Multiplex gene editing and regulation techniques based on CRISPR/Cas system / 生物工程学报

The CRISPR/Cas systems comprising the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an acquired immune system unique to archaea or bacteria. Since its development as a gene editing tool, it has rapidly become a popular research direction in the field of synthetic biology due to its advantages of high efficiency, precision, and versatility. This technique has since revolutionized the research of many fields including life sciences, bioengineering technology, food science, and crop breeding. Currently, the single gene editing and regulation techniques based on CRISPR/Cas systems have been increasingly improved, but challenges still exist in the multiplex gene editing and regulation. This review focuses on the development and application of multiplex gene editing and regulation techniques based on the CRISPR/Cas systems, and summarizes the techniques for multiplex gene editing or regulation within a single cell or within a cell population. This includes the multiplex gene editing techniques developed based on the CRISPR/Cas systems with double-strand breaks; or with single-strand breaks; or with multiple gene regulation techniques, etc. These works have enriched the tools for the multiplex gene editing and regulation and contributed to the application of CRISPR/Cas systems in the multiple fields.

Progress in microbial production of succinic acid / 生物工程学报

Succinic acid is one of the key intermediates in the tricarboxylic acid cycle (TCA)and has huge potentials in biopolymer, food, medicine applications. This article reviews recent research progress in the production of succinic acid by microbial fermentation, including discovery and screening of the succinic-acid-producing microbes, the progress of genetic engineering strategy and metabolic engineering technology for construction of succinic acid-producing strains, and fermentation process control and optimization. Finally, we discussed the limitation of current progress and proposed the future research needs for microbial production of succinic acid.

Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002 / 生物工程学报

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

Succinic acid production with Escherichia coli AFP111 recovered from fermentation / 生物工程学报

During the anaerobic fermentation by Escherichia coli AFP111 for succinic acid production, the viable cell concentration and productivity were decreased with the raising of succinic acid concentration. In order to restore cellular succinic acid productivity and prolong fermentation time, we collected strains and refreshed medium for repetitive succinic acid production. However, productivity is lower than that in the anaerobic fermentation before reusing strains. To enhance the productivity, strains were aerobically cultivated for 3 h in pure water before anaerobic fermentation. The activities of key enzymes were enhanced for better performance in producing succinic acid at anaerobic stage. After three rounds of repetitive fermentations, succinic acid concentration and yield reached to 56.50 g/L and 90% respectively. The succinic acid productivity was 0.81 g/(L x h), which was 13% higher than the repetitive fermentations without aerobic activation of the strains.

Mutating Escherichia coli by atmospheric and room temperature plasmas for succinic acid production from xylose / 生物工程学报

Escherichia coli AFP111 is a spontaneous mutant with mutations in the glucose specific phosphotransferase system (ptsG) in NZN111 (delta pflAB deltaldhA). In AFP111, conversion of xylose to succinic acid generates 1.67 molecule of ATP per xylose. However, the strain needs 2.67 molecule ATP for xylose metabolism. Therefore, AFP111 cannot use xylose due to insufficient ATP under anaerobic condition. Through an atmospheric and room temperature plasma (ARTP) jet, we got a mutant strain named DC111 that could use xylose under anaerobic condition in M9 medium to produce succinic acid. After 72 h, DC111 consumed 10.52 g/L xylose to produce 6.46 g/L succinic acid, and the yield was 0.78 mol/mol. Furthermore, the reaction catalyzed by the ATP-generating PEP-carboxykinase (PCK) was enhanced. The specific activity of PCK was 19.33-fold higher in DC111 than that in AFP111, which made the strain have enough ATP to converse xylose to succinic acid.

Effect of overexpression of nicotinic acid mononucleotide adenylyltransferase on succinic acid production in Escherichia coli NZN111 / 生物工程学报

