酵母甘油代谢工程与基因组重排构建乙醇高产菌株及相关机理研究
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摘要
乙醇是已被大规模生产与应用的可再生生物能源之一。但目前乙醇生产的总体效率效益低。拥有优良的高产酿酒酵母菌株,实现高效率酒精发酵是提高乙醇生产效益的重要途径之一。优良工业酿酒酵母菌株的选育以往主要采用自然筛选和随机诱变育种的传统方法。应用杂交、细胞融合、基因工程等技术构建优良酿酒酵母菌株有颇多论文报道,但获得工业化大规模应用的高水平成果尚鲜见。其主要原因是高产酒精的优良酿酒酵母菌株需具备耐高糖浓度、耐乙醇、耐酸、适应的温度范围宽、生长与发酵速度快、糖醇转化率高等多重优良性能。而这些性能均非单基因所能控制,性能及其发挥所涉及的许多遗传与调控因子、作用途径与机制等尚待研究,单种剧烈的育种手段,常常导致菌株关键性能退化或衰变。因此获得能大规模生产应用的高产优良酿酒酵母菌株,研究难度极大。本文在总结前人经验的基础上,运用诱变、杂交、细胞工程、基因工程与多种方法相结合的基因组重排等方法的单项改进与集成创新,改良、选育、构建高产优良酿酒酵母菌株,并对有关机制进行了研究探索。研究获得如下结果：
1、筛选优良工业酿酒酵母出发菌株诱导三株工业酿酒酵母菌株THA、S25和C87产孢获得90株单倍体。筛选四株高乙醇产量单倍体z1、Z4、Z8和Z9作为后续育种的出发菌株。将两株交配型确定的出发菌株z1和Z4杂交,获得的杂交重组子在230g/L葡萄糖的醪液中发酵。重组子z14乙醇产量最高,达到100.05g/L,比zl和Z4的亲株THA和S25分别提高1.75%和3.04%。
2、利用RAPD-SCAR分子标记筛选细胞融合重组子针对不具有产孢和交配能力的出发菌株z8和Z9,获得菌株特异的RAPD(随机扩增多态性DNA, Random Amplified Polymorphic DNA)-SCAR(特异序列特征性扩增区域,Sequence Characterized Amplified Region)分子标记,替代传统育种的营养缺陷型标记,筛选菌株Z8和Z9的电融合重组子。在含230g/L葡萄糖的醪液中发酵,重组子与出发菌株Z8和Z9相比,乙醇产量提高4.33～8.08%。
3、实验菌株体系的甘油代谢改造基于酵母甘油代谢调控机制,以降低甘油生成、提高乙醇产量为目标。敲除实验菌株4741内源基因FPS1(编码甘油跨膜运输的通道蛋白Fpslp),并同时表达变链球菌Streptococcus mutans的gapN基因(编码NADP+依赖型3-磷酸甘油醛脱氢酶GAPN)获得改造菌株4FG。与对照菌株相比,改造菌株4FG副产物甘油和乙酸产量分别降低21.47%和27.09%,乙醇产量提高9.18%。因此,在实验菌株体系敲除FPS1基因并同时表达异源gapN基因可有效降低副产物甘油和乙酸生成,提高乙醇产量。
4、工业酵母菌株体系的甘油代谢改造对交配型相异的出发菌株Z1(MATα)和Z4(MATa)分别进行甘油代谢改造,应用kanMX和Zeocin抗性标记替代营养缺陷型标记,敲除FPS1基因并整合表达异源gapN基因。将不同抗性标记的改造菌株KFG (MATa fps1ΔgapN-kanMX)和ZFG (MATa fpslΔgapN-Zeocin)进行杂交,G418-Zeocin双抗平板高效筛选获得杂交重组子FG1(MATa/αfps1ΔgapN-kanMX fpslΔgapN-Zeocin)。在含250g/L葡萄糖醪液中发酵,与Z1和Z4的重组子Z14(MATa/α)相比,基因改造重组子FG1的副产物甘油和乙酸产量分别降低18.14%和25.04%,乙醇产量(114.00g/L)提高4.14%。在工业菌株体系进行相同甘油代谢改造可同样改良发酵性状,但效果与实验菌株体系有所差异。
5、通过改进的全基因组重排技术提高菌株乙醇耐受力以提高酵母乙醇耐受力为育种目标,在甘油代谢改造的基础上,利用紫外和EMS两种诱变方式对菌株KFG (MATαfpslΔgapN-kanMX)和ZFG (MATa fpslΔgapN-Zeocin)进行诱变,为后续全基因组重排提供更加多样化的变异菌群。经过两轮全基因组重排及G418-Zeocin双抗平板的高效筛选,获得乙醇耐受力提高的重组子A1。在8%(v/v)乙醇胁迫条件下,重组子A1最大比生长速率比对照菌株FGl提高23.83%。细胞膜完整性分析表明,重组子A1在乙醇胁迫下细胞膜完整性较高,这可能是其耐受力提高的原因之一。同时,高糖(含285g/L葡萄糖)发酵结果表明,重组子A1发酵前30h乙醇生成速率是对照菌株FG1的1.4倍,发酵时间缩短,最终乙醇产量高达117.61g/L,比FG1提高3.91%。
Ethanol is one kind of the most important and well-established reproducible biofuel. However, there are several issues and obstacles reminded in the technology of ethanol production. The achievement of fermentation under ultrahigh ethanol concentration opens a new and important avenue to increase the efficiency of ethanol production, and the basic requirement is the development of Saccharomyces cerevisiae strains with high ethanol-production. Random mutagenesis and screening is the leading method for the development of industrial yeast strains. There are many studies which report hybridization, protoplast fusion and genetic engineering for breeding strains, but few can be applied in a large-scale industrial production. Because the industrial yeasts should maintain mutilple performances, such as stress tolerance, fast growth and fermentation rate, which are based on complex metabolic and regulatory networks. It is difficult to improve complex phenotypes of yeasts for industrial production by single technology. Based on previsious studies, we improved and combined hybridization, protoplast fusion, metabolic engineering and whole genome shuffling for strain improvement, and explored the mechanisam. The results were shown as follows:
