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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611713
• 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著地扩展了大肠杆菌（E.因此它现在是生物制造中最受欢迎的细胞工厂之一。具体而言，基于重组DNA技术的基因工程允许引入外源基因用于附加型表达。基于位点特异性基因敲入和敲除技术的基因组工程可以实现最佳的基因组编辑。代谢工程使得能够控制和调节关键代谢通量以过量产生目标代谢物。合成生物学允许人们移植外来途径来产生非天然代谢物。拟议中的发现资助计划将寻求一种新的方法，将这些生物技术策略整合起来，以实现E。大肠杆菌菌株用于工业用途。 1-丙醇是一种具有多种工业应用的精细化学品，具有替代乙醇作为替代生物燃料的强大潜力。然而，到目前为止，1-丙醇的生产主要依赖于化学合成，并且没有微生物被鉴定为天然的1-丙醇生产者。野生型E.大肠杆菌作为目标产物进行技术示范，所开发的策略可普遍应用于其他高价值代谢产物的生产。最近，我的研究小组在大肠杆菌中发现了一种新的1-丙醇生物合成途径。在大肠杆菌中通过操纵Sbm操纵子的方式表达。这个四基因操纵子（sbm-ygfD-ygfG-ygfH）编码参与钴胺素依赖性代谢途径的各种酶，将琥珀酸脱羧为丙酸。通过表达Sbm操纵子中的某些基因，使琥珀酸沿着大量异化，并与增加各种前体的可用性的关键基因一起，在工程化的E.大肠杆菌菌株成功地证明。为了提高1-丙醇的生产，拟议的研究计划将针对基于生物技术和生物加工策略的新的系统方法，以解决各种问题，以优化该生物转化系统。与工程菌构建相关的基本生物技术问题包括：（1）寻找和鉴定各种微生物中与1-丙醇生物合成相关的新基因;大肠杆菌的基因组工程改造，使其碳通量向1-丙醇生产途径转移;大肠杆菌中敲入和敲除基因组上影响1-丙醇生产的各种关键基因。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、培养基配方、需氧或厌氧培养、替代的廉价碳源，（2）开发用于分批培养、补料分批培养和恒化器培养的操作方案和控制策略，（3）在各种遗传和生物加工背景下代谢通量的数学建模和分析，以识别限制1-丙醇生产的潜在步骤。 拟议的发现研究计划提供了一个独特的培训计划，在新的科学和工业生物技术。学员将获得生物制造未来职业所需的各种高级技能。除了对与生物制造相关的各种新型生物技术有广泛的科学了解外，开发的1-丙醇生产生物菌株和生物工艺可以很容易地转移到加拿大生物工业进行商业化，从而提高加拿大在生物制造和生物燃料方面的技术领先地位。