Escherichia coli NZN111 is a promising strain with ldhA and pflB genes inactivated for the production of succinic acid. However, with these mutations, NAD+ could not be regenerated from NADH, and an unbalanced NADH/NAD+ ratio eliminated cell growth and glucose utilization under anaerobic conditions. Nicotinic acid mononucleotide adenylyltransferase (NAMNAT), encoded by the nadD gene, catalyzes the reaction from nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) during the synthetic pathway of NAD(H). Overexpression of the nadD gene could enhance the concentration of NAD(H) and maintain a suitable NADH/NAD+ ratio. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-nadD, and overexpressed NAMNAT with 1.0 mmol/L of IPTG under anaerobic conditions in sealed bottles. Compared to E. coli NZN111, the concentrations of NAD+ and NADH in the recombinant strain increased by 3.21-fold and 1.67-fold, respectively. The total concentration of NAD(H) was increased by 2.63-fold, and the ratio of NADH/NAD+ decreased from 0.64 to 0.42. The recombinant strain restored the cell growth and glucose utilization under anaerobic conditions. After 72 h, the recombinant strain could consume 14.0 g/L of glucose to produce 6.23 g/L of succinic acid, and the concentration of succinic acid was 19-fold higher than in E. coli NZN111.

Isolation of high osmotic-tolerant mutants of Escherichia coli for succinic acid production by metabolic evolution / 生物工程学报

Succinic acid production was inhibited by high osmotic pressure caused by the accumulation of sodium ions in the process of two-stage fermentation by Escherichia coli using Na2CO3 as the pH regulator. To enhance the resistance of this strain to osmotic stress, the possibility to isolate high NaCl-tolerant mutant strain of Escherichia coli for succinic acid production by metabolic evolution was investigated. The metabolic evolution system was used as a mutant-generating system, allowing the cells to be continuously cultured at the maximum specific growth rate. The mutant strain can grow at maximum rate in the condition of high osmotic by gradually improving the concentration of NaCl in a continuous culture. Then the high osmotic-tolerant mutant strain of E. coli XB4 was selected with NaCl as the osmo-regulator. When using Na2CO3 as the pH regulator, E. coli XB4 was used in a 7.0 L fermenter during two-stage fermentation. After 60 h anaerobic fermentation, the mutant strain XB4 produced 69.5 g/L succinic acid with a productivity of 1.18 g/(L x h), which were increased by 18.6% and 20% compared with that of the parent strain.

Effect of overexpression of malate dehydrogenase on succinic acid production in Escherichia coli NZN111 / 生物工程学报

Escherichia coli NZN111 is a double mutant with lactate dehydrogenase (ldhA) and pyruvate formate-lyase (pflB) inactivated. Under anaerobic conditions, disequilibrium of coenzyme NADH and NAD+ causes Escherichia coli NZN111 losing the glucose utilizing capability. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-mdh and overexpressed the mdh gene with 0.3 mmol/L of IPTG under anaerobic fermentation condition in sealed bottles. The specific malate dehydrogenase (MDH) activity in the recombinant strain was 14.8-fold higher than that in E. coli NZN111. The NADH/ NAD+ ratio decreased from 0.64 to 0.26 and the concentration of NAD+ and NADH increased 1.5-fold and 0.2-fold respectively. Under anaerobic conditions, the recombinant strain possessed the capability of growth and glucose absorption. We took dual-phase fermentation for succinate production. After the dry cell weight (DCW) reached 6.4 g/L under aerobic conditions, the cell culture was changed to anaerobic conditions. After 15 h, 14.75 g/L glucose was consumed and succinic acid reached 15.18 g/L. The yield of succinic acid was 1.03 g/g Glu and the productivity of succinic acid was 1.012 g/(L x h).

Effect of overexpression of nicotinic acid phosphoribosyl transferase on succinic acid production in Escherichia coli NZN111 / 生物工程学报

Escherichia coli strain NZN111 is a promising candidate for the fermentative production of succinate. However, because lactate dehydrogenase and pyruvate formate lyase were inactivated in NZN111, this strain had an unbalanced NADH/NAD+ ratio and could not use glucose under anaerobic conditions. In this study, a recombinant strain E. coli NZN111/pTrc99a-pncB was constructed to overexpress the nicotinic acid phosphoribosyl transferase gene (pncB). Under anaerobic conditions with the addition of 0.5 mmol/L nicotinic acid and 0.3 mmol/L isopropyl beta-D-thiogalactopyranoside (IPTG), the specific nicotinic acid phosphoribosyl transferase (NAPRTase, EC 2.4.2.11) activity in the recombinant strain was 11-fold higher than that in E. coli NZN111, the concentration of NAD(H) was increased by 3.85-fold, especially the concentration of NAD+ was increased by 5.17-fold and NADH/NAD+ was decreased from 0.640 to 0.125. The recombinant strain regained the capability of growth and glucose utilization under anaerobic conditions.