scholarly journals Construction and application of a CRISPR/Cas9-assisted genomic editing system for Corynebacterium glutamicum

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chengzhen Yao ◽  
Xiaoqing Hu ◽  
Xiaoyuan Wang
Keyword(s):  
Corynebacterium Glutamicum ◽  
Cell Factory ◽  
Temperature Sensitive ◽  
Microbial Cell Factory ◽  
Time Saving ◽  
Editing Efficiency ◽  
Highly Efficient ◽  
Native Promoter ◽  
Insertion And Deletion ◽  
Genomic Editing

AbstractCorynebacterium glutamicum is widely used as microbial cell factory for various bioproducts, but its genomic editing efficiency needs to be improved. In this study, a highly efficient CRISPR/Cas9-assisted genomic editing system for C. glutamicum was constructed. This system mainly involves a plasmid and can be used for both gene insertion and deletion in the chromosome of C. glutamicum. The recombinant plasmid for the target gene containing all the editing elements, and first constructed it in E. coli, then purified and transformed it into C. glutamicum. This temperature-sensitive plasmid was cured at high temperature after the genomic editing was completed in C. glutamicum. Using this genetic editing system, the genetic editing efficiency in C. glutamicum ATCC 13032 could reach 95%. The whole work of editing could be done in 8–9 days and showed most time-saving compared to the reported. Using this system, the native promoter of gdhA1 in ATCC 13032 has been replaced with the strong promoter PtacM, and more than 10 genes in ATCC 13032 have been deleted. The results demonstrate that this CRISPR/Cas9-assisted system is highly efficient and very suitable for genome editing in C. glutamicum.

scholarly journals Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lixia Fang ◽  
Jie Fan ◽  
Shulei Luo ◽  
Yaru Chen ◽  
Congya Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Free Fatty Acids ◽  
Stress Responses ◽  
Metabolic Networks ◽  
Target Identification ◽  
High Titer ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Ffa Metabolism

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL−-aidB+-ryfAM−-gadAH−, producing 30.0 g L−1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.

Highly efficient biosynthesis of l-ornithine from mannitol by using recombinant Corynebacterium glutamicum

2021 ◽  
Vol 327 ◽  
pp. 124799
Author(s):  
Qi Sheng ◽  
Xiaoyu Wu ◽  
Yan Jiang ◽  
Zhimin Li ◽  
Fei Wang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Highly Efficient

DEVELOPMENT OF A RAPID CONTINUOUS FLOW SALT LEACHING KIT FOR FABRICATION OF POLY(3-HYDROXYBUTYRIC ACID-CO-3-HYDROXYVALERATE) (PHBV) POROUS 3-D SCAFFOLD

2015 ◽  
Vol 75 (1) ◽  
Author(s):  
Syazwan Aizad ◽  
Badrul Hisham Yahaya ◽  
Saiful Irwan Zubairi
Keyword(s):  
Conventional Method ◽  
Continuous Flow ◽  
Tap Water ◽  
Chemical Properties ◽  
Porous Scaffolds ◽  
Time Saving ◽  
Salt Particle ◽  
Salt Leaching ◽  
Leaching Process ◽  
Highly Efficient

Polyhydroxyalkanoates (PHAs) that are synthesized from bacteria that are predominantly produced by microbial fermentation processes on organic waste, such as palm oil mill effluent (POME), olive oil and kitchen waste, contribute to a sustainable waste management. A great variety of materials from this family can be produced, however the application of PHAs in the production of scaffolds in tissue engineering has been mainly constrained to poly(hydroxybutyrate-co-valerate) (PHBV) due to its highly adjustable physico-chemical properties. One of the common methods in making the 3-D scaffolds is by performing solvent-casting particulate-leaching (SCPL) process, but this process requires a long period of soaking in water to extract the entire salt particle in the 3-D scaffolds. Therefore, the objective of this study is to develop a new method to the conventional method of salt leaching process via a highly efficient continuous flow leaching kit. The salt leaching process was carried out by (1) immersing the 3-D porous scaffolds in a fabricated static container containing tap water and (2) by allowing a pre-setting continuous flow rate of water. The concentration of sodium chloride (NaCl) was calculated periodically for both processes based on the salt standard calibration curve. The results showed that the exhaustive salt leaching of the conventional process occurred at 48 ± 5 hrs with the needs of changing the water twice a day. In contrast, the exhaustive salt leaching process via continuous flow leaching kit occurred at 40 ± 5 mins, 72 times faster than the conventional method (p<0.05). Therefore, the salt leaching process using continuous flow leaching kit can be considered a highly efficient and time saving procedure as compared to the conventional method.  

