Haishan Qi

Rational medium optimization based on comparative metabolic profiling analysis to improve fumaric acid production.

To rationally guide fumaric acid production improvement, metabolic profiling approach was performed to analyze metabolite changes of Rhizopus oryzae FM19 under different fermentation conditions. A correlation between the metabolic profiling and fumaric acid production was revealed by principal component analysis as well as partial least squares. Citric acid, oxaloacetic acid, 2-oxoglutarate, lactic acid, proline, alanine, valine, leucine were identified to be mainly responsible for the metabolism difference, which were involved in the Embden-Meyerhof-Parnas, tricarboxylic acid cycle, amino acid metabolism and fatty acid metabolism. Through the further analysis of metabolites changes together with the above pathways, exogenous addition strategies were developed, which resulted in 14% increase of fumaric acid (up to 56.5 g/L) and less by-products. These results demonstrated that metabolic profiling analysis could be successfully applied to the rational guidance of medium optimization and the productivity improvement of value-added compounds.

Model-Driven Redox Pathway Manipulation for Improved Isobutanol Production in Bacillus subtilis Complemented with Experimental Validation and Metabolic Profiling Analysis

To rationally guide the improvement of isobutanol production, metabolic network and metabolic profiling analysis were performed to provide global and profound insights into cell metabolism of isobutanol-producing Bacillus subtilis. The metabolic flux distribution of strains with different isobutanol production capacity (BSUL03, BSUL04 and BSUL05) drops a hint of the importance of NADPH on isobutanol biosynthesis. Therefore, the redox pathways were redesigned in this study. To increase NADPH concentration, glucose-6-phosphate isomerase was inactivated (BSUL06) and glucose-6-phosphate dehydrogenase was overexpressed (BSUL07) successively. As expected, NADPH pool size in BSUL07 was 4.4-fold higher than that in parental strain BSUL05. However, cell growth, isobutanol yield and production were decreased by 46%, 22%, and 80%, respectively. Metabolic profiling analysis suggested that the severely imbalanced redox status might be the primary reason. To solve this problem, gene udhA of Escherichia coli encoding transhydrogenase was further overexpressed (BSUL08), which not only well balanced the cellular ratio of NAD(P)H/NAD(P)+, but also increased NADH and ATP concentration. In addition, a straightforward engineering approach for improving NADPH concentrations was employed in BSUL05 by overexpressing exogenous gene pntAB and obtained BSUL09. The performance for isobutanol production by BSUL09 was poorer than BSUL08 but better than other engineered strains. Furthermore, in fed-batch fermentation the isobutanol production and yield of BSUL08 increased by 11% and 19%, up to the value of 6.12 g/L and 0.37 C-mol isobutanol/C-mol glucose (63% of the theoretical value), respectively, compared with parental strain BSUL05. These results demonstrated that model-driven complemented with metabolic profiling analysis could serve as a useful approach in the strain improvement for higher bio-productivity in further application.

Engineering Scheffersomyces stipitis for fumaric acid production from xylose.

In this work, Scheffersomyces stipitis, the yeast with excellent xylose-utilizing ability, was firstly engineered for fumaric acid production from xylose with the heterologous reductive pathway from Rhizopus oryzae FM19, and 1.86g/L fumaric acid was produced by the initial strain PSRPMF under the oxygen-limited condition. Furthermore, three strategies were performed to improve the fumaric acid production, including increasing the reductive pathway activity by codon optimization, blocking the fumaric acid conversion in tricarboxylic acid cycle by knocking out the native fumarases, and improving the fumaric acid transportation by overexpressing heterologous transporter. Finally, the strain PSYPMFfS was obtained and the fumaric acid titer reached to 4.67g/L, significantly increased by 37.92-fold than that of the control strain PSPYSS. It was indicated that the S. stipitis was a promising platform for fumaric acid production from xylose.

