During riboflavin biosynthesis in Bacillus subtilis, GTP acts as a precursor supplied by the purine biosynthesis pathway. This study revealed that GTP biosynthesis is tightly regulated by intracellular guanine levels via a guanine-sensing riboswitch, limiting the final riboflavin yield. Using resting cell transformation assays, we confirmed that the transporters NupG and PbuO mediate guanine efflux and influx, respectively. Overexpression of nupG in the original strain BR reduced intracellular guanine levels, resulting in enhanced riboflavin production. These findings indicate that NupG-mediated intracellular guanine efflux could deregulate riboswitch-induced repression of the pur operon. This deregulation increased metabolic flux through the de novo purine biosynthesis pathway and elevated the synthesis of precursor GTP. As a result, engineered strain BR-02 achieved a riboflavin titer of 1,508.22 mg/L, a 15.3% increase over the original strain BR. This study highlights that transporter engineering is an effective metabolic engineering strategy to overcome purine feedback repression in riboflavin biomanufacturing, offering a new rationale for designing microbial cell factories.
Xylose is an abundant carbon source in lignocellulosic biomass, making it extremely important to construct microbial metabolic factories capable of efficiently utilizing xylose. Candida glycerinogenes is a strain with high stress tolerance, and it serves as an efficient platform for energy conversion via lignocellulosic feedstocks. Through transcriptional analysis of genes related to the xylose-highly assimilating strain C. glycerinogenes-cgS35P, it was found that CgGzf3, a transcription factor associated with amino acid metabolism, is involved in the regulation of xylose metabolism. The inhibition of CgGzf3 expression by antisense RNA increased the xylose consumption of the engineered strain by 33.7% and the ethanol yield by 30.4%. Yeast one-hybrid assays confirmed that CgGzf3 directly binds to the promoter regions of key xylose metabolism genes. Antisense RNA inhibits the expression of CgGzf3, which upregulates fermentation metabolism genes and thereby shifts xylose metabolism toward ethanol fermentation. Additionally, the key amino acid biosynthesis genes Arg1, Glt1, and Car2 were upregulated by 1.8-fold, 1.6-fold, and 1.3-fold, respectively. The intracellular amino acid content of the recombinant strain C. glycerinogenes-cgreGzf3 also increased. This study reveals for the first time that the amino acid metabolism transcription factor CgGzf3 is involved in the regulation of xylose metabolism, providing new experimental evidence for the intrinsic connection between xylose metabolism and amino acid metabolism.
The transcription factor CgGzf3 directly affects xylose metabolism by regulating the promoters of key genes, and can indirectly influence xylose assimilation by modulating amino acid anabolic metabolism. Inhibition of CgGzf3 by antisense RNA shifts the overall xylose assimilation toward ethanol fermentation metabolism
Current production of polylactic acid (PLA) relies on agricultural feedstocks. Utilizing solar chemicals produced from CO2 by artificial photosynthesis offers a sustainable alternative for producing biodegradable plastics. However, the direct synthesis of PLA from CO2 within a cell factory remains challenging. Here, we report the direct production of PLA in engineered Escherichia coli from CO2 via ethanol or acetate, which are produced from CO2 electrolysis with solar energy. More importantly, we found that cofeeding ethanol and acetate synergistically enhanced PLA production. The PLA titer under cofeeding condition reached 5-folds and 53-folds of those obtained with ethanol or acetate alone, respectively. This cofeeding effect upregulated the glyoxylate shunt, the Entner-Doudoroff pathway, and serine anabolism, which facilitated efficient lactic acid generation from acetyl-CoA. By further integrating electroreduction and PLA bioproduction, we achieved PLA synthesis from CO2 with a titer of 241 mg/L. This work develops an agriculture-independent approach for bioplastic production from CO2, H2O, and renewable energy.
Rising global energy demands, together with the need for alternative, sustainable, and scalable energy production and storage systems, have attracted industry attention. One such potential solution is bioelectric systems, such as biofuel cells (BFCs) and bio-based batteries (BBBs). BFCs are systems that utilize redox-active biomolecules and biopolymers derived from renewable biological sources such as plants, algae, and bacteria to generate electricity. Generally categorized by their catalysts: microbial biofuel cells and enzymatic biofuel cells (EFCs). BBBs are a subclass of BFCs, which replace an active feeding system with an internal fuel that is discarded once depleted. The varying physical properties, biocompatibility, and operation on renewable biological fuels expanded their use across fields such as waste management, healthcare, agriculture, and robotics. Despite active research and development, regulatory bodies lack policies and regulations governing the production and commercial use of BFC. Efforts to commercialize the technology are held back by technical limitations, economic challenges, and the lack of solid policies surrounding green energy. The failure of previous attempts to commercialize biobatteries has highlighted the gap between experimental feasibility and real-world implementation. Future progress in the field is expected to rely on improved integration of biobatteries into existing hybrid energy systems, advancement in the stability of bio-based materials, and the development of supportive regulatory and market infrastructures.