1. Screening industrial S. cerevisiae yeasts as original strains for industrial breeding. Among the ninety haploids of three industrial strains THA, S25 and C87, four high ethanol-producing strains Z1, Z4, Z8 and Z9 were selected as the original strains for industrial breeding. The hybrids of two haploids Zl and Z4 with mating ability were obtained by hybridization. On a sweet distillery mash (230g/L glucose), the hybrid Z14 with the highest ethanol yield (100.05g/L) increased the ethanol production by 1.75% and 3.04%, compared with the parent strain THA and S25 of haploid Z1 and Z4, respectively.
2. Development and application of RAPD-SCAR markers to identify hybrids of industrial S. cerevisiae. For the original strain Z8 and Z9 without sporulation and mating ability, RAPD (Random Amplified Polymorphic DNA)-SCAR (Sequence Characterized Amplified Region) markers were developed. Instead of traditional auxotrophic markers, the RAPD-SCAR markers were applied to identify the hybrids from protoplast fusion. On a sweet distillery mash (230g/L glucose), hybrids increased ethanol production by 4.33～8.08%, compared with their parent strain Z8 and Z9.
3. Glycerol metabolic engineering in a laboratory strain. To reduce glycerol yield and increase ethanol production, the laboratory strain 4741 was engineered by expressing GAPN (NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase) of Streptococcus mutans and deleting the FPS1 gene (encoding a channel protein Fps1p for glycerol export). The resulting strain 4FG increased the ethanol production by 9.18%, while decreased the yield of glycerol and acetic acid by 21.47% and 27.09%, respectively. The deletion of FPS1 and expression of gapN could be combined to improve the fermentation of yeast strain.
4. Glycerol metabolic engineering in industrial strains. In the original strain Z1 (MATa) and Z4 (MATa), the FPS1 gene was deleted and the gapN gene (encoding GAPN of Streptococcus mutans) was integrated at the locus of the FPS1 gene with kanMX- or Zeocin-resistance marker. The G418-Zeocin drug plate could efficiently select the hybrid FG1 (MATa/αfps1ΔgapN-kanMX fps1ΔgapN-Zeocin) of the engineered strain KFG (MATαfps1ΔgapN-kanMX) and ZFG (MATa fps1ΔgapN-Zeocin). In fermentation medium contaning 250g/L glucose, the ethanol yield of the engineered hybrid FG1 was up to 114.00g/L. Compared with the hybrid Z14(MATa/α) of the original strain Zland Z4, FG1 increased the ethanol production by 4.14%, while decreased the formation of glycerol and acetic acid by 18.14% and 25.04%, respectively.
