Productivity enhancement in L-lysine fermentation using oxygen-enhanced bioreactor and oxygen vector.

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.

Metabolic engineering of Escherichia coli for efficient production of L-5-hydroxytryptophan from glucose.

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 glucose. 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 heterologous 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. CONCLUSION: 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.

Process study of ceramic membrane-coupled mixed-cell fermentation for the production of adenine.

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.

Effect of low-level ultrasound treatment on the production of L-leucine by Corynebacterium glutamicum in fed-batch culture.

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.

Focus properties of cosh-Gaussian beams with the power-exponent-phase vortex.

Vector diffraction theory is used to investigate the focusing properties of cosh-Gaussian beams with the power-exponent-phase vortex. The effects of the decentered parameter, the power order, and the topological charge on the normalized intensity distribution are examined. Results show that intensity distribution in the focal region can be altered significantly by the topological charge, the power order, and the decentered parameter. The pattern of the optical intensity slowly enlarges with the increase of topological charge. The strongest intensity part of the annulus rotates by the changing topological charge. As the power order increases, the intensity distribution is more concentrated. As the decentered parameter increases, there occur multiple relatively strong intensity peaks, and the entire focus pattern extends outward. Some optical gradient force distributions are investigated to illuminate the applications of these alterable foci patterns.

Effective tensor approach for simulating the propagation of partially coherent Hermite-sinh-Gaussian beams through an ABCD optical system in turbulent atmosphere.

An efficient tensor approach is used to simulate the propagation of partially coherent Hermite-sinh-Gaussian (H-ShG) beams through an ABCD optical system in a turbulent atmosphere. Analytical expressions for the average intensity are derived. The properties of the average intensity of the beam are studied numerically. It is found that the average intensity distribution of the beam is almost independent of the structure constant of turbulence when the spatial coherence length is very small and the beam with larger spatial coherence length is less affected by the turbulent atmosphere. Moreover, the transition from an H-ShG beam into a Gaussian beam becomes quicker with smaller beam order and smaller Sh parameters.

A patient-specific haptic drilling simulator based on virtual reality for dental implant surgery.

PURPOSE: In the dental implant surgery, there are inevitable risks due to the anatomically complex operation in the cranio-maxillofacial region. Therefore, there is a trend to use computer-aided technology to simulate the process of the implant surgery. In this study, we present a haptic simulator for trainees to study and rehearse the drilling performance of dental implant surgery. MATERIALS AND METHODS: The dental implant surgery simulator (DISS) is developed based on the haptic force-feedback device Omega.6, and some free open-source software libraries such as Computer Haptics and Active Interface (CHAI3D), Qt and Visualization Toolkit. To achieve the desired effects of drilling, the meshes are subdivided with a recursive algorithm which breaks down a triangular patch into 4 sub-triangles. The drilling operation can be implemented at any specified location of the model. Once the drilling direction is determined, the position and rotation of the haptic device tool are constrained to the orientation through a dimension reduction algorithm. The driller diameter and drill speed are tunable to implement stepwise drilling for the patient-specific models. RESULTS: A patient-specific drilling simulator based on virtual reality for dental implant surgery is presented. The simulation of stepwise drilling was conducted, and three patient-specific models reconstructed by Computed Tomography data were employed to help the novices to find the suitable drilling parameter. CONCLUSION: The obtained results showed that the haptic-based DISS could simulate various dental implant surgeries with different driller diameter and drill speed which takes patient-specific models as input. The evaluation of the DISS proves its good performance and it could provide an effective method to improve the skills and experiences of trainees.

Mechanism study on stability enhancement of adefovir dipivoxil by cocrystallization: Degradation kinetics and structure-stability correlation.

The purpose of this study is to determine the mechanism by which cocrystallization can enhance the stability of adefovir dipivoxil (AD), a diester prodrug of adefovir with known chemical stability problem. Three multi-component crystals of AD with biologically safe coformers, including gallic acid cocrystal hydrate (1:1:1), salicylate salt (1:1), and maleate salt (1:1) were prepared and characterized by thermal analysis, infrared spectroscopy, powder and single crystal X-ray diffraction. DVS measurements and stability tests were applied to evaluate the stability. The new crystalline phases exhibit improved stability compared to pure drug in the order AD gallic acid cocrystal>AD maleate>AD salicylate>AD form I. Degradation kinetics and structure-stability correlation studies demonstrate that the stability enhancement mechanism by cocrystallization involves (1) inhibition of hydrolysis of AD by replacement of drug-drug homosynthons by stronger drug-coformer heterosynthons at adenine fragments; (2) suppression of dimerization of AD by separation of adenine fragments by inserting coformers in crystal lattices; (3) further reducing rates of hydrolysis by forming hydrogen bonds with hydrate water at phosphoryl fragments. This study has important implications for use of cocrystallization approach to some easily degradable drugs in pharmaceutical.