scholarly journals PCR & Go: A Pre-installed Expression Chassis for Facile Integration of Multi-Gene Biosynthetic Pathways

2021 ◽  
Vol 8 ◽  
Author(s):  
Mingming Qi ◽  
Bei Zhang ◽  
Lihong Jiang ◽  
Saijuan Xu ◽  
Chang Dong ◽  
...  
Keyword(s):  
High Efficiency ◽  
Functional Expression ◽  
Single Step ◽  
Yeast Genome ◽  
Biosynthetic Pathways ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Plasmid Library ◽  
Yeast Chromosome ◽  
Β Carotene

The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a “PCR & Go” system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the “PCR & Go” system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the β-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the “PCR & Go” system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.

scholarly journals Overexpression of Thermophilic α-amylase in Bacillus Licheniformis using a High Efficiency Chromosomal Integration and Amplification Strategy

2021 ◽  
Author(s):  
Peili Shen ◽  
Dandan Niu ◽  
Xuelian Liu ◽  
Kangming Tian ◽  
Permaul Kugenthiren ◽  
...  
Keyword(s):  
Bacillus Licheniformis ◽  
High Efficiency ◽  
High Yield ◽  
Chromosomal Integration ◽  
Bacillus Sp ◽  
Strain Development ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
New Strategy ◽  
Amplification Strategy

Abstract Highly efficient preparation of industrially important enzymes depends on development of the genetically stable and high-yield microbial cell factories, which is often a challengeable laboratory hard work. In aims to simplify strain development with high efficiency for enzyme overproduction, a new strategy based on chromosomal integration and amplification in Bacillus sp . was developed. A pair of plasmids, an integrated expression plasmid pUB'-Ex1 and a thermosensitive replicable plasmid pUB-MazF, were constructed. pUB'-Ex1 conditionally self-replicated in Bacillus sp . when the RepF in pUB-MazF expressed. pUB-MazF thermosensitively self-replicated in Bacillus sp . , which was easily cured from the host by inducing MazF expression with IPTG. Bacillus licheniformis BL-UBM that integrated with pUB-MazF was then transformed with pUB'-amyS derived from pUB'-Ex1 by in-frame cloning of amyS encoding a thermophilic α-amylase from Geobacillus stearothermophilus ATCC 31195. The transformant of B. licheniformis BL-UBM with pUB'-amyS was cultivated at 42 o C with the existence of 1 mmol/l IPTG and 500 μg/ml kanamycin and the recombinants with high α-amylase activities were selected. All tested recombinants were extremely high genetic stability. One of which, recombinant BLiS-002, carried five copies of amyS and produced the highest yield of α-amylase. It could yield 50,753 U/ml of α-amylase in a 50-l bioreactor. The strategy developed in this study is of application potential for convenient and quick strain development for industrially important enzyme overexpression.

scholarly journals Methane monooxygenases: central enzymes in methanotrophy with promising biotechnological applications

2021 ◽  
Vol 37 (4) ◽  
Author(s):  
May L. K. Khider ◽  
Trygve Brautaset ◽  
Marta Irla
Keyword(s):  
Animal Feed ◽  
Bacterial Species ◽  
Carbon Sources ◽  
Value Added ◽  
Anthropogenic Pollution ◽  
Functional Expression ◽  
Ongoing Research ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Food Market

AbstractWorldwide, the use of methane is limited to generating power, electricity, heating, and for production of chemicals. We believe this valuable gas can be employed more widely. Here we review the possibility of using methane as a feedstock for biotechnological processes based on the application of synthetic methanotrophs. Methane monooxygenase (MMO) enables aerobic methanotrophs to utilize methane as a sole carbon and energy source, in contrast to industrial microorganisms that grow on carbon sources, such as sugar cane, which directly compete with the food market. However, naturally occurring methanotrophs have proven to be difficult to manipulate genetically and their current industrial use is limited to generating animal feed biomass. Shifting the focus from genetic engineering of methanotrophs, towards introducing metabolic pathways for methane utilization in familiar industrial microorganisms, may lead to construction of efficient and economically feasible microbial cell factories. The applications of a technology for MMO production are not limited to methane-based industrial synthesis of fuels and value-added products, but are also of interest in bioremediation where mitigating anthropogenic pollution is an increasingly relevant issue. Published research on successful functional expression of MMO does not exist, but several attempts provide promising future perspectives and a few recent patents indicate that there is an ongoing research in this field. Combining the knowledge on genetics and metabolism of methanotrophy with tools for functional heterologous expression of MMO-encoding genes in non-methanotrophic bacterial species, is a key step for construction of synthetic methanotrophs that holds a great biotechnological potential.

