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A consolidated bioprocess for cellulosic n-butanol production has been developed by engineering Clostridium cellulovorans to overexpress a bifunctional aldehyde/alcohol dehydrogenase. Rational metabolic engineering is important to further improve butanol production. This study aimed to investigate intracellular metabolism and identify the key regulators of cellulosic butanol formation in C. cellulovorans via integrated Omics and fermentation kinetics data analysis. First, comparative proteomics and metabolomics analyses of wild type and n-butanol producing mutant strain were conducted, which quantified 624 host cell proteins and 474 primary and secondary metabolites. Compared to wild type, most cellulases in cellulolysis were up-regulated, but three glycolysis enzymes and three enzymes in central pathway were down-regulated in the n-butanol producing strain. Second, a dynamic model integrating Omics and fermentation data was developed to identify key regulators in butanol biosynthesis, which were ranked by further metabolic control analysis. Finally, rational metabolic engineering was performed in C. cellulovorans by overexpressing two genes (thl and hbd) identified as important factors limiting butanol biosynthesis, which improved butanol yield and C4/C2 ratio. This study demonstrated a research approach to integrate multi-Omics and fermentation data of C. cellulovorans and guide its rational metabolic engineering, which can also be applied to other microorganisms.

The adoptive transfer of human T cells or genetically-engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. The objective of this study was to develop a novel T cell biomanufacturing platform using stirred-tank bioreactor for large-scale and high-quality cellular production. First, various factors, such as bioreactor parameters, media, supplements, stimulation, seed age, and donors, were investigated. A serum-free fed-batch bioproduction process was developed to achieve 1000-fold expansion within 8 days after first stimulation and another 500-fold expansion with second stimulation. Second, this biomanufacturing process was successfully scaled up in bioreactor with dilution factor of 10, and the robustness and reproducibility of the process was confirmed by the inclusion of different donors’ T cells of various qualities. Finally, T cell quality was monitored using 12 surface markers and 3 intracellular cytokines as the critical quality assessment criteria in early, middle and late stages of cell production. In this study, a new biomanufacturing platform was created to produce reliable, reproducible, high-quality, and large-quantity (i.e. > 5 billion) human T cells in stirred-tank bioreactor. This platform is compatible with the production systems of monoclonal antibodies, vaccines, and other therapeutic cells, which provides not only the proof-of-concept but also the ready-to-use new approach of T cell expansion for clinical immune therapy.

The cellulolytic Clostridium cellulovorans has been engineered to produce n -butanol from low-value lignocellulosic biomass by consolidated bioprocessing (CBP). The objective of this study was to establish a robust cellulosic biobutanol production process using a metabolically engineered C. cellulovorans . First, various methods for the pretreatment of four different corn-based residues, including corn cob, corn husk, corn fiber, and corn bran, were investigated. The results showed that better cell growth and a higher concentration of n -butanol were produced from corn cob that was pretreated with sodium hydroxide. Second, the effects of different carbon sources (glucose, cellulose and corn cob), basal media and culture pH values on butanol production were evaluated in the fermentations performed in 2-L bioreactors to identify the optimal CBP conditions. Finally, the engineered C. cellulovorans produced butanol with final concentration >3 g/L, yield >0.14 g/g, and selectivity >3 g/g from pretreated corn cob at pH 6.5 in CBP. This study showed that the fermentation process engineering of C. cellulovorans enabled a high butanol production directly from agricultural residues.

Chinese hamster ovary (CHO)1 cells have been widely used to express heterologous genes and produce therapeutic proteins in biopharmaceutical industry. Different CHO host cells have distinct cell growth rates and protein expression characteristics. In this study, the expression of about 1,307 host proteins in three sublines, i.e. CHO K1, CHO S and CHO/dihydrofolate reductase (dhfr)−, were investigated and compared using proteomic analysis. The proteins involved in cell growth, glycolysis, tricarboxylic acid cycle, transcription, translation and glycosylation were quantitated using Liquid chromatography tandem-mass spectrometry (LC-MS/MS). The key host cell proteins that regulate the kinetics of cell growth and the magnitude of protein expression levels were identified. Furthermore, several rational cell engineering strategies on how to combine the desired features of fast cell growth and efficient production of therapeutic proteins into one new super CHO host cell have been proposed.

