利用廉价并来源丰富的纤维素资源生产燃料乙醇目前遇到的主要挑战之一，是纤维素酶的成本居高不下。生物联合加工工艺将纤维素酶的产生、糖化和乙醇发酵集中在同一种微生物，降低了生产成本，但重组菌株的酶蛋白合成和分泌效率还很低。重组酿酒酵母菌株中酶的合成和分泌对菌株的代谢造成了负担，但关于该过程中胁迫因素的分子机理，以及如何通过提高外源蛋白表达的胁迫耐受性提高菌株的酶活还未进行研究。本课题将研究胁迫相关基因对重组酿酒酵母菌株蛋白表达和胁迫耐受性的影响，并研究胁迫耐性基因提高重组蛋白纤维素酶酶活和分泌的分子机制，进一步评价所构建的可高产纤维素酶和具有较强环境胁迫耐受性的重组菌株发酵不同纤维素原料生产燃料乙醇的能力。本合作研究不仅将加深我们对酵母中表达外源蛋白相关的环境胁迫相关基因功相关基因功能的理解相关基因功能的理解，还将获得更高效利用纤维素原料生产燃料乙醇的酿酒酵母重组菌株。

Development of alternative and sustainable energy to replace finite fossil fuel resources has become a global demand. Ethanol derived from biomass has been identified as a prime candidate for liquid fuel for the transport sector due to established production technology and minimal impact on existing supply chain infrastructure. However, the commercialization of lignocellulosic ethanol faces significant challenges, primarily stemming from its recalcitrance to cellulase and the lack of low-cost technologies to overcome this obstacle. Thermo-chemical pretreatment of lignocellulose has become accepted due to its ease of execution, however, chemicals released during this process may contribute to inhibitory fermentation conditions. To reduce the production cost, consolidated bioprocess (CBP) has been proposed which uses recombinant yeast cells with the capability of heterologous cellulosic enzyme production, therefore the cost of enzymes required to degrade the biomass substrates can be greatly reduced. However, so far the efficiency of CBP is still not high. The project aims to develop yeast strains that can withstand the harsh conditions involved in producing fuel ethanol from biomass, and efficient cellulosic ethanol production using the engineered strains. Metabolic engineering of the yeast Saccharomyces cerevisiae will be employed to improve stress tolerance of yeast strains. Furthermore, process optimization will also be studied to improve production of cellulosic ethanol. In the recent studies of our group, various genes that showed positive effect on stress tolerance to various enviromental factors (including high concentration of ethanol and acetic acid, as well as high temperature and oxidative stress) were identified, which include genes encoding zinc finger protein for global regulation of celular metabolism and genes encoding metabolic enzymes, as well as genes with unknown functions. The function of such genes in improvement of stres tolernace of heterologous protein production will be explored, and the mechanims of the improved enzyme production and secretion will be studied. In addition, genes that are identified that improve protein expression will be engineered into recombinant yeast strains that produce cellulosic enzymes, and process optimization will also be studied. In South Africa, efforts will be made to engineer genes previously identified by the Chinese group that improve strain robustness into the recombinant CBP yeast strains. Strains with improved robustness to process inhibitors and improved enzyme production ability will then be tested on various substrates that will be used at industrial level to produce biofuel. The joint research proposed in this study will not only deepen our understanding of gene functions on the stress tolerance associated with overexpression of non-native protein expression in yeast, but also will yield efficient yeast strains for cellulosic ethanol production.

发展生物炼制技术，利用木质纤维素资源生产能源和化学品，是解决能源供应安全以及环境保护问题的有效途径之一。但是，目前纤维素生产成本过高是限制木质纤维素生物转化经济性的重要原因。利用生物联合加工技术将产酶、酶解和糖化整合在一起，有利于降低生产成本，但是目前重要的挑战是酿酒酵母纤维素酶分泌水平很有限。本课题组前期研究发现，多个关键基因过表达可提高酿酒酵母环境胁迫耐性，而酿酒酵母生产纤维素酶也对细胞产生一定的胁迫，但是，目前对利用环境胁迫耐性相关基因改造重组酵母提高纤维素酶分泌还没有进行深入研究。本课题对酿酒酵母环境胁迫耐性研究过程中发现的关键基因对提高重组酿酒酵母纤维素酶分泌的作用进行研究，发现了多个可促进纤维素酶分泌的新功能基因。此外，利用环境胁迫耐性基因改造生物联合加工重组酵母菌株，有效提高了纤维素乙醇的生产效率。本课题所获得的结果，不仅丰富了酿酒酵母环境胁迫响应和耐受性的分子机理，也对进一步提高木质纤维素生物转化和生物炼制水平奠定了基础。