Cao Weijia,Ma Weichao,Zhang Bowen,Wang Xin,Chen Kequan,Li Yan,Ouyang Pingkai
The development of efficient microbial processes for pinocembrin production has attracted considerable attention. However, pinocembrin biosynthetic efficiency is greatly limited by the low availability of the malonyl-CoA cofactor in Escherichia coli. Fatty acid biosynthesis is the only metabolic process in E. coli that consumes malonyl-CoA; therefore, we overexpressed the fatty acid biosynthetic pathway enzymes β-ketoacyl-ACP synthase III (FabH) and β-ketoacyl-ACP synthase II (FabF) alone and in combination, and investigated the effect on malonyl-CoA. Interestingly, overexpressing FabH, FabF or both enzymes in E. coli BL21 (DE3) decreased fatty acid synthesis and increased cellular malonyl-CoA levels 1.4-, 1.6-, and 1.2-fold, respectively. Furthermore, pinocembrin production was increased 10.6-, 31.8-, and 5.87-fold in recombinant strains overexpressing FabH, FabF and both enzymes, respectively. Overexpression of FabF, therefore, triggered the highest pinocembrin production and malonyl-CoA levels. The addition of cerulenin further increased pinocembrin production in the FabF-overexpressing strain, from 25.8 to 29.9 mg/L. These results demonstrated that overexpressing fatty acid synthases can increase malonyl-CoA availability and improve pinocembrin production in a recombinant E. coli host. This strategy may hold promise for the production of other important natural products in which cellular malonyl-CoA is rate limiting.
3-Hydroxypropionic acid (3-HP) is a versatile platform chemical with broad applications in biodegradable plastics and superabsorbent polymers. Acetate derived from waste biomass and industrial off-gases can serve as a sustainable C2 feedstock for 3-HP production. However, the efficient conversion of acetate to 3-HP remains constrained by insufficient intracellular bicarbonate supply and competition with fatty acid biosynthesis for malonyl-CoA. To address these issues, carboxylation efficiency was enhanced in E. coli by co-expressing carbonic anhydrase (CA) with sodium-dependent bicarbonate transporters (BicA) and supplementation with sodium bicarbonate. Further overexpression of fabF was used to inhibit the initiation of fatty acid biosynthesis, reducing competition for malonyl-CoA. The engineered strain achieved a 3-HP yield of 0.73 g/g in shake flask culture, approaching the theoretical maximum without chemical inhibitors. In a fed-batch bioreactor, the 3-HP titer reached 20.55 g/L with a yield of 0.56 g/g (0.37 mol/mol), corresponding to 74.67% of the theoretical maximum yield. This bioprocess enables efficient valorization of sustainable C2 feedstocks, offering a promising pathway for the industrial production of bio-based platform chemicals.
Sun Tao,Li Shubin,Song Xinyu,Pei Guangsheng,Diao Jinjin,Cui Jinyu,Shi Mengliang,Chen Lei,Zhang Weiwen
Background Photosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO 2 . Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts. Results Aiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium Synechocystis sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in Escherichia coli . Both regulatory tools functioned well in regulating exogenous reporter gene lacZ and endogenous glgC gene in Synechocystis sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD 730 nm ) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch. Conclusions In this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for Synechocystis sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.
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