙醇发酵的工业酿酒酵母、提高乙醇发酵效率奠定了基础。
Flocculation of Saccharomyces cerevisiae strains has raised great interest in industry due to its advantage in biomass recovery by sedimentation instead of centrifugation that is required by regular non-flocculating yeast, which consequently saves capital investment on centrifuges and energy consumption on centrifuge operation. However, constitutive flocculation of yeast cells has the disadvantage of slow growth and ethanol fermentation caused by mass transfer limitation. Therefore, inducible flocculation of yeast cells with the flocculation onset near the end of the fermentation is preferred. Moreover, improved ethanol tolerance ensures high yeast viability under high gravity fermentation conditions, and thus is important for fuel ethanol production to improve ethanol titer and save energy consumption for downstream processes such as ethanol distillation and stillage treatment. In this work, a platform for developing inducible flocculating yeast strains was established, and molecular mechanism underlying improved ethanol tolerance by zinc supplementation was investigated by transcriptomic and proteomic analysis, through which key genes involved in stress tolerance were proposed for developing robust industrial yeast strains for efficient ethanol fermentation.
The flocculation phenotypes of S. cerevisiae were controlled by the expression of FLO genes, and thus the gene FLO1was cloned from the self-flocculating yeast SPSC01by PCR amplification, which was subsequently integrated into the chromosome of a non-flocculating industrial yeast Sc4126under the control of the trehalose-6-phosphate synthase1(TPS1) promoter from SPSC01, endowing the genetically modified strain an inducible flocculating phenotype in response to ethanol concentration. As a result, the flocculation of the recombinant strain was weak at low ethanol concentration, but was improved with ethanol accumulation. Almost all yeast cells flocculated at the end of the fermentation, which were recovered by sedimentation. Compared to the constitutive flocculation developed with the same host, no mass transfer limitation was observed with the inducible flocculating strain, which significantly improved its growth and ethanol fermentation performance. When TPS1cloned from the model yeast strain S288c with less STREs (stress response element) in the promoter region than that from SPSC01was employed, the response of the transformant's flocculation to ethanol stress was attenuated, retarding the occurrence of yeast flocculation, and in the meantime the flocculation strength was weakened, which further improved its growth and ethanol fermentation.
Ethanol tolerance of yeast cells is regulated by multiple genes, and thus can be improved by engineering global transcriptional factors. In the previous studies, it was found that zinc supplementation improved ethanol tolerance of the self-flocculating yeast SPSC01. Further study was performed in this work to reveal the mechanism underlying this phenomenon, providing potential targets in the key pathways for metabolic engineering. When continuous ethanol fermentation with the medium containing150g/L glucose was performed under0.05g/L zinc sulfate supplementation conditions, improved yeast viability to ethanol-shock treatment was observed, and production of by-products such as acetic acid and pyruvate acid was decreased. It was also found that the intracellular ROS level was down-regulated, and nitrogen and ergosterol contents were both increased. Transcriptomic and proteomic analysis revealed a total of330differentially expressed genes in transcriptional levels and71differentially expressed genes in translational levels, indicating the global effect of Zn2+on the metabolism of yeast cells. The inconsistency of the transcriptional and translational variations provides insights on the abundant post-transcriptional or post-translational regulations of genes affected by Zn2+, which include the synthesis of ribosome proteins, glycolysis, ergosterol synthesis and phospholipids metabolism. On the other hand, ROS response and intracellular redox balance, as well as zinc homeostasis were also identified to be impacted by the zinc supplementation. Activation or repression of several transcriptional factors was proposed as potential targets for engineering yeast cells with improved ethanol tolerance. Moreover,94genes with unknown functions associated with the response of Zn2were also identified, which will be further studied for their roles in ethanol tolerance.
Finally, artificial transcription factor technique employing zinc finger protein was proved to efficiently alter the global network of yeast metabolism, which results in breeding stable industrial yeasts with improved ethanol tolerance, and provides basis for further developing robust industrial yeasts for more efficient ethanol fermentation.
引文
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