Introduction: L-lysine is a bulk product. In industrial production using high-biomass fermentation, the high density of bacteria and the intensity of production require sufficient cellular respiratory metabolism for support. Conventional bioreactors often have difficulty meeting the oxygen supply conditions for this fermentation process, which is not conducive to improving the sugar-amino acid conversion rate. In this study, we designed and developed an oxygen-enhanced bioreactor to address this problem.Methods: This bioreactor optimizes the aeration mix using an internal liquid flow guide and multiple propellers.Results: Compared with a conventional bioreactor, it improved the kLa from 367.57 to 875.64 h-1, an increase of 238.22%. The results show that the oxygen supply capacity of the oxygen-enhanced bioreactor is better than that of the conventional bioreactor. Its oxygenating effect increased the dissolved oxygen in the middle and late stages of fermentation by an average of 20%. The increased viability of Corynebacterium glutamicum LS260 in the mid to late stages of growth resulted in a yield of 185.3 g/L of L-lysine, 74.57% conversion of lysine from glucose, and productivity of 2.57 g/L/h, an increase of 11.0%, 6.01%, and 8.2%, respectively, over a conventional bioreactor. Oxygen vectors can further improve the production performance of lysine strains by increasing the oxygen uptake capacity of microorganisms. We compared the effects of different oxygen vectors on the production of L-lysine from LS260 fermentation and concluded that n-dodecane was the most suitable. Bacterial growth was smoother under these conditions, with a 2.78% increase in bacterial volume, a 6.53% increase in lysine production, and a 5.83% increase in conversion. The different addition times of the oxygen vectors also affected the final yield and conversion, with the addition of oxygen vectors at 0 h, 8 h, 16 h, and 24 h of fermentation increasing the yield by 6.31%, 12.44%, 9.93%, and 7.39%, respectively, compared to fermentation without the addition of oxygen vectors. The conversion rates increased by 5.83%, 8.73%, 7.13%, and 6.13%, respectively. The best results were achieved by adding oxygen vehicles at the 8th hour of fermentation, with a lysine yield of 208.36 g/L and a conversion rate of 83.3%. In addition, n-dodecane significantly reduced the amount of foam produced during fermentation, which is beneficial for fermentation control and equipment.Conclusion: The new oxygen-enhanced bioreactor improves oxygen transfer efficiency, and oxygen vectors enhance the ability of cells to take up oxygen, which effectively solves the problem of insufficient oxygen supply during lysine fermentation. This study provides a new bioreactor and production solution for lysine fermentation.
Various process intensification methods were proposed to improve the yield, quality, and safety of fermented products. Here, we report the enhancement of L-leucine production by Corynebacterium glutamicum CP using ultrasound-assisted fed-batch fermentation. Response surface methodology was employed to optimize the sonication conditions. At an ultrasonic power density of 94 W/L, frequency of 25 kHz, interval of 31 min, and duration of 37 s, C. glutamicum CP produced 52.89 g/L of L-leucine in 44 h, representing a 21.6% increase compared with the control. The production performance of L-leucine was also improved under ultrasonic treatment. Moreover, the effects of ultrasound treatment on the fermentation performance of L-leucine were studied in terms of cell morphology, cell membrane permeability, and enzyme activity. The results indicate that ultrasonication is an efficient method for the intensification of L-leucine production by C. glutamicum CP.