5. Improving ethanol tolerance in S. cerevisiae by the improved whole genome shuffling. Strain A1 was obtained after two rounds of genome shuffling of UV and EMS mutants derived from the engineered strain KFG and ZFG. Under the stress of 8%(v/v) ethanol, maximum specific growth rate of shuffled strain A1 increased 23.83%, compared with the control strain FG1. The protection of cell membrane integrality maybe the reason of the improved ethanol tolerance in strain A1. In fermentation medium contaning 285g/L glucose, ethanol yield rate of the shuffled strain A1 was 1.4-fold than that of the control strain FG1 during the first 30h. Moreover, the ethanol production of the shuffled strain A1 was up to 117.61g/L, and increased by 3.91%, compared with the control strain FG1.
1、筛选优良工业酿酒酵母出发菌株诱导三株工业酿酒酵母菌株THA、S25和C87产孢获得90株单倍体。筛选四株高乙醇产量单倍体z1、Z4、Z8和Z9作为后续育种的出发菌株。将两株交配型确定的出发菌株z1和Z4杂交,获得的杂交重组子在230g/L葡萄糖的醪液中发酵。重组子z14乙醇产量最高,达到100.05g/L,比zl和Z4的亲株THA和S25分别提高1.75%和3.04%。
2、利用RAPD-SCAR分子标记筛选细胞融合重组子针对不具有产孢和交配能力的出发菌株z8和Z9,获得菌株特异的RAPD(随机扩增多态性DNA, Random Amplified Polymorphic DNA)-SCAR(特异序列特征性扩增区域,Sequence Characterized Amplified Region)分子标记,替代传统育种的营养缺陷型标记,筛选菌株Z8和Z9的电融合重组子。在含230g/L葡萄糖的醪液中发酵,重组子与出发菌株Z8和Z9相比,乙醇产量提高4.33～8.08%。
3、实验菌株体系的甘油代谢改造基于酵母甘油代谢调控机制,以降低甘油生成、提高乙醇产量为目标。敲除实验菌株4741内源基因FPS1(编码甘油跨膜运输的通道蛋白Fpslp),并同时表达变链球菌Streptococcus mutans的gapN基因(编码NADP+依赖型3-磷酸甘油醛脱氢酶GAPN)获得改造菌株4FG。与对照菌株相比,改造菌株4FG副产物甘油和乙酸产量分别降低21.47%和27.09%,乙醇产量提高9.18%。因此,在实验菌株体系敲除FPS1基因并同时表达异源gapN基因可有效降低副产物甘油和乙酸生成,提高乙醇产量。
4、工业酵母菌株体系的甘油代谢改造对交配型相异的出发菌株Z1(MATα)和Z4(MATa)分别进行甘油代谢改造,应用kanMX和Zeocin抗性标记替代营养缺陷型标记,敲除FPS1基因并整合表达异源gapN基因。将不同抗性标记的改造菌株KFG (MATa fps1ΔgapN-kanMX)和ZFG (MATa fpslΔgapN-Zeocin)进行杂交,G418-Zeocin双抗平板高效筛选获得杂交重组子FG1(MATa/αfps1ΔgapN-kanMX fpslΔgapN-Zeocin)。在含250g/L葡萄糖醪液中发酵,与Z1和Z4的重组子Z14(MATa/α)相比,基因改造重组子FG1的副产物甘油和乙酸产量分别降低18.14%和25.04%,乙醇产量(114.00g/L)提高4.14%。在工业菌株体系进行相同甘油代谢改造可同样改良发酵性状,但效果与实验菌株体系有所差异。
5、通过改进的全基因组重排技术提高菌株乙醇耐受力以提高酵母乙醇耐受力为育种目标,在甘油代谢改造的基础上,利用紫外和EMS两种诱变方式对菌株KFG (MATαfpslΔgapN-kanMX)和ZFG (MATa fpslΔgapN-Zeocin)进行诱变,为后续全基因组重排提供更加多样化的变异菌群。经过两轮全基因组重排及G418-Zeocin双抗平板的高效筛选,获得乙醇耐受力提高的重组子A1。在8%(v/v)乙醇胁迫条件下,重组子A1最大比生长速率比对照菌株FGl提高23.83%。细胞膜完整性分析表明,重组子A1在乙醇胁迫下细胞膜完整性较高,这可能是其耐受力提高的原因之一。同时,高糖(含285g/L葡萄糖)发酵结果表明,重组子A1发酵前30h乙醇生成速率是对照菌株FG1的1.4倍,发酵时间缩短,最终乙醇产量高达117.61g/L,比FG1提高3.91%。
Ethanol is one kind of the most important and well-established reproducible biofuel. However, there are several issues and obstacles reminded in the technology of ethanol production. The achievement of fermentation under ultrahigh ethanol concentration opens a new and important avenue to increase the efficiency of ethanol production, and the basic requirement is the development of Saccharomyces cerevisiae strains with high ethanol-production. Random mutagenesis and screening is the leading method for the development of industrial yeast strains. There are many studies which report hybridization, protoplast fusion and genetic engineering for breeding strains, but few can be applied in a large-scale industrial production. Because the industrial yeasts should maintain mutilple performances, such as stress tolerance, fast growth and fermentation rate, which are based on complex metabolic and regulatory networks. It is difficult to improve complex phenotypes of yeasts for industrial production by single technology. Based on previsious studies, we improved and combined hybridization, protoplast fusion, metabolic engineering and whole genome shuffling for strain improvement, and explored the mechanisam. The results were shown as follows:
