Ya-Jie Tang

Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing.

Engineering cellular metabolism for improved production of valuable chemicals requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for β-carotene overproduction. We collectively tested 33 genomic modifications and constructed more than 100 genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained15 targeted mutations and produced 2.0 g/L β-carotene in fed-batch fermentation.

Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum.

Investigations on Ganoderma lucidum fermentation suggested that the responses of the cell growth and metabolites biosynthesis to pH and dissolved oxygen tension (DOT) were different. The ganoderic acid (GA) production of 321.6 mg/L was obtained in the pH-shift culture by combining a 4-day culture at pH 3.0 with the following 6-day culture at pH 4.5, which was higher by 45% and 300% compared with the culture at pH 3.0 and 4.5, respectively. The GA production of 487.1mg/L was achieved in the DOT-shift culture by combining a 6-day culture at 25% of DOT with a following 6-day culture at 10% of DOT, which was higher by 43% and 230% compared with the culture at 25% and 10% of DOT, respectively. A fed-batch fermentation process by combining the above-mentioned pH-shift and DOT-shift strategies resulted in a significant synergistic enhancement of GA accumulation up to 754.6 mg/L, which is the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactor.

Significance of inoculation density control in production of polysaccharide and ganoderic acid by submerged culture of Ganoderma lucidum

Abstract Control of inoculation density was significant for cell growth, morphology, and production of polysaccharide and ganoderic acid in submerged culture of the higher fungus Ganoderma lucidum . A maximal cell concentration of 15.7 g dry cell weight (DW)/l was obtained at an inoculation density of 330 mg DW/l. For inoculation density within the range of 70–670 mg DW/l, a large inoculation density led to a small pellet size and high production of extracellular and intracellular polysaccharides, while a relatively big pellet size and high accumulation of ganoderic acid were observed at a low inoculation density. It was also shown that small pellet size resulted in high polysaccharide production, while large pellet size led to high production of ganoderic acid.

Tuber melanosporum fermentation medium optimization by Plackett-Burman design coupled with Draper-Lin small composite design and desirability function

A novel method using Plackett-Burman design (PBD) coupled with Draper-Lin small composite design (SCD) and desirability function (DF) was developed to optimize Tuber melanosporum fermentation medium. Firstly, sucrose, yeast extract, peptone and Mg(2+) were identified to be key medium components by PBD. Secondly, in order to evaluate the relationships between responses and the identified key components, mathematical models were developed by SCD. Finally, by using DF, the responses were optimized simultaneously and the optimal concentration was located to be 73 g/L sucrose, 11 g/L yeast extract, 8 g/L peptone, and 46 mM Mg(2+). Under the identified optimal conditions, the maximal biomass, the production of extracellular (EPS) and intracellular (IPS) polysaccharides was 25.10 + or - 0.12 g/L, 3.88 + or - 0.23 g/L and 2.87 + or - 0.32 g/L, respectively, which agreed with the predicted values well. Compared with the basic medium, the biomass, the production of EPS and IPS was enhanced by 54.4%, 71.7% and 124.2%, respectively.

Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides

For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35-5 g/L when its residual level decreased to 10-5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72+/-2.57 g DW/L, 7.09+/-0.62 and 4.43+/-0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.

Scale-Up of a Liquid Static Culture Process for Hyperproduction of Ganoderic Acid by the Medicinal Mushroom Ganoderma lucidum

Scale‐up of a liquid static culture process was studied for hyperproduction of ganoderic acid (GA) by a famous Chinese traditional medicinal mushroom, Ganoderma lucidum. Initial volumetric oxygen transfer coefficient (KLa) and area of liquid surface per liquid volume (As) were identified as key factors affecting cell growth and GA accumulation in liquid static cultures of G. lucidum, on the basis of which a multilayer static bioreactor was designed. At a low initial KLa level of 2.1 h‐1, a thick layer of white mycelia was formed on the liquid surface, and an optimal production of total GA (i.e., GA production in the liquid and on the liquid surface) was obtained. Both the formation of white mycelia and production of GA on the liquid surface were enhanced with an increase of As within the range as investigated (0.24–1.53 cm2/mL). At an As value of 0.90 cm2/mL, the total GA production reached maximum. A successful scale‐up from a 20‐mL static T‐flask to a 7.5‐L three‐layer static bioreactor was achieved based on initial KLa. The maximum biomass (20.8 ± 0.1 g DW/L), GA content (4.96 ± 0.13 mg/100 mg DW), and total GA production (976 ± 35 mg/L) were attained in static bioreactors. Not only GA content but also its production obtained in this work were the highest ever reported.

