优化细胞自絮凝强化生物炼制生产强度和产物浓度及生物量低成本采收

Biomass resources are renewable and environmentally friendly. However, low productivity and product titer associated with biomass conversion make the biorefinery not economically competitive. Low cell density within bioreactors and inhibitory effect on microbial cells exerted by elevated product titer are root reasons for these phenomena. When self-flocculated, not only are cells self-immobilized within bioreactors for high density to improve productivity, but most importantly their tolerance to product inhibition is significantly improved, and high product titer can thus be achieved. In addition, cells can be recovered by cost-effective sedimentation, which is significant for large scale production of microalgae as feedstock for biorefinery.. In this project, self-flocculating Saccharomyces cerevisiae and Zymomonas mobilis are selected as model systems for eukaryotic and prokaryotic microbes, and molecular mechanism underlying their self-flocculation and enhanced tolerance to product inhibition associated with the morphological change will be investigated. Genes responsible for their self-flocculation will be mined from the genomics with functional FLO genes identified, and their expression and regulation controlled at genetic and epigenetic levels will be further elucidated for genetic manipulations. It is hypothesized that the improved tolerance of microbial cells to product inhibition and other environmental stresses is due to enhanced quorum sensing (QS), since the self-flocculation of microbial cells provides extremely high local cell density, and in the meantime creates a unique microenvironment for more efficient transport of signal molecules and signal transduction as well, which will be examined experimentally by identifying signal molecules that mediate the QS process and stress response. At the end, platforms for engineering microbial cells with the self-flocculating phenotype will be established, which would lay a foundation for developing novel technologies to improve productivity and product titer for biorefinery processes as well as harvest microalgae cost-effectively as the potential feedstock for biorefinery.

生物质可再生且环境友好，对可持续发展具有重要意义。然而，生物炼制过程生产强度低和产物浓度低的问题突出，不仅导致反应器容积规模大，设备投资高，而且产品分离能耗高并产生大量废水。此外，微藻作为第三代生物炼制原料，大规模培养后采收成本高是制约其开发利用的瓶颈。细胞密度低是反应器生产强度低的主要原因，而产物对细胞强抑制制约其浓度提高。细胞自絮凝不仅可以在反应器中自固定化提高细胞密度，而且细胞对高浓度产物抑制耐受性显著增强，同时还可以实现自沉降采收。. 本项目以乙醇发酵性能优良自絮凝酿酒酵母和运动发酵单胞菌为真核和原核微生物模式体系，解析细胞自絮凝及导致环境胁迫耐受性增强分子机制，建立改造非絮凝菌株，赋予可控自絮凝表型的平台技术，进而在分子、细胞、群体和过程工程多尺度调控优化细胞自絮凝，为解决生物炼制过程生产强度低和产物浓度低及微藻大规模培养后采收成本高突出问题创新技术开发，奠定科学基础。

微生物发酵转化是生物炼制的核心，但目标产物生产强度低和浓度低的“双低”问题突出，不仅导致反应器容积规模大，设备投资高，下游产品分离能耗高，而且产生大量废水。反应器中细胞密度低是其生产强度低的主要原因，而高浓度产物抑制限制产物浓度提高。细胞自絮凝可以使其在生物反应器中自固定化提高细胞密度，同时能够提高细胞对高浓度产物抑制的耐受性。本项目以自絮凝酿酒酵母和运动单胞菌为真核和原核微生物模式体系，解析细胞自絮凝及导致环境胁迫耐受性增强分子机制，建立改造非絮凝菌株，赋予可控自絮凝表型平台技术，为创新技术开发，解决生物炼制过程“双低”问题奠定科学基础。此外，以秸秆为代表的木质纤维素类生物质是生物炼制基础原料，主要组分纤维素和半纤维素水解糖是微生物发酵产品生产所必须，而纤维素酶是这类水解糖制备的瓶颈。.对自絮凝酿酒酵母菌株，在前期工作解析其自絮凝分子机制基础上，通过比较基因组和转录组分析，发现并鉴明了调控高浓度乙醇及高温胁迫响应的关键基因，构建了耐高浓度乙醇和高发酵温度的工程菌株。鉴明了导致运动单胞菌自絮凝的物质基础为纤维素微丝，阐明了运动单胞菌自絮凝菌株中纤维素合成酶催化结构域BcsA编码基因上游短核苷酸序列缺失突变使其融合到该基因后对BcsA催化功能的影响，是纤维素微丝生物合成及细胞自絮凝的必要条件，而编码催化c-di-GMP降解酶基因的突变导致信使分子胞内积累，激活胞内纤维素生物合成，是运动单胞菌细胞自絮凝的充分条件，建立了改造非絮凝菌株，赋予其可控自絮凝表型技术平台。开发了基于自行合成廉价高效可溶性诱导物流加的液体深层发酵技术，发酵液中滤纸酶活超过90FPU/mL，可以支撑秸秆类生物质生物炼制过程原位产酶直接应用；鉴明了里氏木霉纤维素酶合成调控新转录因子，为理性设计改造菌株奠定了科学基础。.取得的学术成果在国际期刊发表论文21篇，技术成果申请发明专利5项（授权3项）；博士后出站4人，博士生毕业9人，硕士生毕业10人，建立了高水平从事生物炼制基础研究和创新技术开发团队。