Construction of an Acetate Metabolic Pathway to Enhance Electron Generation of Engineered Shewanella oneidensis

Background: Microbial fuel cells (MFCs) are a novel bioelectrochemical devices that can use exoelectrogens as biocatalyst to convert various organic wastes into electricity. Among them, acetate, a major component of industrial biological wastewater and by-product of lignocellulose degradation, could release eight electrons per mole when completely degraded into CO2 and H2O, which has been identified as a promising carbon source and electron donor. However, Shewanella oneidensis MR-1, a famous facultative anaerobic exoelectrogens, only preferentially uses lactate as carbon source and electron donor and could hardly metabolize acetate in MFCs, which greatly limited Coulombic efficiency of MFCs and the capacity of bio-catalysis.Results: Here, to enable acetate as the sole carbon source and electron donor for electricity production in S. oneidensis, we successfully constructed three engineered S. oneidensis (named AceU1, AceU2, and AceU3) by assembling the succinyl-CoA:acetate CoA-transferase (SCACT) metabolism pathways, including acetate coenzyme A transferase encoded by ato1 and ato2 gene from G. sulfurreducens and citrate synthase encoded by the gltA gene from S. oneidensis, which could successfully utilize acetate as carbon source under anaerobic and aerobic conditions. Then, biochemical characterizations showed the engineered strain AceU3 generated a maximum power density of 8.3 ± 1.2 mW/m2 with acetate as the sole electron donor in MFCs. In addition, when further using lactate as the electron donor, the maximum power density obtained by AceU3 was 51.1 ± 3.1 mW/m2, which approximately 2.4-fold higher than that of wild type (WT). Besides, the Coulombic efficiency of AceU3 strain could reach 12.4% increased by 2.0-fold compared that of WT, which demonstrated that the engineered strain AceU3 can further utilize acetate as an electron donor to continuously generate electricity.Conclusion: In the present study, we first rationally designed S. oneidensis for enhancing the electron generation by using acetate as sole carbon source and electron donor. Based on synthetic biology strategies, modular assembly of acetate metabolic pathways could be further extended to other exoelectrogens to improve the Coulombic efficiency and broaden the spectrum of available carbon sources in MFCs for bioelectricity production.

A new strategy to improve ramie degumming based on removal of the xylan branched structure

Ramie fiber is known as the “king of natural fibers,” and the key to its wide application is efficient and green manufacturing. Microbial degumming has gradually become a hot area of research due to its environmental protection and mild operating conditions. However, some gummy materials remain after microbial degumming. Xylan is the main component of residual gums; its acetylated branched chains create the space barrier that makes the removal of hemicellulose difficult during ramie degumming. An acetyl xylan esterase (AXE) was obtained from Bacillus pumilus and characterized to solve this problem. Its optimum temperature and pH were 35°C and 8.0, respectively, and it had good temperature and pH stability. These properties were consistent with the conditions of ramie degumming and they laid a foundation for the application of AXE in ramie degumming. Besides, an engineered strain with a high activity of AXE was constructed successfully on the basis of the wild-type degumming strain Pectobacterium carotovorum HG-49 and used for ramie degumming. The removal rate of hemicellulose and total gums by the engineered strain increased by 4.89% and 2.53%, respectively, compared with that of the wild-type strain. Moreover, the role of this AXE in ramie degumming was further proven by X-ray diffraction and scanning electron microscopy. This study showed that AXE played an important role in the removal of hemicellulose in the degumming process of ramie fibers, thus providing a promising degumming strategy for ramie and other bast fiber plants.

Efficient Conversion of Glycerol to Ethanol by an Engineered Saccharomyces cerevisiae Strain

Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, inefficient conversion of glycerol to ethanol by natural Saccharomyces cerevisiae limits its use in these processes. Here, we have developed an efficient glycerol-converting yeast strain by genetically modifying the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by an O 2 -dependent dynamic shuttle and abolishing both glycerol phosphorylation and biosynthesis in S. cerevisiae D452-2 strain, as well as vigorous expression of whole genes in the DHA-pathway ( Candid utilis glycerol facilitator, Ogataea polymorpha glycerol dehydrogenase, endogenous dihydroxyacetone kinase, and triosephosphate isomerase). The engineered strain showed conversion efficiencies (CE) up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with production rate >1 g/L −1 h −1 when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain converted a mixture of glycerol and glucose into bioethanol (>86 g/L) with 92.8% CE. To the best of our knowledge, this is the highest reported titer of bioethanol produced from glycerol and glucose. Notably, we developed a glycerol-utilizing transformant from parent strain, which cannot utilize glycerol as a sole carbon source. The developed strain converted glycerol to ethanol with a productivity of 0.44 g/L −1 h −1 on minimal medium under semi-aerobic conditions. Our findings will promote the utilization of glycerol in eco-friendly biorefineries and integrate bioethanol and plant-oil industries. IMPORTANCE With the development of efficient lignocellulosic biorefineries, glycerol has attracted attention as an eco-friendly biomass-derived solvent that can enhance the dissociation of lignin and cell wall polysaccharides during the pretreatment process. Co-conversion of glycerol with the sugars released from biomass after glycerolysis increases the resources for ethanol production and lowers the burden of component separation. However, low conversion efficiency from glycerol and sugars limits the industrial application of this process. Therefore, the generation of an efficient glycerol-fermenting yeast will promote the applicability of integrated biorefineries. Hence, metabolic flux control in yeast grown on glycerol will lead to the generation of cell factories that produce chemicals, which will boost biodiesel and bioethanol industries. Additionally, the use of glycerol-fermenting yeast will reduce global warming and generation of agricultural waste, leading to the establishment of a sustainable society.

Biodegradation of p-nitrophenol by engineered strain

Abstractp-Nitrophenol (PNP) is an important environmental pollutant and can causes significant environmental and health risks. Compared with the traditional methods, biodegradation is a useful one to completely remove the harmful pollutants from the environment. Here, an engineered strain was first constructed by introducing PNP biodegradation pathway via the hydroquinone (HQ) pathway into Escherichia coli. In the engineered strain BL-PNP, PNP was completely degraded to β-ketoadipate and subsequently enter the metabolites of multiple anabolic pathways. The high tolerance and rapid degradation ability to PNP enable the engineered strain to have the potential to degrade toxic substances. The engineered strain created in this study can be used as a functional strain for bioremediation of PNP and potential toxic intermediates, and the method of assembling aromatic hydrocarbons metabolic pathway can be used to eradicate nitroaromatic pollutants in the environment.

Production enhancement of the glycopeptide antibiotic A40926 produced by an engineered N. gerenzanensis lcu1

Objective To enhance the production of A40926 by implementing a strategy of the combination of genetically engineered strain construction and medium optimization. Results The engineered strain of Nonomuraea gerenzanensis presented an increment of 30.6 percent in A40926 production compared with that of the parent strain. Subsequently, an assembling medium, which was defined as M9 medium and mainly comprised glucose, maltodextrin, soybean meal, peptone, L-valine, and other inorganic salts, was determined as the optimal medium among the tested nine media. The optimum concentration of medium components was glucose 10 g/l, maltodextrin 37.9 g/l, soybean meal 34.5 g/l, peptone 30.0 g/l, and L-valine 4.3 g/l, respectively. The optimized medium was verified experimentally, and A40926 yield increased significantly from 257 mg/l to 332 mg/l, as compared to the non-optimized medium. The strategy brought a significant increase of A40926 yield by 65.2 percent. Conclusions The engineered mutant with the genetic attributes of the co-expression of the dbv3 and dbv20 genes and the deletion of the dbv23 gene could obviously enhance the production of A40926. In addition, the optimization of medium was an effective and essential tool for the improvement of the secondary metabolites in Actinomyces.

Biodegradation of P-Nitrophenol by Engineered Strain

p-Nitrophenol (PNP) is an important environmental pollutant and can causes significant environmental and health risks. Compared with the traditional methods, biodegradation is a useful one to completely remove the harmful pollutants from the environment. Here, an engineered strain was first constructed by introducing PNP biodegradation pathway via the hydroquinone (HQ) pathway into Escherichia coli. In the engineered strain BL-PNP, PNP was completely degraded to β-ketoadipate and subsequently enter the metabolites of multiple anabolic pathways. The high tolerance and rapid degradation ability to PNP enable the engineered strain to have the potential to degrade toxic substances. The engineered strain created in this study can be used as a functional strain for bioremediation of PNP and potential toxic intermediates, and the method of assembling aromatic hydrocarbons metabolic pathway can be used to eradicate nitroaromatic pollutants in the environment.