scholarly journals Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenning Liu ◽  
Xue Zhang ◽  
Dengwei Lei ◽  
Bin Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Phosphopantetheinyl Transferase ◽  
Chromosome Engineering ◽  
Microbial Cell Factory ◽  
E Coli ◽  
Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

scholarly journals Changes in Oxygen Availability during Glucose-Limited Chemostat Cultivations of Penicillium chrysogenum Lead to Rapid Metabolite, Flux and Productivity Responses

Metabolites ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 45
Author(s):  
Qi Yang ◽  
Wenli Lin ◽  
Jiawei Xu ◽  
Nan Guo ◽  
Jiachen Zhao ◽  
...  
Keyword(s):  
Steady State ◽  
Dissolved Oxygen ◽  
Penicillium Chrysogenum ◽  
Large Scale ◽  
Scale Up ◽  
Redox Status ◽  
Oxygen Availability ◽  
Industrial Scale ◽  
Cell Factory ◽  
Microbial Cell Factory

Bioreactor scale-up from the laboratory scale to the industrial scale has always been a pivotal step in bioprocess development. However, the transition of a bioeconomy from innovation to commercialization is often hampered by performance loss in titer, rate and yield. These are often ascribed to temporal variations of substrate and dissolved oxygen (for instance) in the environment, experienced by microorganisms at the industrial scale. Oscillations in dissolved oxygen (DO) concentration are not uncommon. Furthermore, these fluctuations can be exacerbated with poor mixing and mass transfer limitations, especially in fermentations with filamentous fungus as the microbial cell factory. In this work, the response of glucose-limited chemostat cultures of an industrial Penicillium chrysogenum strain to different dissolved oxygen levels was assessed under both DO shift-down (60% → 20%, 10% and 5%) and DO ramp-down (60% → 0% in 24 h) conditions. Collectively, the results revealed that the penicillin productivity decreased as the DO level dropped down below 20%, while the byproducts, e.g., 6-oxopiperidine-2-carboxylic acid (OPC) and 6-aminopenicillanic acid (6APA), accumulated. Following DO ramp-down, penicillin productivity under DO shift-up experiments returned to its maximum value in 60 h when the DO was reset to 60%. The result showed that a higher cytosolic redox status, indicated by NADH/NAD+, was observed in the presence of insufficient oxygen supply. Consistent with this, flux balance analysis indicated that the flux through the glyoxylate shunt was increased by a factor of 50 at a DO value of 5% compared to the reference control, favoring the maintenance of redox status. Interestingly, it was observed that, in comparison with the reference control, the penicillin productivity was reduced by 25% at a DO value of 5% under steady state conditions. Only a 14% reduction in penicillin productivity was observed as the DO level was ramped down to 0. Furthermore, intracellular levels of amino acids were less sensitive to DO levels at DO shift-down relative to DO ramp-down conditions; this difference could be caused by different timescales between turnover rates of amino acid pools (tens of seconds to minutes) and DO switches (hours to days at steady state and minutes to hours at ramp-down). In summary, this study showed that changes in oxygen availability can lead to rapid metabolite, flux and productivity responses, and dynamic DO perturbations could provide insight into understanding of metabolic responses in large-scale bioreactors.