Higher-level production of ascomycin (FK520) by Streptomyces hygroscopicus var. ascomyceticus irradiated by femtosecond laser

Femtosecond laser irradiation technology was employed for the first time to improve the ascomycin (FK520) yield of Streptomyces hygroscopicus var. ascomyceticus NT2-11, which is an N-methyl-N-nitro-N-nitrosoguanidine (NTG)-induced strain derived from S. hygroscopicus (ATCC14891). The mutant FS35 with high and stable FK520 production capacity was then obtained in the optimal irradiation conditions (25 mW for 6 min) by the Titanium sapphire laser system (810 nm, 76 MHz, 150 fs). The FK520 production capacity of FS35 was 45% higher than that of the parental strain NT2-11. Moreover, under the optimal fermentation conditions, FK520 fermentation titer of FS35 reached 300 mg/L and the intrinsic kinetics of FS35 and NT2-11 were investigated comparatively in 3 phases. The mathematical models provided a good description of FK520 fermentation process for both strains and valuable information for optimizing operation and pilotplant enlargement research. The comparative studies on parameters of the models confirmed the advantages in production and the decrease of substrate inhibition through femtosecond laser irradiation. Therefore, femtosecond laser irradiation provides a promising way to enhance the production of FK520 in S. hygroscopicus.

Comparative metabolomic-based metabolic mechanism hypothesis for microbial mixed cultures utilizing cane molasses wastewater for higher 2-phenylethanol production.

The mixed microbes coculture method in cane molasses wastewater (CMW) was adopted to produce 2-phenylethanol (2-PE). Comparative metabolomics combined with multivariate statistical analysis was performed to profile the differences of overall intracellular metabolites concentration for the mixed microbes cocultured under two different fermentation conditions with low and high 2-PE production. In total 102 intracellular metabolites were identified, and 17 of them involved in six pathways were responsible for 2-PE biosynthesis. After further analysis of metabolites and verification by feeding experiment, an overall metabolic mechanism hypothesis for the microbial mixed cultures (MMC) utilizing CMW for higher 2-PE production was presented. The results demonstrated that the branches of intracellular pyruvate metabolic flux, as well as the flux of phenylalanine, tyrosine, tryptophan, glutamate, proline, leucine, threonine, and oleic acid, were closely related to 2-PE production and cell growth, which provided theoretical guidance for domestication and selection of species as well as medium optimization for MMC metabolizing CMW to enhance 2-PE yield.

Poly(carboxybetaine methacrylate)-functionalized magnetic composite particles: A biofriendly support for lipase immobilization

In this work, poly(carboxybetaine methacrylate) as an extremely hydrophilic polymer was modified on superparamagnetic Fe3O4 nanoparticles (pCB-Fe3O4), which were employed to immobilize porcine pancreatic lipase. The properties of immobilized lipase were investigated in comparison with the free enzyme counterpart. Enzymatic stability, reusability, and activity of the immobilized lipase were found significantly superior to that of the free lipase. In particular, at an elevated temperature of 60°C, the immobilized lipase retained 50% of its initial activity after 150min, while the free enzyme displayed only one-third activity of the immobilized enzyme. Besides, the immobilized lipase retained >60% of its initial activity after 7 cycles. Furthermore, the value of Kcat/Km indicated the catalytic efficiency of the immobilized lipase was increased by 50% compared to that of the free one. The pCB-Fe3O4 particles displayed non-cytotoxicity, while the naked Fe3O4 particles caused only 50% viability of NIH 3T3 cells. These results showed that pCB-Fe3O4 composite particles had higher efficiency and improved stability for lipase immobilization, which are more promising for industrial scale up of biocatalytic systems with excellent biocompatibility.

Integration of parallel 13C‐labeling experiments and in silico pathway analysis for enhanced production of ascomycin

Herein, the hyper‐producing strain for ascomycin was engineered based on 13C‐labeling experiments and elementary flux modes analysis (EFMA). First, the metabolism of non‐model organism Streptomyces hygroscopicus var. ascomyceticus SA68 was investigated and an updated network model was reconstructed using 13C‐ metabolic flux analysis. Based on the precise model, EFMA was further employed to predict genetic targets for higher ascomycin production. Chorismatase (FkbO) and pyruvate carboxylase (Pyc) were predicted as the promising overexpression and deletion targets, respectively. The corresponding mutant TD‐FkbO and TD‐ΔPyc exhibited the consistency effects between model prediction and experimental results. Finally, the combined genetic manipulations were performed, achieving a high‐yield ascomycin engineering strain TD‐ΔPyc‐FkbO with production up to 610 mg/L, 84.8% improvement compared with the parent strain SA68. These results manifested that the integration of 13C‐labeling experiments and in silico pathway analysis could serve as a promising concept to enhance ascomycin production, as well as other valuable products. Biotechnol. Bioeng. 2017;114: 1036–1044.

