High-yield porphyrin production through metabolic engineering and biocatalysis.

Porphyrins and their derivatives find extensive applications in medicine, food, energy and materials. In this study, we produced porphyrin compounds by combining Rhodobacter sphaeroides as an efficient cell factory with enzymatic catalysis. Genome-wide CRISPRi-based screening in R. sphaeroides identifies hemN as a target for improved coproporphyrin III (CPIII) production, and exploiting phosphorylation of PrrA further improves the production of bioactive CPIII to 16.5 g L-1 by fed-batch fermentation. Subsequent screening and engineering high-activity metal chelatases and coproheme decarboxylase results in the synthesis of various metalloporphyrins, including heme and the anti-tumor agent zincphyrin. After pilot-scale fermentation (200 L) and setting up the purification process for CPIII (purity >95%), we scaled up the production of heme and zincphyrin through enzymatic catalysis in a 5-L bioreactor, with CPIII achieving respective enzyme conversion rates of 63% and 98% and yielding 10.8 g L-1 and 21.3 g L-1, respectively. Our strategy offers a solution for high-yield bioproduction of heme and other valuable porphyrins with substantial industrial and medical applications.

Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides.

The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides, has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides. In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q10 (Q10) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, P rsp _ 7571 and P rsp _ 6124 , showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a P rsp _ 7571 -derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides. Finally, as a demonstration, the synthetic pathway of Q10 was modulated by the selected promoter T334* in JDW-710; the Q10 yield in shake-flasks increased 28% and the production reached 226 mg/L. These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides-derived MCFs.

Modeling and Quantification of Impact of Psychological Factors on Rehabilitation of Stroke Patients.

The brain damage could lead to the loss of the central nervous system, so a stroke patient may lose the function of dominating his/her body. The rehabilitation aims to maximize the potential to restore a patient who has an impairment. Traditional rehabilitation is to train a patient's muscles and joints under the guidance of doctors to improve the strength of muscles and restore the motor function of joints. However, stroke patients are usually depressed, lonely, and irritable, and they might easily generate negative emotions during a rehabilitation process. With a sole goal of helping patients restore their body functions from the physiology perspective, the traditional rehabilitation took little consideration on the impact of rehabilitation, which is reflected and measured from the perspective of emotions. Therefore, we suggest adding affective regulation to the stroke rehabilitation; in such a way, the patients' exercise could be completed with high intrinsic motivation, and the performance of the rehabilitation process can be enhanced. Two main contributions in the presented works are: 1) the expanded emotional model to represent the status of stroke patients where the impact of psychological factors can be taken into consideration and 2) the quantifiable measurement of rehabilitation performance as well as the corresponding design of experiments to verify the positive impact of psychological adjustment on human subjects. Note that due to the limited conditions, the experimental verification was performed on healthy college students. Since our work focused on modeling and quantification of psychological factors, it is reasonable to expend our work to other human subjects including stoke patients.