Engineering the CBB cycle and hydrogen utilization pathway of Ralstonia eutropha H16 for improved autotrophic growth and PHB production

2020 ◽  
Author(s):  
Zhongkang Li ◽  
Muzi Hu ◽  
Bin Xiong ◽  
Dongdong Zhao ◽  
Chunzhi Zhang ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Ralstonia Eutropha ◽  
Biological Research ◽  
Industrial Biotechnology ◽  
Cell Factory ◽  
Autotrophic Growth ◽  
Microbial Cell Factory ◽  
Knock Out ◽  
Ralstonia Eutropha H16 ◽  
Living Organisms

Abstract CO 2 is fixed by all living organisms with an autotrophic metabolism, among which the Calvin-Benson-Bassham ( CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO 2 fixation efficiency of cells is an important subject of biological research with significant application potential. In this work, the autotrophic microbe Ralstonia eutropha H16 was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha , we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found while both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were f unctional d uring autotrophic growth of R. eutropha. And SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system and RbcX, the culture OD 600 of engineered strain increased 89.15% after 72 hours of autotrophic growth, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and improved upon original CBB pathway. Meanwhile, expression of hydrogenases were optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD 600 to 93.4% at 72 hours. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.71%. To our best knowledge, this is the first report of successfully engineering the CBB pathway of R. eutropha for improved activity , and is one of only a few cases where the efficiency of CO 2 assimilation pathway was improved. Our work demonstrates that R. eutropha is an extremely useful platform for studying and engineering the CBB for applications in more important organisms, such as agricultural crops, and a potential microbial cell factory to develop industrial biotechnology for sequestrating CO 2 .

Soil: Microbial Cell Factory for Assortment with Beneficial Role in Agriculture

2019 ◽  
pp. 63-92 ◽  
Author(s):  
Pratiksha Singh ◽  
Rajesh Kumar Singh ◽  
Mohini Prabha Singh ◽  
Qi Qi Song ◽  
Manoj K. Solanki ◽  
...  
Keyword(s):  
Microbial Cell ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Soil Microbial ◽  
Beneficial Role

scholarly journals Klebsiella pneumoniae—A Useful Pathogenic Strain for Biotechnological Purposes: Diols Biosynthesis under Controlled and Uncontrolled pH Levels

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 293 ◽  
Author(s):  
Laura Mitrea ◽  
Dan Cristian Vodnar
Keyword(s):  
Klebsiella Pneumoniae ◽  
Ph Value ◽  
Cell Factory ◽  
Anaerobic Conditions ◽  
Liquid Media ◽  
Microbial Cell Factory ◽  
Substrate Consumption ◽  
Main Carbon Source ◽  
Ph Level ◽  
Main Carbon

Despite being a well-known human pathogen, Klebsiella pneumoniae plays a significant role in the biotechnology field, being considered as a microbial cell factory in terms of valuable chemical biosynthesis. In this work, Klebsiella pneumoniae DSMZ 2026 was investigated for its potential to biosynthesize 1,3-propanediol (PDO) and 2,3-butanediol (BDO) during batch fermentation under controlled and uncontrolled pH levels. The bacterial strain was cultivated at a bioreactor level, and it was inoculated in 2 L of specific mineral broth containing 50 g/L of glycerol as the main carbon source. The process was conducted under anaerobic conditions at 37 °C and 180 RPM (rotations per minute) for 24 h. The effect of pH oscillation on the biosynthesis of PDO and BDO was investigated. Samples were taken every 3 h and specific tests were performed: pH measurement, main substrate consumption, PDO and BDO production. The cell morphology was analyzed on both solid and liquid media. After 24 h of cultivation, the maximum concentrations of PDO and BDO were 28.63 ± 2.20 g/L and 18.10 ± 1.10 g/L when the pH value was maintained at 7. Decreased concentrations of PDO and BDO were achieved (11.08 ± 0.14 g/L and 7.35 ± 0.00 g/L, respectively) when the pH level was not maintained at constant values. Moreover, it was identified the presence of other metabolites (lactic, citric, and succinic acids) in the cultivation media at the beginning of the process, after 12 h and 24 h of cultivation.

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