Metabolic engineering最新文献

Acetivibrio thermocellus (formerly Clostridium thermocellum) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PP_i-dependent 6-phosphofructokinase (PP_i-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PP_i-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable in vitro ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PP_i can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PP_i-generating mechanism to drive glycolysis, as PP_i-Pfk can simply use whatever PP_i is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPP_i-Pfk strain, unable to valorize PP_i, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PP_i. Together with other mutations, this resulted in an A. thermocellus strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/g_x/h. These findings are both relevant for fundamental insight into dual ATP/PP_i Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of A. thermocellus for consolidated bioprocessing.

For mammalian synthetic biology research, multiple orthogonal and tunable gene expression systems have been developed, among which the tetracycline (Tet)-inducible system is a key tool for gain-of-function mutations. Precise and long-lasting regulation of genetic circuits is necessary for the effective use of these systems in genetically engineered stable cell lines. However, current cell line development strategies, which depend on either random or site-specific integration along with antibiotic selection, are unpredictable and unsustainable, limiting their widespread use. To overcome these issues, we aimed to establish a Robust Overexpression via Site-specific integration of Effector (ROSE) system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated streamlined Tet-On3G-inducible master cell line (MCL) development platform. ROSE MCLs equipped with a landing pad facilitated the transcriptional regulation of various effector genes via recombinase-mediated cassette exchange. Long-term investigation revealed that the modular design of genetic payloads and integration sites significantly affected the induction capacity and stability, with ROSE MCLs exhibiting exceptional induction performance. To demonstrate the versatility of our platform, we explored its efficiency for the precise regulation of selection stringency, manufacturing of therapeutic antibodies with tunable expression levels and timing, and transcription factor engineering. Overall, this study demonstrated the effectiveness and reliability of the ROSE platform, highlighting its potential for various biological and biotechnological applications.