Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, 47907, USA.
Microbial Chemistry, Department of Chemistry - Ångström, Uppsala University, 751 20, Uppsala, Sweden; Section of Plant Biochemistry, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
Metab Eng. 2019 Dec;56:77-84. doi: 10.1016/j.ymben.2019.08.014. Epub 2019 Aug 27.
Photosynthetic microorganisms are increasingly being investigated as a sustainable alternative to existing bio-industrial processes, converting CO into desirable end products without the use of carbohydrate feedstock. The Calvin-Benson-Bassham (CBB) cycle is the main pathway of carbon fixation metabolism in photosynthetic organisms. In this study, we analyzed the metabolic fluxes in two strains of the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) that overexpressed fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) and transketolase (TK), respectively. These two potential carbon flux control enzymes in the CBB cycle had previously been shown to improve biomass accumulation when overexpressed under air and low light (15 μmol m s) conditions (Liang and Lindblad, 2016). We measured the growth rates of Synechocystis under atmospheric and high (3% v/v) CO conditions at 80 μmol m s. Surprisingly, the cells overexpressing transketolase (tktA) demonstrated no significant increase in growth rates when CO was increased, suggesting an altered carbon flux distribution and a potential metabolic bottleneck in carbon fixation. Moreover, the tktA strain had an increased susceptibility to oxidative stress under high light as revealed by its chlorotic phenotype under high light conditions. In contrast, the fructose-1,6/sedoheptulose-1,7-bisphosphatase (70glpX) and wild-type cells demonstrated increases in growth rates as expected. To investigate the disparate phenotypical responses of these different Synechocystis strains, isotopically non-stationary metabolic flux analysis (INST-MFA) was used to estimate the carbon flux distribution of tktA, 70glpX, and a kanamycin-resistant control (Km), under atmospheric conditions. In addition, untargeted label-free proteomics, which can detect changes in relative enzymatic abundance, was employed to study the possible effects caused by overexpressing each enzyme. Fluxomic and proteomic results indicated a decrease in oxidative pentose phosphate pathway activity when either FBP/SBPase or TK were overexpressed, resulting in increased carbon fixation efficiency. These results are an example of the integration of multiple omic-level experimental techniques and can be used to guide future metabolic engineering efforts to improve performances and efficiencies.
光合微生物作为现有生物工业过程的可持续替代物，正在越来越受到关注，它们可以在不使用碳水化合物原料的情况下将 CO 转化为理想的终产物。卡尔文-本森-巴斯汉姆（Calvin-Benson-Bassham，CBB）循环是光合生物中碳固定代谢的主要途径。在这项研究中，我们分析了两种单细胞蓝藻集胞藻 PCC 6803（集胞藻）菌株的代谢通量，这两种菌株分别过表达了果糖-1,6/七碳糖-1,7-双磷酸酶（FBP/SBPase）和转酮醇酶（TK）。这两种 CBB 循环中的潜在碳通量控制酶之前已经被证明在空气和低光（15 μmol m s）条件下过表达时可以提高生物量积累（Liang 和 Lindblad，2016）。我们在 80 μmol m s 的大气和高（3% v/v）CO 条件下测量了集胞藻的生长速率。令人惊讶的是，过表达转酮醇酶（tktA）的细胞在 CO 增加时并没有显著提高生长速率，这表明碳通量分布发生了改变，碳固定可能存在代谢瓶颈。此外，高光照下 tktA 菌株的表型为黄化，这表明其对氧化应激的敏感性增加。相比之下，果糖-1,6/七碳糖-1,7-双磷酸酶（70glpX）和野生型细胞的生长速率如预期的那样增加。为了研究这些不同集胞藻菌株的不同表型反应，我们使用非稳态同位素标记代谢通量分析（INST-MFA）来估计 tktA、70glpX 和卡那霉素抗性对照（Km）在大气条件下的碳通量分布。此外，还采用无靶向标记的蛋白质组学来研究过表达每种酶可能引起的变化，蛋白质组学可以检测相对酶丰度的变化。通量组学和蛋白质组学的结果表明，当 FBP/SBPase 或 TK 过表达时，氧化戊糖磷酸途径的活性降低，导致碳固定效率提高。这些结果是整合多种组学水平的实验技术的一个例子，可以用于指导未来的代谢工程努力，以提高性能和效率。