In order to solve the problems of high complexity, many by-products, high pollution and difficult extraction of the existing adenine production process, in this study, ceramic membrane-coupled mixed cell fermentation was used to produce adenine while reducing the synthesis of by-products and simplifying the production process of adenine. Nucleoside hydrolase (encoded by the rihC gene) was used to produce adenine by coordinated fermentation with the adenosine-producing bacterium Bacillus Subtilis XGL. The adenosine hydrolase (AdHy)-expressing strain Escherichia coli BL21-AdHy was successfully employed and the highest activity of the crude enzyme solution was found by orthogonal experiments at 170 W power, 42% duty cycle, and 8 min of sonication. The highest AdHy activity was found after 18 h of induction incubation. E. coli BL21-AdHy was induced for 18 h and sonicated under the above ultrasonic conditions and the resulting crude enzyme solution was used for co-fermentation of the strain and enzyme. Moreover, 15% (v/v) of the AdHy crude enzyme solution was added to fermentation of B. subtilis XGL after 35 h. Finally, the whole fermentation system was dialyzed using coupled ceramic membranes for 45 and 75 h, followed by the addition of fresh medium. In contrast, the AdHy crude enzyme solution was added after 35, 65, and 90 h of B. subtilis fermentation, with three additions of 15, 15, and 10% of the B. subtilis XGL fermentation system. The process was validated in a 5 L fermenter and 14 ± 0.25 g/L of adenine was obtained, with no accumulation of adenosine and d-ribose as by-products. The enzymatic activity of the AdHy crude solution treated with ultrasound was greatly improved. It also reduced the cellular activity of E. coli BL21-AdHy and reduced effects on bacterial co-fermentation. Membrane-coupled dialysis solved the problem of decreased yield due to poor bacterial survival and decreased viability, and eliminated inhibition of the product synthesis pathway by adenosine. The batch addition of crude enzyme broth allowed the continuous conversion of adenosine to adenine. This production method provides the highest yield of biologically produced adenine reported to date, reduces the cost of adenine production, and has positive implications for the industrial production of adenine by fermentation. And it provides a reference for producing other high-value-added products made by fermentation.
Background 5-hydroxytryptophan (5-HTP), the direct biosynthetic precursor of the neurotransmitter 5-hydroxytryptamine, has been shown to have unique efficacy in the treatment of a variety of disorders, including depression, insomnia, and chronic headaches, and is one of the most commercially valuable amino acid derivatives. However, microbial fermentation for 5-HTP production continues to face many challenges, including low titer/yield and the presence of the intermediate L-tryptophan (L-Trp), owing to the complexity and low activity of heterologous expression in prokaryotes. Therefore, there is a need to construct an efficient microbial cell factory for 5-HTP production. Results We describe the systematic modular engineering of wild-type Escherichia coli for the efficient fermentation of 5-HTP from sugars. First, a xylose-induced T7 RNA polymerase-PT7 promoter system was constructed to ensure the efficient expression of each key heterologous pathway in E. coli. Next, a new tryptophan hydroxylase mutant was used to construct an efficient tryptophan hydroxylation module, and the cofactor tetrahydrobiopterin synthesis and regeneration pathway was expressed in combination. The L-Trp synthesis module was constructed by modifying the key metabolic nodes of tryptophan biosynthesis, and the de novo synthesis of 5-HTP was achieved. Finally, the NAD(P)H regeneration module was constructed by the moderate expression of the heterologous GDHesi pathway, which successfully reduced the surplus of the intermediate L-Trp. The final engineered strain HTP11 was able to produce 8.58 g/L 5-HTP in a 5-L bioreactor with a yield of 0.095 g/g glucose and a maximum real-time productivity of 0.48 g/L/h, the highest values reported by microbial fermentation. Conclusions In this study, we demonstrate the successful design of a cell factory for high-level 5-HTP production, combined with simple processes that have potential for use in industrial applications in the future. Thus, this study provides a reference for the production of high-value amino acid derivatives using a systematic modular engineering strategy and a basis for an efficient engineered strain development of 5-HTP high-value derivatives.
Background: Various process intensifications are proposed to improve process efficiency, quality, and safety of fermented products. With the market significantly growing, the production process of L-leucine needs significant improvements to increased productivity and yield. Ultrasound, a process intensification, is an emerging and fast-growing technology due to its wide range of applications in food science and fermentation industry. Results: By optimizing the time points and parameters of ultrasound, the biomass of Corynebacterium glutamicum CP was significantly improved. The response surface methodology was adopted to create prediction profiler model and optimize sonication conditions. C. glutamicum CP fermentation at conditions of ultrasonic power of 94 W/L, frequency of 25 kHz, interval of 31 min, and duration of 37 s produced 52.89 g/L of L-leucine in 44 h, increased by 21.6% compared with the control. The production performance of L-leucine was also improved under ultrasonic treatment. Moreover, the cell morphology of C. glutamicum CP were more irregular, elongated, and swollen cells under the ultrasonic condition. The cell permeability of C. glutamicum CP and the activity of key enzymes with ultrasonic treatment were significantly higher than the control. Conclusions: We successfully obtain the optimum ultrasonic conditions to produce L-leucine in fed-batch culture. The results indicate that ultrasonication is an efficient intensification process for enhancing fermentation to produce L-leucine from C. glutamicum CP.
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