A facile modification to improve the biocompatibility and adsorbability of activated carbon with zwitterionic hydrogel

In this work, poly(carboxybetaine methacrylate) hydrogel (pCBMA) was employed to modify the activated carbon (AC) for improving the biocompatibility and adsorption capacity of AC in biological environments. First, size-controlled hydrogel beads and hydrogel coated AC (pCBMA-AC) were fabricated with a homemade device, and the preparation conditions were optimized. Then the physical and biological properties of pCBMA-AC with different diameters were investigated. 2 mm pCBMA-AC dispalyed excellent stability with leakage rate only 0.16% after 72 h shaking incubation, as well as remarkable biocompatibility with merely 0.13% hemolysis rate and 3.41% cell death, while 14.72% and 70.11% for the bare AC, respectively, indicating the acceptable lower hemolysis and cytotoxicity according to ISO 10993. Furthermore, the adsorption capacities of pCBMA-AC were evaluated in biological environments with methylene blue as model molecules. The pCBMA-AC displayed 93.50% and 97.32% adsorption rates in BSA solution and FBS, respectively, but only 70.33% and 40.26% for the uncoated AC. These results indicated that pCBMA endows AC remarkable biocompatibility and adsorption capacity, which could extend the applications of AC in biological environments.

Biocompatible magnetic nanoparticles grafted by poly(carboxybetaine acrylamide) for enzyme immobilization

Herein, the zwitterionic material poly (carboxybetaine acrylamide) was grafted onto iron oxide to obtain biocompatible magnetic nanoparticles Fe3O4-pCBAA which were employed to immobilize enzymes. The nanocomplxes Fe3O4-pCBAA were characterized using scanning electron microscopy (SEM), dynamic light scattering (DLS), zeta potential, Fourier transform-infrared (FT-IR) spectra and energy dispersive X-ray spectrometry (EDX). The urease as a model enzyme was immobilized with the novel supports and the properties of immobilized urease were further investigated in comparison with the free urease counterpart. The immobilized urease exhibited excellent thermodynamic and chemical stability. Particularly, 60% of initial activity was remained after being stored at 70 °C for 2 h while the free urease only remained 30%. Besides, the relative activity of immobilized enzyme was 1.7 times that of free ones after disposed in ethanol and 2-propanol for 2 h, and 7 times in DMF. Moreover, immobilized urease retained >80% of its initial activity after 5 cycles. In addition, the immobilization carrier Fe3O4-pCBAA displayed famous biocompatibility, and the immobilized urease performed better in complex biological samples, which were >85% and <60% of its initial activity for the immobilized and dissociative urease, respectively, in 20% and 25% of serum. These results confirm that the nanoparticles Fe3O4-pCBAA are biofriendly and efficient supports for enzyme immobilization and potential for practical applications in bio-microenvironments.

Metabolomics assisted metabolic network modeling and network wide analysis of metabolites in microbiology

Abstract Metabolomics is the science of qualitatively and quantitatively analyzing low molecular weight metabolites occur in a given biological system. It provides valuable information to elucidate the functional roles and relations of different metabolites in a metabolic pathway. In recent years, a large amount of research on microbial metabolomics has been conducted. It has become a useful tool for achieving highly efficient synthesis of target metabolites. At the same time, many studies have been conducted over the years in order to integrate metabolomics data into metabolic network modeling, which has yielded many exciting results. Additionally, metabolomics also shows great advantages in analyzing the relationship of metabolites network wide. Integrating metabolomics data into metabolic network construction and applying it in network wide analysis of cell metabolism would further improve our ability to control cellular metabolism and optimize the design of cell factories for the overproduction of valuable biochemicals. This review will examine recent progress in the application of metabolomics approaches in metabolic network modeling and network wide analysis of microbial cell metabolism.