Enzymatic saccharification of sugarcane bagasse by N-methylmorpholine-N-oxide-tolerant cellulase from a newly isolated Galactomyces sp. CCZU11-1

Based on the enrichment culture strategy, a novel N-methylmorpholine-N-oxide (NMMO)-tolerant cellulase-producing strain Galactomyces sp. CCZU11-1 was isolated from soil samples. After the optimization of culture condition, the highest FPA (13.4 U/mL) and CMCase (24.5 U/mL) were obtained. In both culture and reaction media containing NMMO 25% (w/v), the cellulase from Galactomyces sp. CCZU11-1 still had good activity. Furthermore, high saccharification rate was obtained in aqueous-NMMO media. Moreover, the fermentability of the hydrolyzates, obtained after enzymatic in situ saccharification of the NMMO-pretreated sugarcane bagasse, was evaluated using Saccharomyce scerevisiae. In conclusion, Galactomyces sp. CCZU11-1 is a promising candidate as high NMMO-tolerant cellulase producer and has potential application in future.

Determination of 5α-androst-16-en-3α-ol in truffle fermentation broth by solid-phase extraction coupled with gas chromatography–flame ionization detector/electron impact mass spectrometry

A novel method using solid-phase extraction coupled with gas chromatography and flame ionization detector (FID)/electron impact mass spectrometry (EIMS) was developed for the determination of 5alpha-androst-16-en-3alpha-ol (androstenol), a steroidal compound belonging to the group of musk odorous 16-androstenes, in truffle fermentation broth. Comparison studies between FID and EIMS indicated two detectors gave similar quantitative results. The highest androstenol concentration of 123.5 ng/mL was detected in Tuber indicum fermentation broth, while no androstenol was found in Tuber aestivum fermentation broth. For the first time, this work confirmed the existence of androstenol in the truffle fermentation broth, which suggested truffle fermentation is a promising alternative for androstenol production on a large scale.

Activation of glyoxylate pathway without the activation of its related gene in succinate-producing engineered Escherichia coli

For the first time, glyoxylate pathway in the biosynthesis of succinate was activated without the genetic manipulations of any gene related with glyoxylate pathway. Furthermore, the inactivation of succinate biosynthesis by-products genes encoding acetate kinase (ackA) and phosphotransacetylase (pta) was proven to be the key factor to activate glyoxylate pathway in the metabolically engineered Escherichia coli under anaerobic conditions. In order to enhance the succinate biosynthesis specifically, the genes (i.e., ldhA, ptsG, ackA-pta, focA-pflB, adhE) that disrupt by-products biosynthesis pathways were combinatorially deleted, while the E. coli malate dehydrogenase (MDH) was overexpression. The highest succinate production of 150.78 mM was obtained with YJ003 (ΔldhA, ptsG, ackA-pta), which were 5-folds higher than that obtained with wild type control strain DY329 (25.13 mM). For further understand the metabolic response as a result of several genetic manipulations, an anaerobic stoichiometric model that takes into account the glyoxylate pathway have successfully been implemented to estimate the intracellular fluxes in various recombinant E. coli. The fraction to the glyoxylate pathway from OAA in DY329 was 0 and 31% in YJ003, which indicated that even without the absence of the iclR mutation; the glyoxylate pathway was also activated by deleting the by-products biosynthetic genes, and to be responsible for the higher succinate yields. For further strengthen glyoxylate pathway, a two-stage fed-batch fermentation process was developed by using a 600 g l(-1) glucose feed to achieve a cell growth rate of 0.07 h(-1) in aerobic fermentation, and using a 750 g l(-1) glucose feed to maintain the residual glucose concentration around 40 g l(-1) when its residual level decreased to 10gl(-1) in anaerobic fermentation. The best mutant strain YJ003/pTrc99A-mdh produces final succinate concentration of 274 mM by fed-batch culture, which was 10-folds higher than that obtained with wild type control strain DY329. This study discovered that glyoxylate pathway could be activated by deleting glyoxylate pathway irrelevant genes (i.e., genes encoding acetate and lactate) and consequently the succinate biosynthesis was effectively improved. This work provides useful information for the modification of metabolic pathway to improve succinate production.

Enhancement of validamycin A production by addition of ethanol in fermentation of Streptomyces hygroscopicus 5008.

The effect of ethanol on the production of the important agro-antibiotic validamycin A (Val-A) in medium containing agricultural by-products was investigated. Under the optimal condition of ethanol addition, the maximal Val-A production titer reached 18 g/L, which increased by 60% compared to the control. To provide an insight into cell response to ethanol, the intracellular reactive oxygen species (ROS), gene transcription and enzyme activity were determined. Intracellular ROS as the molecular signal was increased in the ethanol condition. Global regulators afsR and glnR were involved in regulation of Val-A biosynthesis, and the transcription of eight Val-A structural genes was enhanced. The activity of glucose-6-phosphate dehydrogenase (G6PD) was enhanced while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was inhibited. A signal transduction cascade from cell signal response to activated transcription of Val-A biosynthetic genes and enhanced antibiotic production is proposed. The information can be helpful for the improvement of large-scale fermentation.