Mycolicibacterium cell factory for the production of steroid-based drug intermediates

2021 ◽  
pp. 107860
Author(s):  
Anqi Zhao ◽  
Xiaoqin Zhang ◽  
Yamei Li ◽  
Zhi Wang ◽  
Yongkun Lv ◽  
...  
Keyword(s):  
Cell Factory ◽  
Drug Intermediates

scholarly journals Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395

2021 ◽  
Vol 22 (2) ◽  
pp. 480
Author(s):  
Jongrae Kim ◽  
Kwang Suk Chang ◽  
Sangmuk Lee ◽  
EonSeon Jin
Keyword(s):  
Genome Editing ◽  
Chlorella Vulgaris ◽  
Negative Selection ◽  
Screening Strategy ◽  
Feed Additive ◽  
Cell Factory ◽  
Dna Transformation ◽  
Research Groups ◽  
A Genome ◽  
Food And Feed

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

scholarly journals NaCl Promotes the Efficient Formation of Haematococcus pluvialis Nonmotile Cells under Phosphorus Deficiency

Marine Drugs ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. 337
Author(s):  
Feng Li ◽  
Ning Zhang ◽  
Yulei Zhang ◽  
Qingsheng Lian ◽  
Caiying Qin ◽  
...  
Keyword(s):  
Haematococcus Pluvialis ◽  
Phosphorus Deficiency ◽  
Cell Formation ◽  
Animal Nutrition ◽  
Cell Factory ◽  
Cell Type ◽  
Negative Effects ◽  
Related Factors ◽  
Motile Cells ◽  
Application Prospects

Natural astaxanthin helps reduce the negative effects caused by oxidative stress and other related factors, thereby minimizing oxidative damage. Therefore, it has considerable potential and broad application prospects in human health and animal nutrition. Haematococcus pluvialis is considered to be the most promising cell factory for the production of natural astaxanthin. Previous studies have confirmed that nonmotile cells of H. pluvialis are more tolerant to high intensity of light than motile cells. Cultivating nonmotile cells as the dominant cell type in the red stage can significantly increase the overall astaxanthin productivity. However, we know very little about how to induce nonmotile cell formation. In this work, we first investigated the effect of phosphorus deficiency on the formation of nonmotile cells of H. pluvialis, and then investigated the effect of NaCl on the formation of nonmotile cells under the conditions of phosphorus deficiency. The results showed that, after three days of treatment with 0.1% NaCl under phosphorus deficiency, more than 80% of motile cells had been transformed into nonmotile cells. The work provides the most efficient method for the cultivation of H. pluvialis nonmotile cells so far, and it significantly improves the production of H. pluvialis astaxanthin.

scholarly journals Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Marine Drugs ◽  
2021 ◽  
Vol 19 (5) ◽  
pp. 239
Author(s):  
Anguo Li ◽  
Ruihao Huang ◽  
Chaogang Wang ◽  
Qunju Hu ◽  
Hui Li ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Chlamydomonas Reinhardtii ◽  
Penaeus Monodon ◽  
Total Soluble Protein ◽  
Cell Factory ◽  
Gram Positive Bacteria ◽  
Resistance Selection ◽  
Expression Cluster ◽  
Potential Strategy

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.

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.

Recent advances in droplet microfluidics for enzyme and cell factory engineering

2021 ◽  
pp. 1-23
Author(s):  
Jianhua Yang ◽  
Ran Tu ◽  
Huiling Yuan ◽  
Qinhong Wang ◽  
Leilei Zhu
Keyword(s):  
Droplet Microfluidics ◽  
Cell Factory ◽  
Recent Advances

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 Nanocellulose-based mechanically stable immobilization matrix for enhanced ethylene production: A framework for photosynthetic solid-state cell factories

2021 ◽  
Author(s):  
Ville Rissanen ◽  
Sindhujaa Vajravel ◽  
Sergey Kosourov ◽  
Suvi Arola ◽  
Eero Kontturi ◽  
...  
Keyword(s):  
Solid State ◽  
Ethylene Production ◽  
Cell Immobilization ◽  
Cell Factory ◽  
Cell Factories ◽  
Solid State Cell ◽  
Immobilization Matrix

Cell immobilization is a promising approach to create efficient photosynthetic cell factories for sustainable chemicals production. Here, we demonstrate a novel photosynthetic solid-state cell factory design for sustainable biocatalytic ethylene...

scholarly journals Plasticity of Supporting Cells in a Stem Cell Factory

Immunity ◽  
2010 ◽  
Vol 33 (3) ◽  
pp. 291-293 ◽  
Author(s):  
Paul W. Kincade
Keyword(s):  
Stem Cell ◽  
Cell Factory ◽  
Supporting Cells
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