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Background: Clear cell renal cell carcinoma (ccRCC) represents the most prevalent subtype, accounting for nearly 80% of all RCC cases. Recent research has shown that high expression of circular non-coding RNA (circRNA) is associated with poor prognosis in patients with renal clear cell carcinoma (ccRCC), however, the underlying mechanism remains unclear. Methods: After analysing self-sequenced renal cancer and paracancer circRNA sequencing data and comparing it with the GEO public database, we discovered that circASAP1 expression was significantly up-regulated in renal cancers. We also tested circASAP1 levels in 102 renal cancer patients and found that high expression of circASAP1 was associated with poor prognosis and metastasis. The interaction between circASAP1, HNRNPC and their downstream target genes was confirmed through experiments such as RNA pull-down, RIP and fluorescence in situ hybridisation. A series of in vitro and in vivo functional experiments were performed to verify the effects of circASAP1 on RCC proliferation and metastasis. Results: Circular RNA sequencing analysis revealed that circASAP1 expression was markedly elevated in ccRCC, with a significant association observed between elevated circASAP1 expression and poor prognosis and metastasis. Actinomycin D, RNase R, as well as fluorescence in situ hybridization (FISH) analyses revealed the ring structure and cytoplasmic localization of circASAP1. High circASAP1 expression was associated with ccRCC cell proliferative viability, invasion, and metastasis in CCK-8, transwell, plate cloning, and EdU experiments. Interaction of circASAP1 with HNRNPC and their downstream target genes was confirmed by RNA pull-down, RNA immunoprecipitation, FISH, silver staining, and mass spectrometry. Experiments using truncated isoforms demonstrated that amino acids 16–87 of HNRNPC bound circASAP1. Proteins altered by circASAP1 were enriched in the ferroptosis pathway on the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Conclusions: The relationship between circRNA and the ASAP1/HNRNPC/GPX4 axis was demonstrated by experimental data, which was further confirmed by rescue experiments. circASAP1 influenced tumor growth and ferroptosis in animal experiments and predicted the prognosis of patients with ccRCC. The circASAP1/HNRNPC/GPX4 axis provides novel directions and potential targets for RCC treatment. [ABSTRACT FROM AUTHOR]

Background: Corynebacterium crenatum SYPA 5 is the industrial strain for L-arginine production. Poly-β-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and L-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the L-arginine biosynthesis in C. crenatum SYPA 5., Results: C. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of L-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L L-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key L-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9-3.0 times compared with the parent strain., Conclusions: Accumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time L-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular L-arginine and intracellular PHB simultaneously. All of these have a potential biotechnological application as a strategy for high-yield L-arginine.

Background: Acetoin reductase (Acr) catalyzes the conversion of acetoin to 2,3-butanediol (2,3-BD) with concomitant oxidation of NADH to NAD(+). Therefore, intracellular 2,3-BD production is likely governed by the quantities of rate-limiting factor(s) Acr and/or NADH. Previously, we showed that a high level of Acr was beneficial for 2,3-BD accumulation., Results: Metabolic engineering strategies were proposed to redistribute carbon flux to 2,3-BD by manipulating NADH levels. The disruption of NADH oxidase (YodC, encoded by yodC) by insertion of a formate dehydrogenase gene in Bacillus subtilis was more efficient for enhancing 2,3-BD production and decreasing acetoin formation than the disruption of YodC by the insertion of a Cat expression cassette. This was because the former resulted in the recombinant strain AFY in which an extra NADH regeneration system was introduced and NADH oxidase was disrupted simultaneously. On fermentation by strain AFY, the highest 2,3-BD concentration increased by 19.9 % while the acetoin titer decreased by 71.9 %, relative to the parental strain. However, the concentration of lactate, the main byproduct, increased by 47.2 %. To further improve carbon flux and NADH to 2,3-BD, the pathway to lactate was blocked using the insertional mutation technique to disrupt the lactate dehydrogenase gene ldhA. The resultant engineered strain B. subtilis AFYL could efficiently convert glucose into 2,3-BD with little acetoin and lactate accumulation., Conclusions: Through increasing the availability of NADH and decreasing the concentration of unwanted byproducts, this work demonstrates an important strategy in the metabolic engineering of 2,3-BD production by integrative recombinant hosts.

Background: Bacillus amyloliquefaciens B10-127 exhibited an excellent ability for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD) from biodiesel-derived glycerol. However, the accumulation of by-products (acetoin, acetoin, lactate and succinate) and the 2,3-BD yield remains prohibitively low for commercial production., Results: Several strategies were developed to manipulate the carbon flux to 2,3-BD branch in a designed B. amyloliquefaciens. Firstly, extra copies of NADH/NAD(+) regeneration system were introduced into B. amyloliquefaciens by co-overproduction of glycerol dehydrogenase and acetoin reductase, which resulting in improvement of 2,3-BD production and suppression of by-products accumulation. Subsequently, the transcriptional regulator ALsR under the control of a moderate promoter PbdhA was introduced into B. amyloliquefaciens, which increased carbon flux to 2,3-BD branch. Finally, a three-stage dissolved oxygen control strategy were proposed based on analysis of the characteristic of 2,3-BD fermentation, and a two-stage pH control strategy were proposed based on different pH preferences of ACR for reduction and oxidation. Following these strategies, a high titer (102.3 g/L), yield (0.44 g/g), and productivity (1.16 g/L/h) of 2,3-BD were achieved., Conclusions: To our knowledge, this is the highest reported 2,3-BD production using biodiesel-derived glycerol as substrate, and this designed B. amyloliquefaciens should be an excellent candidate for producing 2,3-BD on an industrial scale.