scholarly journals Towards a Synthetic Biology Toolset for Metallocluster Enzymes in Biosynthetic Pathways: What We Know and What We Need

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6930
Author(s):  
Helena Shomar ◽  
Gregory Bokinsky
Keyword(s):  
Synthetic Biology ◽  
Protein Interactions ◽  
Enzyme Stability ◽  
Functional Expression ◽  
Specific Protein ◽  
Live Cells ◽  
Proper Function ◽  
Biosynthetic Pathways ◽  
Renewable Materials ◽  
Cell Factories

Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein–protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.

scholarly journals Animal Fat as a Substrate for Production of n-6 Fatty Acids by Fungal Solid-State Fermentation

2021 ◽  
Vol 9 (1) ◽  
pp. 170
Author(s):  
Ondrej Slaný ◽  
Tatiana Klempová ◽  
Volha Shapaval ◽  
Boris Zimmermann ◽  
Achim Kohler ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Solid State ◽  
Solid State Fermentation ◽  
Carotene Content ◽  
Animal Fat ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Wide Range ◽  
By Products ◽  
Β Carotene

The method of solid-state fermentation (SSF) represents a powerful technology for the fortification of animal-based by-products. Oleaginous Zygomycetes fungi are efficient microbial cell factories used in SSF to valorize a wide range of waste and rest cereal materials. The application of this fermentation technique for utilization and biotransformation of animal-based materials represents a distinguished step in their treatment. In this study, for the first time, the strain Umbelopsis isabellina CCF2412 was used for the bioconversion of animal fat by-products to the fermented bioproducts enriched with n-6 polyunsaturated fatty acids, mainly γ-linolenic acid (GLA). Bioconversion of both cereals and the animal fat by-product resulted in the production of fermented bioproducts enriched with not just GLA (maximal yield was 6.4 mg GLA/g of fermented bioproduct), but also with high yields of glucosamine. Moreover, the fermentation on the cornmeal matrix led to obtaining bioproduct enriched with β-carotene. An increased amount of β-carotene content improved the antioxidant stability of obtained fermented bioproducts. Furthermore, the application of Fourier-transform infrared spectroscopy for rapid analysis and characterization of the biochemical profile of obtained SSF bioproducts was also studied.

scholarly journals Metabolic Engineering of Microbial Cell Factories for Biosynthesis of Flavonoids: A Review

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4522
Author(s):  
Hanghang Lou ◽  
Lifei Hu ◽  
Hongyun Lu ◽  
Tianyu Wei ◽  
Qihe Chen
Keyword(s):  
Biological Activity ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Antibacterial Properties ◽  
Plant Secondary Metabolites ◽  
Biosynthetic Pathways ◽  
Expression Systems ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Anti Cancer

Flavonoids belong to a class of plant secondary metabolites that have a polyphenol structure. Flavonoids show extensive biological activity, such as antioxidative, anti-inflammatory, anti-mutagenic, anti-cancer, and antibacterial properties, so they are widely used in the food, pharmaceutical, and nutraceutical industries. However, traditional sources of flavonoids are no longer sufficient to meet current demands. In recent years, with the clarification of the biosynthetic pathway of flavonoids and the development of synthetic biology, it has become possible to use synthetic metabolic engineering methods with microorganisms as hosts to produce flavonoids. This article mainly reviews the biosynthetic pathways of flavonoids and the development of microbial expression systems for the production of flavonoids in order to provide a useful reference for further research on synthetic metabolic engineering of flavonoids. Meanwhile, the application of co-culture systems in the biosynthesis of flavonoids is emphasized in this review.

scholarly journals The Sensory Significance of Apocarotenoids in Wine: Importance of Carotenoid Cleavage Dioxygenase 1 (CCD1) in the Production of β-Ionone

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2779 ◽  
Author(s):  
John J. B. Timmins ◽  
Heinrich Kroukamp ◽  
Ian T. Paulsen ◽  
Isak S. Pretorius
Keyword(s):  
Large Scale ◽  
Consumer Preferences ◽  
Carotenoid Cleavage Dioxygenase ◽  
Making Sense ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Reduced Costs ◽  
Key Drivers ◽  
Food And Drink ◽  
Β Carotene

