干酪乳杆菌利用糖蜜合成乙醇的丙酮酸代谢关键酶基因的调控

A plentiful soybean molasses is produced from domestic soybean industry. The by-product of soybean protein concentrate rich in carbohydrates could be used as bioethanol fermentation substrate. At present, the efficiency of production ethanol from soybean molasses by yeast is not satisfactory to bioethanol industry as yeast is incapable of metabolizing raffinose and Stachyose tetrahydrate in soybean molasses. The majority of soybean molasses is utilized as cheap feed or sewerage disposal resulting in wasting valuable resource and contaminating environment. Therefore, it is imperative to develop a microorganism to ferment soybean molasses to synthesize bioethanol. Some lactic acid bacteria strains could not only ferment main carbohydrates of soybean molasses and also have complete ethanol synthesis pathways. Thus it is great potential for lactic acid bacteria applied as catalysts for biomass energy. Generally, most of pyruvate from carbon metabolite flux is converted into lactic acid by the wild type of lactic acid bacterium strain, rather than ethanol, so ethanol production rate is low. In view of this problem, a Lactobacillus casei strain was screened which is characterized with ethanol high-yield on molasses medium. Accordingly, the objective of this project is to realize the key enzyme genes of metabolism pathways of pyruvate converted into ethanol of L. casei strain and their regulation mechanisms. The genomics and metabolomics technology will be used to study the relationship between ethanol production capacity and genetic structure of L. casei, the influence of metabolic conditions on gene expression, the construction of genetic engineering strain by the knockout of L-ldh1 gene linked to lactic acid production and insertion of the PET cassette. The purpose of the project is to illustrate the key enzyme genes of pyruvate converted into ethanol and their expression mechanism, to regulate ethanol metabolism of pyruvate and to construct an ethanol high-yield engineering strain. This subject lays foundation for development and utilization of new biofuel starter cultures. It has important significance for bioethanol production industry.

我国大豆加工业产生大量的糖蜜副产物，其富含碳水化合物，可作为生产乙醇的底物。由于酵母菌不能有效利用糖蜜使其转化为乙醇，目前只能作为低廉的饲料或废弃，造成资源浪费和环境污染。故开发能充分利用糖蜜并使其转化为乙醇的微生物是亟待解决的问题。某些乳酸菌能有效利用大豆糖蜜并具有合成乙醇的完整通路，但通常将大部分丙酮酸转化为乳酸，故乙醇得率低。本项目基于前期获得的高产乙醇的干酪乳杆菌，围绕其碳代谢途径中丙酮酸转化为乙醇关键酶及调控机制，应用基因组学与代谢组学技术，重点研究干酪乳杆菌产乙醇能力与基因结构的关系；培养条件对丙酮酸代谢关键酶基因表达的影响；敲除LDH基因及优化密码子构建工程菌株。目的阐明丙酮酸转化为乙醇的代谢途径关键酶基因及其表达机制，调控菌株的丙酮酸代谢转化为乙醇，构建能利用糖蜜高产乙醇的工程菌株。项目成果对生物质能源新型发酵剂的开发与利用提供理论支持，对生物乙醇制备工业具有重大意义。

大豆糖蜜中丰富的碳水化合物可作为微生物生产乙醇的底物。与其他乙醇生产菌株相比，某些乳酸菌具有利用大豆糖蜜生产生物乙醇的优势：底物选择宽泛、较强的醇耐受力和生产乙醇的完整通路。但乳酸菌通常将大部分丙酮酸转化为乳酸。本项目以前期获得的高产乙醇的工程菌株干酪乳杆菌12A E1为对照材料，分析菌株的醇耐受、碳水化合物代谢及乙醇生产的能力；应用基因组学与代谢组学技术，研究干酪乳杆菌产乙醇能力与基因结构的关系，目的是阐明菌株将碳水化合物转化为丙酮酸后生成乙醇的代谢关键酶基因及其表达机制。结果表明，在pH 6.0的条件下，接种菌株到5°BX的糖蜜中，37℃厌氧发酵36 h后乙醇产量可达到13.77g/L，糖利用率约为78.60%；应用荧光定量PCR技术检测碳源代谢关键酶的基因表达水平发现，菌株对碳源的利用具有先后顺序，且某些条件会抑制菌株对某种碳源的代谢；采用梅里埃API 50 CHL测试菌株的糖代谢特征发现，干酪乳杆菌具有代谢五碳糖和低聚糖的能力；在发酵实验结果中，菌株均可利用大豆糖蜜的主要碳水化合物水苏糖、棉子糖和蔗糖；依据菌株的全基因组序列，分析比较各菌株的代谢途径和代谢关键酶信息，验证菌株的代谢能力，阐述菌株间代谢差异的来源，设计出丙酮酸代谢调控的方案。本研究在分子水平上，阐述了丙酮酸代谢合成乙醇的机制，确定了通过调控丙酮酸代谢途径使干酪乳杆菌成为合成生物乙醇发酵剂的可行性。本项目为乳酸菌合成生物乙醇产业化应用提供理论依据和技术支持，项目成果对生物质能源新型发酵剂的开发与利用提供理论支持，对生物乙醇制备工业具有重大意义。