Metabolic engineering最新文献

Adeno-associated viruses (AAV) are promising vectors for gene therapy due to their efficacy in vivo. However, there is room for improvement to address key limitations such as the pre-existing immunity to AAV in patients, high-dose toxicity, and relatively low efficiency for some cell types. This study introduces a metabolic engineering approach, using knockout of the enzyme phosphatidylserine synthase 1 (PTDSS1) to increase the abundance of extracellular vesicle-enclosed AAV (EV-AAV) relative to free AAV in the supernatant of producer cells, simplifying downstream purification processes. The lipid-engineered HEK293T-ΔPTDSS1 cell line achieved a 42.7-fold enrichment of EV-AAV9 compared to free AAV9 in the supernatant. The rational genetic strategy also led to a 300-fold decrease of free AAV in supernatant compared to wild-type HEK293T. The membrane-engineered EV-AAV9 (mEV-AAV9) showed unique envelope composition alterations, including cholesterol enrichment and improved transduction efficiency in human AC16 cardiomyocytes by 1.5-fold compared to conventional EV-AAV9 and by 11-fold compared to non-enveloped AAV9. Robust in-vivo transduction four weeks after intraparenchymal administration of mEV-AAV9 was observed in the murine brain. This study shows promise in the potential of lipid metabolic engineering strategies to improve the efficiency and process development of enveloped gene delivery vectors.

Lactate metabolism plays a critical role in mammalian cell bioprocessing, influencing cellular performance and productivity. The transition from lactate production to consumption, known as lactate metabolic shift, is highly beneficial and has been shown to extend culture lifespan and enhance productivity, yet its molecular drivers remain poorly understood. Here, we have explored the mechanisms that underpin this metabolic shift through two case studies, illustrating environmental- and genetic-driven factors. We characterised these study cases at process, metabolic and transcriptomic levels. Our findings indicate that glutamine depletion coincided with the timing of the lactate metabolic shift, significantly affecting cell growth, productivity and overall metabolism. Transcriptome analysis revealed dynamic regulation the ATF4 pathway, involved in the amino acid (starvation) response, where glutamine depletion activates ATF4 gene and its targets. Manipulating ATF4 expression through overexpression and knockdown experiments showed significant changes in metabolism of glutamine and lactate, impacting cellular performance. Overexpression of ATF4 increased cell growth and glutamine consumption, promoting a lactate metabolic shift. In contrast, ATF4 downregulation decreased cell proliferation and glutamine uptake, leading to production of lactate without any signs of lactate shift. These findings underscore a critical role for ATF4 in regulation of glutamine and lactate metabolism, related to phasic patterns of growth during CHO cell culture. This study offers unique insight into metabolic reprogramming during the lactate metabolic shift and the molecular drivers that determine cell status during culture.

Production of recombinant proteins is regarded as an important breakthrough in the field of biomedicine and industrial biotechnology. Due to the complexity of the protein secretory pathway and its tight interaction with cellular metabolism, the application of traditional metabolic engineering tools to improve recombinant protein production faces major challenges. A systematic approach is required to generate novel design principles for superior protein secretion cell factories. Here, we applied a proteome-constrained genome-scale protein secretory model of the yeast Saccharomyces cerevisiae (pcSecYeast) to simulate α-amylase production under limited secretory capacity and predict gene targets for downregulation and upregulation to improve α-amylase production. The predicted targets were evaluated using high-throughput screening of specifically designed CRISPR interference/activation (CRISPRi/a) libraries and droplet microfluidics screening. From each library, 200 and 190 sorted clones, respectively, were manually verified. Out of them, 50% of predicted downregulation targets and 34.6% predicted upregulation targets were confirmed to improve α-amylase production. By simultaneously fine-tuning the expression of three genes in central carbon metabolism, i.e. LPD1, MDH1, and ACS1, we were able to increase the carbon flux in the fermentative pathway and α-amylase production. This study exemplifies how model-based predictions can be rapidly validated via a high-throughput screening approach. Our findings highlight novel engineering targets for cell factories and furthermore shed light on the connectivity between recombinant protein production and central carbon metabolism.

Plecomacrolides, such as concanamycins and bafilomycins, are potent and specific inhibitors of vacuolar-type ATPase. Concanamycins are 18-membered macrolides with promising therapeutic potential against multiple diseases, including viral infection, osteoporosis, and cancer. Due to the complexity of their total synthesis, the production of concanamycins is only achieved through microbial fermentation. However, the low titers of concanamycin A and its analogs in the native producing strains are a significant bottleneck for scale-up, robust structure-activity relationship studies, and drug development. To address this challenge, we designed a library of engineered Streptomyces strains for the overproduction of concanamycin A-C by combining the overexpression of target regulatory genes with the optimization of fermentation media. Integration of two endogenous regulators from the concanamycin biosynthetic gene cluster (cms) and one heterologous regulatory gene from the bafilomycin biosynthetic gene cluster significantly increased production of concanamycin A and its less abundant analog concanamycin B in Streptomyces eitanensis. The highest titers reported to date were observed in the engineered S. eitanensis DHS10676, which produced over 900 mg/L of concanamycin A and 300 mg/L of concanamycin B. Heterologous overexpression of the identified target regulatory genes across a panel of Streptomyces spp. harboring a putative concanamycin biosynthetic gene cluster confirmed its identity, and significantly improved concanamycin A production in all tested strains. Strain engineering, optimization of fermentation, and extraction purification protocols enabled swift access to these structurally complex plecomacrolides for semi-synthetic medicinal chemistry-based approaches. Together, this work established a platform for robust overproduction of concanamycin analogs across species.