1. Screening industrial S. cerevisiae yeasts as original strains for industrial breeding. Among the ninety haploids of three industrial strains THA, S25 and C87, four high ethanol-producing strains Z1, Z4, Z8 and Z9 were selected as the original strains for industrial breeding. The hybrids of two haploids Zl and Z4 with mating ability were obtained by hybridization. On a sweet distillery mash (230g/L glucose), the hybrid Z14 with the highest ethanol yield (100.05g/L) increased the ethanol production by 1.75% and 3.04%, compared with the parent strain THA and S25 of haploid Z1 and Z4, respectively.
2. Development and application of RAPD-SCAR markers to identify hybrids of industrial S. cerevisiae. For the original strain Z8 and Z9 without sporulation and mating ability, RAPD (Random Amplified Polymorphic DNA)-SCAR (Sequence Characterized Amplified Region) markers were developed. Instead of traditional auxotrophic markers, the RAPD-SCAR markers were applied to identify the hybrids from protoplast fusion. On a sweet distillery mash (230g/L glucose), hybrids increased ethanol production by 4.33～8.08%, compared with their parent strain Z8 and Z9.
3. Glycerol metabolic engineering in a laboratory strain. To reduce glycerol yield and increase ethanol production, the laboratory strain 4741 was engineered by expressing GAPN (NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase) of Streptococcus mutans and deleting the FPS1 gene (encoding a channel protein Fps1p for glycerol export). The resulting strain 4FG increased the ethanol production by 9.18%, while decreased the yield of glycerol and acetic acid by 21.47% and 27.09%, respectively. The deletion of FPS1 and expression of gapN could be combined to improve the fermentation of yeast strain.
4. Glycerol metabolic engineering in industrial strains. In the original strain Z1 (MATa) and Z4 (MATa), the FPS1 gene was deleted and the gapN gene (encoding GAPN of Streptococcus mutans) was integrated at the locus of the FPS1 gene with kanMX- or Zeocin-resistance marker. The G418-Zeocin drug plate could efficiently select the hybrid FG1 (MATa/αfps1ΔgapN-kanMX fps1ΔgapN-Zeocin) of the engineered strain KFG (MATαfps1ΔgapN-kanMX) and ZFG (MATa fps1ΔgapN-Zeocin). In fermentation medium contaning 250g/L glucose, the ethanol yield of the engineered hybrid FG1 was up to 114.00g/L. Compared with the hybrid Z14(MATa/α) of the original strain Zland Z4, FG1 increased the ethanol production by 4.14%, while decreased the formation of glycerol and acetic acid by 18.14% and 25.04%, respectively.
5. Improving ethanol tolerance in S. cerevisiae by the improved whole genome shuffling. Strain A1 was obtained after two rounds of genome shuffling of UV and EMS mutants derived from the engineered strain KFG and ZFG. Under the stress of 8%(v/v) ethanol, maximum specific growth rate of shuffled strain A1 increased 23.83%, compared with the control strain FG1. The protection of cell membrane integrality maybe the reason of the improved ethanol tolerance in strain A1. In fermentation medium contaning 285g/L glucose, ethanol yield rate of the shuffled strain A1 was 1.4-fold than that of the control strain FG1 during the first 30h. Moreover, the ethanol production of the shuffled strain A1 was up to 117.61g/L, and increased by 3.91%, compared with the control strain FG1.
引文
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