Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization.

BACKGROUND: Microbial engineering aims to enhance the ability of bacteria to produce valuable products, including vitamin B6 for various applications. Numerous microorganisms naturally produce vitamin B6, yet the metabolic pathways involved are rigorously controlled. This regulation by the accumulation of vitamin B6 poses a challenge in constructing an efficient cell factory. RESULTS: In this study, we conducted transcriptome and metabolome analyses to investigate the effects of the accumulation of pyridoxine, which is the major commercial form of vitamin B6, on cellular processes in Escherichia coli. Our omics analysis revealed associations between pyridoxine and amino acids, as well as the tricarboxylic acid (TCA) cycle. Based on these findings, we identified potential targets for fermentation optimization, including succinate, amino acids, and the carbon-to-nitrogen (C/N) ratio. Through targeted modifications, we achieved pyridoxine titers of approximately 514 mg/L in shake flasks and 1.95 g/L in fed-batch fermentation. CONCLUSION: Our results provide insights into pyridoxine biosynthesis within the cellular metabolic network for the first time. Our comprehensive analysis revealed that the fermentation process resulted in a remarkable final yield of 1.95 g/L pyridoxine, the highest reported yield to date. This work lays a foundation for the green industrial production of vitamin B6 in the future.

Multiple Cofactor Engineering Strategies to Enhance Pyridoxine Production in Escherichia coli.

Pyridoxine, also known as vitamin B6, is an essential cofactor in numerous cellular processes. Its importance in various applications has led to a growing interest in optimizing its production through microbial biosynthesis. However, an imbalance in the net production of NADH disrupts intracellular cofactor levels, thereby limiting the efficient synthesis of pyridoxine. In our study, we focused on multiple cofactor engineering strategies, including the enzyme design involved in NAD+-dependent enzymes and NAD+ regeneration through the introduction of heterologous NADH oxidase (Nox) coupled with the reduction in NADH production during glycolysis. Finally, the engineered E. coli achieved a pyridoxine titer of 676 mg/L in a shake flask within 48 h by enhancing the driving force. Overall, the multiple cofactor engineering strategies utilized in this study serve as a reference for enhancing the efficient biosynthesis of other target products.

Engineering and finetuning expression of SerC for balanced metabolic flux in vitamin B6 production.

Vitamin B6 plays a crucial role in cellular metabolism and stress response, making it an essential component for growth in all known organisms. However, achieving efficient biosynthesis of vitamin B6 faces the challenge of maintaining a balanced distribution of metabolic flux between growth and production. In this study, our focus is on addressing this challenge through the engineering of phosphoserine aminotransferase (SerC) to resolve its redundancy and promiscuity. The enzyme SerC was semi-designed and screened based on sequences and predicted kcat values, respectively. Mutants and heterologous proteins showing potential were then fine-tuned to optimize the production of vitamin B6. The resulting strain enhances the production of vitamin B6, indicating that different fluxes are distributed to the biosynthesis pathway of serine and vitamin B6. This study presents a promising strategy to address the challenge posed by multifunctional enzymes, with significant implications for enhancing biochemical production through engineering processes.

Protein engineering and iterative multimodule optimization for vitamin B6 production in Escherichia coli.

Vitamin B6 is an essential nutrient with extensive applications in the medicine, food, animal feed, and cosmetics industries. Pyridoxine (PN), the most common commercial form of vitamin B6, is currently chemically synthesized using expensive and toxic chemicals. However, the low catalytic efficiencies of natural enzymes and the tight regulation of the metabolic pathway have hindered PN production by the microbial fermentation process. Here, we report an engineered Escherichia coli strain for PN production. Parallel pathway engineering is performed to decouple PN production and cell growth. Further, protein engineering is rationally designed including the inefficient enzymes PdxA, PdxJ, and the initial enzymes Epd and Dxs. By the iterative multimodule optimization strategy, the final strain produces 1.4 g/L of PN with productivity of 29.16 mg/L/h by fed-batch fermentation. The strategies reported here will be useful for developing microbial strains for the production of vitamins and other bioproducts having inherently low metabolic fluxes.

Decreased expression of long non-coding RNA MEG3 acts as a potential predictor biomarker in progression and poor prognosis of osteosarcoma.

INTRODUCTION: Long non-coding RNA MEG3 (lncRNA MEG3) has been showed to involve in a variety of cancers. However, the association between lncRNA MEG3 expression level and the prognosis of osteosarcoma is still unclear. METHODS: The expression levels of lncRNA MEG3 in osteosarcoma tissues and adjacent non-tumor tissues were detected using quantitative real-time PCR (qRT-PCR). Differences in patient survival were determined using the Kaplan-Meier method and a log-rank test. A Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values. RESULTS: Our findings showed that expression of lncRNA MEG3 was clearly lower in osteosarcoma tissues compared with adjacent non-tumor tissues. The expression of lncRNA MEG3 was associated with clinical stage and distant metastasis (P<0.05). Kaplan-Meier analysis showed that patients with low lncRNA MEG3 expression had a shorter overall survival (log-rank test, P<0.05). Furthermore, multivariate analysis revealed that decreased expression of lncRNA MEG3, advanced clinical stage and distant metastasis were all independent predictors to overall survival of osteosarcoma patients. CONCLUSIONS: Downregulation of lncRNA MEG3 was associated with poor overall survival of osteosarcoma. LncRNA MEG3 could be a useful biomarker for progression and prognosis of osteosarcoma.