Yao Ruilian, Li Jiawei, Feng Lei, Zhang Xuehong, Hu Hongbo
1State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240 China.
2Instrumental Analysis Center, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240 China.
Biotechnol Biofuels. 2019 Feb 13;12:29. doi: 10.1186/s13068-019-1372-4. eCollection 2019.
Bioprocessing offers a sustainable and green approach to manufacture various chemicals and materials. Development of bioprocesses requires transforming common producer strains to cell factories. C metabolic flux analysis (C-MFA) can be applied to identify relevant targets to accomplish the desired phenotype, which has become one of the major tools to support systems metabolic engineering. In this research, we applied C-MFA to identify bottlenecks in the bioconversion of glycerol into acetol by . Valorization of glycerol, the main by-product of biodiesel, has contributed to the viability of biofuel economy.
We performed C-MFA and measured intracellular pyridine nucleotide pools in a first-generation acetol producer strain (HJ06) and a non-producer strain (HJ06C), and identified that engineering the NADPH regeneration is a promising target. Based on this finding, we overexpressed encoding NAD kinase or encoding membrane-bound transhydrogenase either individually or in combination with HJ06, obtaining HJ06N, HJ06P and HJ06PN. The step-wise approach resulted in increasing the acetol titer from 0.91 g/L (HJ06) to 2.81 g/L (HJ06PN). To systematically characterize and the effect of mutation(s) on the metabolism, we also examined the metabolomics and transcriptional levels of key genes in four strains. The pool sizes of NADPH, NADP and the NADPH/NADP ratio were progressively increased from HJ06 to HJ06PN, demonstrating that the sufficient NADPH supply is critical for acetol production. Flux distribution was optimized towards acetol formation from HJ06 to HJ06PN: (1) The carbon partitioning at the DHAP node directed gradually more carbon from the lower glycolytic pathway through the acetol biosynthetic pathway; (2) The transhydrogenation flux was constantly increased. In addition, C-MFA showed the rigidity of upper glycolytic pathway, PP pathway and the TCA cycle to support growth. The flux patterns were supported by most metabolomics data and gene expression profiles.
This research demonstrated how C-MFA can be applied to drive the cycles of design, build, test and learn implementation for strain development. This succeeding engineering strategy can also be applicable for rational design of other microbial cell factories.
生物加工为制造各种化学品和材料提供了一种可持续且绿色的方法。生物加工过程的开发需要将常见的生产菌株转化为细胞工厂。碳代谢通量分析（C-MFA）可用于识别实现所需表型的相关靶点，已成为支持系统代谢工程的主要工具之一。在本研究中，我们应用C-MFA来识别甘油转化为丙酮醇生物转化过程中的瓶颈。甘油作为生物柴油的主要副产物，其增值有助于生物燃料经济的可行性。
我们在第一代丙酮醇生产菌株（HJ06）和非生产菌株（HJ06C）中进行了C-MFA并测量了细胞内吡啶核苷酸库，确定工程化NADPH再生是一个有前景的靶点。基于这一发现，我们单独或与HJ06组合过表达编码NAD激酶的 或编码膜结合转氢酶的 ，获得了HJ06N、HJ06P和HJ06PN。这种逐步方法导致丙酮醇滴度从0.91 g/L（HJ06）提高到2.81 g/L（HJ06PN）。为了系统地表征突变对代谢的影响，我们还检测了四种菌株中关键基因的代谢组学和转录水平。从HJ06到HJ06PN，NADPH、NADP的库大小和NADPH/NADP比值逐渐增加，表明充足的NADPH供应对丙酮醇生产至关重要。从HJ06到HJ06PN，通量分布朝着丙酮醇形成方向优化：（1）二羟丙酮磷酸（DHAP）节点处的碳分配逐渐引导更多来自糖酵解下游途径的碳通过丙酮醇生物合成途径；（2）转氢通量持续增加。此外，C-MFA显示糖酵解上游途径、磷酸戊糖途径（PP途径）和三羧酸循环（TCA循环）对支持生长的刚性。通量模式得到了大多数代谢组学数据和基因表达谱的支持。
本研究展示了C-MFA如何应用于驱动菌株开发的设计、构建、测试和学习实施循环。这种成功的工程策略也可应用于其他微生物细胞工厂的合理设计。