The objective of this study was to improve the production of butyric acid by process optimization using the metabolically engineered mutant of Clostridium tyrobutyricum (PAK-Em). First, the free-cell fermentation at pH 6.0 produced butyric acid with concentration of 38.44 g/L and yield of 0.42 g/g. Second, the immobilizedcell fermentations using fibrous-bed bioreactor (FBB) were run at pHs of 5.0, 5.5, 6.0, 6.5 and 7.0 to optimize fermentation process and improve the butyric acid production. It was found that the highest titer of butyric acid, 63.02 g/L, was achieved at pH 6.5. Finally, the metabolic flux balance analysis was performed to investigate the carbon rebalance in C. tyrobutyricum. The results show both gene manipulation and fermentation pH change redistribute carbon between biomass, acetic acid and butyric acid. This study demonstrated that high butyric acid production could be obtained by integrating metabolic engineering and fermentation process optimization. Open image in new window

Butanol is a promising biofuel with high energy intensity and can be used as gasoline substitute. It can be produced as a sustainable energy by microorganisms (such as Clostridia) from low-value biomass. However, the low productivity, yield and selectivity in butanol fermentation are still big challenges due to the lack of an efficient butanol-producing host strain. In this article, we systematically review the host cell engineering of Clostridia, focusing on (1) various strategies to rebalance metabolic flux to achieve a high butanol production by regulating the metabolism of carbon, redox or energy, (2) the challenges in pathway manipulation, and (3) the application of proteomics technology to understand the intracellular metabolism. In addition, the process engineering is also briefly described. The objective of this review is to summarize the previous research achievements in the metabolic engineering of Clostridium and provide guidance for future novel strain construction to effectively produce butanol. Open image in new window

Biobutanol is a sustainable green biofuel that can substitute for gasoline. Carbon flux has been redistributed in Clostridium tyrobutyricum via metabolic cell engineering to produce biobutanol. However, the lack of reducing power hampered the further improvement of butanol production. The objective of this study was to improve butanol production by rebalancing redox. Firstly, a metabolically-engineered mutant CTC-fdh-adhE2 was constructed by introducing heterologous formate dehydrogenase (fdh) and bifunctional aldehyde/alcohol dehydrogenase (adhE2) simultaneously into wild-type C. tyrobutyricum. The mutant evaluation indicated that the fdh-catalyzed NADH-producing pathway improved butanol titer by 2.15-fold in the serum bottle and 2.72-fold in the bioreactor. Secondly, the medium supplements that could shift metabolic flux to improve the production of butyrate or butanol were identified, including vanadate, acetamide, sodium formate, vitamin B12 and methyl viologen hydrate. Finally, the free-cell fermentation produced 12.34 g/L of butanol from glucose using the mutant CTC-fdh-adhE2, which was 3.88-fold higher than that produced by the control mutant CTC-adhE2. This study demonstrated that the redox engineering in C. tyrobutyricum could greatly increase butanol production.

Aims Currently, there is no direct method for detecting Clostridium thermocellum in the insoluble medium. In this study, a quantitative real-time PCR assay was developed for the direct growth detection of C. thermocellum at the single-cell level in lignocellulosic biomasses. Methods and Results The assay targeted the cipA gene and was able to distinguish C. thermocellum from other species with good reproducibility which quantitative detection limit was 10 cell equivalents (CE) per reaction. OD600-based counting and qPCR quantification of C. thermocellum cultured in soluble medium were compared and an excellent consistency was revealed, indicating the appropriateness of the developed qPCR method. Analysis based on yellow affinity substrate and fermentation products may incorrectly estimate its population. Conclusions The developed assay can serve as a specific, sensitive and reproducible method for the detection of C. thermocellum in lignocellulosic biomass at the single-cell level. Significance and Impact of the Study With the ability to rapidly detect C. thermocellum, this method will contribute substantially to the understanding of the lignocellulosic biomass degradation mechanism. Moreover, it can also be applied to detect C. thermocellum growth in certain co-culture system for the understanding of the metabolic interactions.