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

现代生物技术的进步极大地扩展了大肠杆菌的能力，使其成为最受欢迎的生物制造细胞工厂之一。具体地说，基于重组DNA技术的基因工程允许引入外源基因来进行异体表达。基因组工程，基于特定部位的基因敲入和敲除技术，使最佳的基因组编辑成为可能。代谢工程能够控制和调节关键的代谢通量，以过量生产目标代谢物。合成生物学允许人们嫁接外来途径来产生非天然代谢物。拟议的发现资助计划将寻求一种新的方法，整合这些生物技术战略，以实现用于工业目的的大肠杆菌菌株的“基因剪裁”的最终目标。 1-丙醇是一种具有多种工业用途的精细化学品，具有很强的替代乙醇作为生物燃料的潜力。然而，到目前为止，1-丙醇的生产主要依靠化学合成，还没有一种微生物被鉴定为天然的1-丙醇产生菌。野生型大肠杆菌的非天然正丙醇代谢物将作为技术示范的目标产品，所开发的策略可普遍应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变位酶(SBM)操纵子，在大肠杆菌中发现了一种新的生物合成正丙醇的方法。这个四基因操纵子(SBM-ygfD-ygfG-ygfH)编码多种酶，参与依赖钴胺的代谢途径，使琥珀酸脱羧化为丙酸。通过在SBM操纵子中表达琥珀酸广泛异化的某些基因，以及提高各种前体的利用率的关键基因，成功地在工程菌中证明了异源生产正丙醇。为了提高1-丙醇的产量，拟议的研究计划将以一种基于生物技术和生物加工战略的新的系统方法为目标，以解决各种问题来优化这一生物转化系统。与菌株构建相关的基本生物技术问题包括：(1)从各种微生物中寻找和鉴定参与生物合成正丙醇的新基因；(2)对大肠杆菌进行代谢工程，以驱动中心碳流向正丙醇生产途径；(3)对大肠杆菌进行基因组工程，以敲除基因组上影响正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)培养条件的一般特征，如pH、温度、培养基配方、好氧或厌氧培养、替代廉价碳源；(2)为分批、补料和恒化器培养制定操作规程和控制策略；(3)在不同遗传和生物处理背景下对代谢通量进行数学建模和分析，以确定限制1-丙醇生产的潜在步骤。 拟议的发现号研究计划在新型科学和工业生物技术方面提供了独特的培训计划。受训人员将获得未来生物制造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生物技术有广泛的科学了解外，开发的用于生产1-丙醇的生物菌株和生物工艺可以很容易地转移到加拿大生物产业进行商业化，从而增强加拿大在生物制造和生物燃料方面的技术领先地位。

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.