Engineer Escherichia coli for 1-propanol production by integrating novel biotechnological and bioprocessing strategies

通过整合新颖的生物技术和生物加工策略，改造大肠杆菌以生产 1-丙醇

• 批准号: RGPIN-2014-05568
• 负责人: Chou, CPerry
• 金额: $ 2.55万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=637956
• 关键词: Engineer; Escherichia; coli; propanol; production

Advances in modern biotechnology have significantly extended the capacity of the bacterium Escherichia coli (E. coli) so that it is now one of the most popular cell factories for biomanufacturing. Specifically, genetic engineering based on recombinant DNA technology allows the introduction of foreign genes for episomal expression. Genomic engineering, based on site-specific gene knock-in and knock-out technology, enables optimum genomic editing. Metabolic engineering enables both control and tuning of key metabolic fluxes to overproduce target metabolites. Synthetic biology allows one to graft foreign pathways to produce non-natural metabolites. The proposed Discovery Grant program will pursue a novel approach that integrates these biotechnological strategies to realize the ultimate goal of “genetic tailoring” of E. coli strains for industrial purposes. 1-Propanol is a fine chemical with various industrial applications and has strong potential to replace ethanol as an alternative biofuel. However, up to now, 1-propanol production primarily relies on chemical synthesis and no microorganisms have been identified as a natural 1-propanol producer. This non-native metabolite of 1-propanol for wild-type E. coli will be used as the target product for technological demonstration and the developed strategies can be generically applied to produce other high-value metabolites. Recently, my research group identified a novel biosynthesis of 1-propanol in E. coli by manipulating the sleeping beauty mutase (Sbm) operon. This four-gene operon (sbm-ygfD-ygfG-ygfH) encodes various enzymes involved in a cobalamin-dependent metabolic pathway for decarboxylation of succinate into propionate. By expressing certain genes within the Sbm operon for extensive dissimilation of succinate along with key genes for increasing the availability of various precursors, heterologous production of 1-propanol in engineered E. coli strains was successfully demonstrated. To enhance 1-propanol production, the proposed research program will target a new systematic approach based on biotechnological and bioprocessing strategies to address various issues to optimize this biotransformation system. Fundamental biotechnological issues associated with strain construction include: (1) search and identification of novel genes involved in biosynthesis of 1-propanol from various microorganisms, (2) metabolic engineering of E. coli to drive the central carbon flux towards 1-propanol production pathway, (3) genomic engineering of E. coli to knock in and knock out various key genes affecting 1-propanol production on the genome. On the other hand, applied bioprocessing issues associated with the cultivation system include: (1) generic characterization of cultivation conditions, such as pH, temperature, medium recipe, aerobic or anaerobic cultivation, alternative cheap carbon sources, (2) development of operating protocols and control strategies for batch, fedbatch, and chemostat cultivations, (3) mathematical modeling and analysis of metabolic fluxes under various genetic and bioprocessing backgrounds to identify potential steps limiting 1-propanol production. The proposed Discovery research program provides a unique training program in novel scientific and industrial biotechnologies. Trainees will obtain a wide range of advanced skills required for future careers in biomanufacturing. In addition to extensive scientific understanding of various novel biotechnologies associated with biomanufacturing, the developed biological strains and bioprocess for 1-propanol production can be readily transferred to the Canadian bio-industry for commercialization, boosting Canada’s technological leadership in biomanufacturing and biofuels.

现代生物技术的进步显着扩大了细菌大肠杆菌（大肠杆菌）的能力，因此它现在是生物制造最受欢迎的细胞工厂之一。基于重组DNA技术的特定基因工程允许引入外源基因以进行偶发表达。基于基于特定地点的基因敲入和敲除技术的基因组工程可实现最佳基因组编辑。代谢工程可以控制和调整关键代谢通量，以使靶向代谢产物过度生产。合成生物学使人们可以将外国途径移植以产生非天然代谢产物。拟议中的发现赠款计划将采用一种新颖的方法，将这些生物技术策略整合在一起，以实现大肠杆菌菌株“基因剪裁”出于工业目的的最终目标。 1-丙醇是一种具有各种工业应用的优质化学物质，并且具有替代乙醇作为替代生物燃料的强大潜力。但是，到现在为止，1-丙醇的产生主要依赖化学合成，并且没有将微生物鉴定为天然的1-丙醇生产剂。这种用于野生型大肠杆菌的1-丙醇的非本地代谢产物将用作技术演示的目标产品，并且开发的策略通常可用于生产其他高价值代谢物。最近，我的研究小组通过操纵“睡美人突变酶（SBM）”歌剧来确定大肠杆菌中1-丙醇的新生物合成。这款四基因歌剧（SBM-YGFD-YGFG-YGFH）编码参与钴胺素依赖性代谢途径的各种酶，用于将琥珀酸酯脱羧为丙酸。通过在SBM歌剧中表达某些基因，以与关键基因以及提高各种前体的可用性相同的广泛伪造，从而成功地证明了工程大肠杆菌菌株中1-丙醇的异源产生。为了增强1-丙醇的生产，拟议的研究计划将基于生物技术和生物处理策略采用新的系统方法，以解决各种问题，以优化这种生物转化系统。与应变构造相关的基本生物技术问题包括：（1）搜索和鉴定来自各种微生物的1-丙醇的新基因，（2）大肠杆菌的代谢工程，以将中央碳的代谢工程推向1-丙醇生产途径，（（3）大肠杆菌的基因组生产和敲除基因的基因组合基因组基因组合。另一方面，与培养系统相关的应用生物处理问题包括：（1）培养条件的一般表征，例如pH，温度，中等食谱，有氧或厌氧培养，替代性廉价碳源，（2）在批处理，FedBatch和化学分析（3）基因生成和化学分析（3）的批量生产，（3）的廉价碳源，（2）背景以识别限制1-丙醇产生的潜在步骤。拟议的发现研究计划提供了新型科学和工业生物技术的独特培训计划。学员将获得未来生物制造业所需的广泛的高级技能。除了对与生物制造相关的各种新型生物技术的广泛科学理解外，开发的生物学菌株和生物生产的生物普罗维斯可以很容易地转移到加拿大的生物学生物中，以促进加拿大加拿大在加拿大的技术领导地位，从而在生物制造和生物制造中提高技术领导力。