Olfactory cues are key drivers of our multisensory experiences of food and drink. For example, our perception and enjoyment of the flavour and taste of a wine is primarily steered by its aroma. Making sense of the underlying smells that drive consumer preferences is integral to product innovation as a vital source of competitive advantage in the marketplace, which explains the intense interest in the olfactory component of flavour and the sensory significance of individual compounds, such as one of the most important apocarotenoids for the bouquet of wine, β-ionone (violet and woody notes). β-Ionone is formed directly from β-carotene as a by-product of the actions of carotenoid cleavage dioxygenases (CCDs). The biological production of CCDs in microbial cell factories is one way that important aroma compounds can be generated on a large scale and with reduced costs, while retaining the ‘natural’ moniker. The CCD family includes the CCD1, CCD2, CCD4, CCD7 and CCD8; however, the functions, co-dependency and interactions of these CCDs remain to be fully elucidated. Here, we review the classification, actions and biotechnology of CCDs, particularly CCD1 and its action on β-carotene to produce the aromatic apocarotenoid β-ionone.

scholarly journals Enzymatic Hydrolysate of Cinnamon Waste Material as Feedstock for the Microbial Production of Carotenoids

2021 ◽  
Vol 18 (3) ◽  
pp. 1146
Author(s):  
Stefano Bertacchi ◽  
Stefania Pagliari ◽  
Chiara Cantù ◽  
Ilaria Bruni ◽  
Massimo Labra ◽  
...  
Keyword(s):  
Industrial Sector ◽  
Waste Material ◽  
Fungal Cell ◽  
Enzymatic Hydrolysate ◽  
Carbon And Nitrogen ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Separate Hydrolysis And Fermentation ◽  
Additional Processing ◽  
Microbial Cultivation

In the context of the global need to move towards circular economies, microbial cell factories can be employed thanks to their ability to use side-stream biomasses from the agro-industrial sector to obtain additional products. The valorization of residues allows for better and complete use of natural resources and, at the same time, for the avoidance of waste management to address our needs. In this work, we focused our attention on the microbial valorization of cinnamon waste material after polyphenol extraction (C-PEW) (Cinnamomum verum J.Presl), generally discarded without any additional processing. The sugars embedded in C-PEW were released by enzymatic hydrolysis, more compatible than acid hydrolysis with the subsequent microbial cultivation. We demonstrated that the yeast Rhodosporidium toruloides was able to grow and produce up to 2.00 (±0.23) mg/L of carotenoids in the resulting hydrolysate as a sole carbon and nitrogen source despite the presence of antimicrobial compounds typical of cinnamon. To further extend the potential of our finding, we tested other fungal cell factories for growth on the same media. Overall, these results are opening the possibility to develop separate hydrolysis and fermentation (SHF) bioprocesses based on C-PEW and microbial biotransformation to obtain high-value molecules.

scholarly journals Protein-Engineered Polymers Functionalized with Antimicrobial Peptides for the Development of Active Surfaces

2021 ◽  
Vol 11 (12) ◽  
pp. 5352
Author(s):  
Ana Margarida Pereira ◽  
Diana Gomes ◽  
André da Costa ◽  
Simoni Campos Dias ◽  
Margarida Casal ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Recombinant Dna ◽  
Biological Properties ◽  
Resistant Bacteria ◽  
Recombinant Dna Technology ◽  
Free Standing ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Antimicrobial Materials

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.

Developing fourth-generation biofuels secreting microbial cell factories for enhanced productivity and efficient product recovery; a review

Fuel ◽  
2021 ◽  
Vol 298 ◽  
pp. 120858
Author(s):  
Sana Malik ◽  
Ayesha Shahid ◽  
Chen-Guang Liu ◽  
Aqib Zafar Khan ◽  
Muhammad Zohaib Nawaz ◽  
...  
Keyword(s):  
Product Recovery ◽  
Microbial Cell ◽  
Fourth Generation ◽  
Microbial Cell Factories ◽  
Cell Factories

scholarly journals Complete biosynthesis of a sulfated chondroitin in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Abinaya Badri ◽  
Asher Williams ◽  
Adeola Awofiranye ◽  
Payel Datta ◽  
Ke Xia ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Scale Up ◽  
Production Methods ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Microbial Product ◽  
Dry Cell Weight ◽  
One Step ◽  
Dry Cell

AbstractSulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

Sign in / Sign up

Export Citation Format

Share Document