Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011

2012 ◽  
Vol 97 (7) ◽  
pp. 3053-3062 ◽  
Author(s):  
SangJoon Mo ◽  
Sung-Kwon Lee ◽  
Ying-Yu Jin ◽  
Chung-Hun Oh ◽  
Joo-Won Suh
Keyword(s):  
Metabolic Engineering ◽  
Random Mutagenesis ◽  
Combined Approach ◽  
Streptomyces Sp

scholarly journals Random Mutagenesis of Clostridium cellulolyticum by Using a Tn1545 Derivative

2010 ◽  
Vol 76 (13) ◽  
pp. 4546-4549 ◽  
Author(s):  
Jean-Charles Blouzard ◽  
Odile Valette ◽  
Chantal Tardif ◽  
Pascale de Philip
Keyword(s):  
Cell Wall ◽  
Metabolic Engineering ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Random Mutagenesis ◽  
Cell Wall Degradation ◽  
Clostridium Cellulolyticum ◽  
Plant Cell Wall Degradation ◽  
Random Insertion

ABSTRACT Further understanding of the plant cell wall degradation system of Clostridium cellulolyticum and the possibility of metabolic engineering in this species highlight the need for a means of random mutagenesis. Here, we report the construction of a Tn1545-derived delivery tool which allows monocopy random insertion within the genome.

Improvement of thermostability of cholesterol oxidase from streptomyces Sp. SA-COO by random mutagenesis

2021 ◽  
pp. 106028
Author(s):  
Aliakbar Fazaeli ◽  
Saeed Ebrahimi Fana ◽  
Abolfazl Golestani ◽  
Mahdi Aminian
Keyword(s):  
Cholesterol Oxidase ◽  
Random Mutagenesis ◽  
Streptomyces Sp

scholarly journals Application of Random Mutagenesis and Synthetic FadR Promoter for de novo Production of ω-Hydroxy Fatty Acid in Yarrowia lipolytica

2021 ◽  
Vol 9 ◽  
Author(s):  
Beom Gi Park ◽  
Junyeob Kim ◽  
Eun-Jung Kim ◽  
Yechan Kim ◽  
Joonwon Kim ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Metabolic Engineering ◽  
Palmitic Acid ◽  
Yarrowia Lipolytica ◽  
Cell Sorting ◽  
De Novo ◽  
Random Mutagenesis ◽  
Oleaginous Yeasts ◽  
Synthetic Promoters ◽  
Base Strain

As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.

Biosurfactant produced by a Rhodococcus erythropolis mutant as an oil spill response agent

2016 ◽  
Vol 51 (2) ◽  
pp. 97-105 ◽  
Author(s):  
Qinhong Cai ◽  
Baiyu Zhang ◽  
Bing Chen ◽  
Tong Cao ◽  
Ze Lv
Keyword(s):  
Metabolic Engineering ◽  
Solvent Extraction ◽  
Oil Spill ◽  
Parent Strain ◽  
Rhodococcus Erythropolis ◽  
Random Mutagenesis ◽  
Oil Spill Response ◽  
Oily Water ◽  
Layer Chromatography ◽  
Better Than

Biosurfactants have been considered as superior alternatives to currently used surfactants as they are generally more biodegradable, less toxic, and better at enhancing biodegradation. However, the application of biosurfactants is limited by the availability of economic biosurfactants and the corresponding producers that can work effectively. Hyperproducers generated by metabolic engineering of biosurfactant producers are highly desired to overcome this obstacle. A Rhodococcus erythropolis SB-1A strain was isolated from offshore oily water samples. One of its mutants derived by random mutagenesis with ultraviolet radiation, producing high levels of biosurfactants, was selected by the oil spreading technique. The mutant produces biosurfactants with critical micelle dilutions approximately four times those of the parent strain. The results obtained with thin layer chromatography indicated the produced biosurfactant remained unchanged between the mutant and the parent strain. In addition, the produced biosurfactants were recovered with solvent extraction and applied as the oil spill response agents. Based on the baffled flask test (BFT) results, the dispersion efficiency of the biosurfactants produced by the mutant is higher than that induced by the parent strain. When compared with Corexit dispersants, it was found that the produced biosurfactants performed better than Corexit 9527 and were comparable with Corexit 9500.

scholarly journals Combining Random Mutagenesis and Metabolic Engineering for Enhanced Tryptophan Production in Synechocystis sp. Strain PCC 6803

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Arnav Deshpande ◽  
Jeremiah Vue ◽  
John Morgan
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Amino Acid ◽  
Animal Feed ◽  
Random Mutagenesis ◽  
Genetically Engineered ◽  
Chorismate Mutase ◽  
Content Type ◽  
Step Process ◽  
Pcc 6803

ABSTRACT Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroGfbr and trpEfbr, were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroGfbr and trpEfbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach. IMPORTANCE Aromatic amino acids such as tryptophan are primarily used as additives in the animal feed industry and are typically produced using genetically engineered heterotrophic organisms such as Escherichia coli. This involves a two-step process, where the substrate such as molasses is first obtained from plants followed by fermentation by heterotrophic organisms. We have engineered photoautotrophic cyanobacterial strains by a combination of random mutagenesis and metabolic engineering. These strains grow on CO2 as the sole carbon source and utilize light as the sole energy source to produce tryptophan, thus converting the two-step process into a single step. Our results show that combining random mutagenesis and metabolic engineering was superior to either approach alone. This study also builds a foundation for further engineering of cyanobacteria for industrial tryptophan production.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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