Efficient biosynthesis of 1,3-PDO, a key bio-based chemical, depends on precise regulation of the host metabolic network. In this study, a heterologous CRISPR-Cas12a genome editing system was established and systematically optimized in Klebsiella pneumoniae, enabling efficient and stable genome editing (75-100% efficiency). Using on this platform, by-product pathways were reduced through multi-gene deletions (frdA, poxB, adhE, ldhA, glpK, ptsG and dhaM) to enhance the yield of 1,3-PDO from glycerol. By heterologously expressing GPD1/GPP2 and optimizing promoter, a metabolic network for the "glucose-glycerol-1,3-PDO" pathways was reconstructed. Cometabolism studies indicated that low concentrations of xylose and arabinose as co-substrates enhanced conversion efficiency of glycerol, whereas glucose alleviated metabolic competition. Under cometabolism of glucose and glycerol, the engineered strain K. pneumoniae S2 ΔABEAKGM-1-2 produced 1003.7 mmol/L (76.4 g/L) of 1,3-PDO with a yield of 0.83 mol/mol glycerol, an overall molar yield of 0.78 mol/mol based on total substrate consumption, and a productivity of 27.9 mmol/L/h. When lignocellulosic hydrolysate was used as co-substrate, 981.1 mmol/L (74.7 g/L) of 1,3-PDO was produced with a yield of 0.77 mol/mol glycerol and a productivity of 27.3 mmol/L/h. This study achieved efficient redirection of carbon flux toward 1,3-PDO through systematic metabolic engineering, providing valuable strain resources and technical guidance for the sustainable and cost-effective biomanufacturing of bio-based 1,3-PDO.
Li Wang, Ming-Yang Zhao, Yuan-Ming Ye et al.· Bioresource Technology· 0 citations
Branched-chain fatty acids (BCFAs), naturally synthesized by Gram-positive bacteria, are promising feedstocks for the production of advanced biofuels. However, efficient BCFA biosynthesis in Gram-negative bacteria such as Escherichia coli remains challenging because of insufficient supply of branched-chain acyl-CoA precursors, poor compatibility of the endogenous fatty acid synthesis pathway with branched-chain substrates, and limited cellular robustness toward non-native fatty acids. In this study, we first engineered an orthogonal isovaleryl-CoA biosynthetic pathway in E. coli and demonstrated its functionality in supporting BCFA production. Importantly, we identified a strong synergistic interaction between the isovaleryl-CoA pathway and the branched-chain α-keto acid dehydrogenase pathway, and thereby established a dual-route strategy for precursor supply. To further enhance BCFA production, we rewired central carbon metabolism by eliminating competing pathways and introducing a non-oxidative glycolysis pathway to increase acetyl-CoA availability while minimizing byproduct formation. We additionally optimized the fatty acid biosynthetic module through expression of a highly active 'TesA (R65C) variant and the transcriptional regulator FadR, and enhanced cellular robustness via introduction of N138H mutation into PcnB and overexpression of the stress resistance associated genes rfaY and yafL. The final engineered strain produced 2.96 g/L BCFAs (approximately 10-fold higher than the previously reported titers), representing 55% of total fatty acids, with a yield of 0.08 g/g glucose. Overall, this work established a dual-precursor supply strategy combined with systems metabolic engineering for BCFA production, providing a foundation for the development of sustainable bioprocesses for advanced branched-chain biofuels.
Mengfan Hu, Cai Feng, Mingjun Li et al.· Metabolic Engineering· 0 citations
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