丁二烯产量和工艺路线受到石油资源瓶颈和污染环境的限制，以可再生资源C1（甲醇或甲烷）为中心的生物基路线是清洁和可持续生产的创新替代策略，然而目前微生物同化C1效率低，制约目的代谢途径产率。提高C1催化转化就要解决两个核心问题：（1）一碳中间代谢物（甲酸和甲醛）胞内低效同化传递；（2）同化一碳的有效利用。为此本项目提出以甲醇为出发底物，以前期研究的具有工业价值的甲基微菌Methylomicrobium alcaliphilum 20Z为底盘，以合成丁二烯为目标，通过基因组定向进化重构一碳中间代谢物的组合同化传递区域，最少化二氧化碳生成，提高乙酰辅酶A合成以匹配丁二烯合成通量需求；同时途径工程组装丁二烯合成途径，精细调控启动子强度和核糖体结合位点等基因元器件，将高效同化一碳有效导入丁二烯合成。本项目提出丁二烯高效清洁生产的创新技术，同时延伸和增值甲醇工业的产业链，拓展石化产品绿色生产新方向。

Butadiene is an important petrochemical and the production is limited due to fossil resources and industry effect on environment. Renewable C1 resources (i.e. methanol or methane) have great potential for development of green technology platform for butadiene by bacteria. However, the current low efficiency of C1 assimilation of microbial biocatalysis restricts the yield and titer of butadiene. The approach to increase C1 biocatalysis efficiency is to solve the essential problems of (1) low efficiency of one-carbon intermediates assimilation and (2) Utilization of the assimilated carbons in a high yield butadiene production. To this end, we propose to engineer a previously studied gammaproteobacterial Methylomicrobium alcaliphilum 20Z to biocatalyze methanol conversion to butadiene. The methodologies of directed evolution of genome with a high throughput screening will be carried out to construct an efficient combination assimilation pathway of one carbon. This will enhance the flux into the precursor metabolite of acetyl-CoA for butadiene synthesis and in addition minimize the release of greenhouse gas, CO2. Next, the heterologous pathway of butadiene will be assembled and genetic regulatory elements including promoters and ribosome binding site (RBS) will be optimized for maximum flux from acetyl-CoA to butadiene. This proposal provides a novel biotechnology and ideas in engineered bacteria for highly efficient methane-to-butadiene conversion. The proposed strategies will allow the industry to utilize methanol which is currently produced in excess capacity in China. Ultimately, the technologies provide the new and green direction of the petrochemicals production.