agrobacterium-tumefaciens-mediated-genetic-engineering-green

<em>Agrobacterium tumefaciens</em>-Mediated Genetic Engineering of Green Microalgae, <em>Chlorella vulgaris</em>

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

University of Waterloo

Metabolic engineering is the practice of using directed genetic manipulations to rewire cellular metabolism primarily with the aim to transform the organism into a single-celled chemical factory. Using biological processes, we can produce more complex chemicals in a more sustainable way. This is particularly important for chemicals which are hard to synthesize using traditional chemistry. However, cells have evolved for growth and must be engineered to produce a single chemical at commercially viable levels. This review focuses on the strategies used to rewire cellular metabolism to produce chemicals using isoprenoid production in Escherichia coli as an example that illustrates many of the challenges faced in metabolic engineering.

Metabolic engineering of Escherichia coli for the production of isoprenoids.

Polyunsaturated fatty acids (PUFAs) play a significant role in the modulation and prevention of various diseases, and hence are attracting increasing attention from the biotech industry. Thraustochytrids are marine heterokonts that exhibit robust growth rates, high PUFA content, and more specifically, a large percentage of omega-3 fatty acids like docosahexaenoic acid (DHA). Recently, ionic liquids (ILs) have been shown to improve the efficiency of organic solvent extraction of oils from wet oleaginous yeast and microalgae under mild conditions. Two ILs, the imidazolium 1-ethyl-3-methylimidazolium ethylsulfate [C₂mim][EtSO₄] IL and the phosphonium (tetrabutylphosphonium propanoate [P][Prop]) IL were assessed for their ability to facilitate extraction of PUFA-containing lipids from a  sp. (T18) through efficient cell wall disruption. The oil extracted after IL pretreatment was further characterized with respect to fatty acid methyl ester (FAME) composition, while the effects of process parameters, such as the ratio of ionic liquid to co-solvent, the mass ratio of microalgae to the mixture of ionic liquid, and type of co-solvent were also investigated for both ILs. The results indicate that these ILs can disrupt the cells of  sp. when mixed with a co-solvent (methanol), and facilitated the recovery of oils over a large degree of dewatered  biomass (0⁻77.2 wt% water) in a short period of time (60 min) at ambient temperature, hence demonstrating a water compatible, low-energy, lipid recovery method. The lipid recovery was not affected by repeated usage of recycled ILs (tested up to five times).

Efficient Extraction of a Docosahexaenoic Acid (DHA)-Rich Lipid Fraction from  sp. Using Ionic Liquids.

Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-d-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far.

Cell free biosynthesis of isoprenoids from isopentenol.

Microalgae can accumulate large proportions of their dry cell weight as storage lipids when grown under appropriate nutrient limiting conditions. While a high ratio of carbon to nitrogen is often cited as the primary mode of triggering lipid accumulation in microalgae, fast optimization strategies to increase lipid production for mixotrophic cultivation have been difficult to developed due to the low cell densities of algal cultures, and consequently the limited amount of biomass available for compositional analysis. Response surface methodologies provide a power tool for assessing complex relationships such as the interaction between the carbon source and nitrogen source. A 15 run Box-Behnken design performed in shaker flasks was effective in studying the effect of carbon, nitrogen, and magnesium on the growth rate, maximum cell density, lipid accumulation rate, and glucose consumption rate. Using end-point dry cell weight and total lipid content as assessed by direct transesterification to FAME, numerical optimization resulted in a significant increase in lipid content from 18.5 ± 0.76% to 37.6 ± 0.12% and a cell density of 5.3 ± 0.1 g/L to 6.1 ± 0.1 g/L between the centre point of the design and the optimized culture conditions. The presented optimization process required less than 2 weeks to complete, was simple, and resulted in an overall lipid productivity of 383 mg/L·d.

Fast media optimization for mixotrophic cultivation of Chlorella vulgaris.

Production of hydrophobic or lipophilic compounds via biosynthesis in microbes is fraught with challenges. Since they cannot transverse the cell membrane and exit the cell, they can accumulate in intracellular hydrophobic microenvironments like the internal hydrophobic layer of the cell membrane. As specific yield of these products increases, cytotoxic effects become apparent causing production of the biomolecule to cease or even cell death. Strategies that have evolved in recent years to engineer greater storage capacity in microbes producing hydrophobic compounds will be discussed.

Strategies for production of hydrophobic compounds.

The development of cascade cell-free systems reduces the requirement for extensive metabolic engineering and optimization to increase in vivo pathway flux. For continuous operation and increased stability, direct enzyme entrapment during reactor fabrication by three-dimensional (3D)-printing allows for simple immobilization procedures without enzyme-specific optimization. In this study, the isopentenol utilization pathway (IUP) was selected for the synthesis of amorphadiene, an antimalaria drug precursor, using a 3D-printed, sequentially immobilized, microfluidic reactor. As an initial proof-of-concept, alkaline phosphatase (ALP) was entrapped in a poly(methyl methacrylate) (PMMA)-based matrix during stereolithographic 3D-printing and was kinetically characterized. No significant shift of the kinetically modeled substrate binding affinity was observed during immobilization and continuous operation of an entrapped ALP microfluidic reactor displayed high stability. The IUP enzymes retained moderate activity during entrapment (6.6%-9.6%) relative to the free enzyme solutions, however the sequentially immobilized IUP microfluidic reactor was severely limited by low pathway flux due to the use of stereolithographic 3D-printing which significantly diluted enzyme concentrations for printing. Although this study demonstrated the use of additive manufacturing for the synthesis of amorphadiene using a complex five-enzyme cascade microfluidic reactor, stereolithographic enzyme entrapment remains limited in scope and dependent on advancements to additive manufacturing technologies.

Kinetic characterization and modeling of sequentially entrapped enzymes in 3D-printed PMMA microfluidic reactors for the synthesis of amorphadiene via the isopentenol utilization pathway.

Editorial overview: Chemical Biotechnology.

Microalgae is emerging as a promising platform for the production of recombinant proteins of therapeutic importance. Since microalgae are safe for oral consumption and some microalgal species can form extracellular vesicles, they can potentially be used as edible vaccines or drug-delivery vehicles in addition to their status as naturally rich sources of nutraceutical bioproducts. This article focuses on recent advances in the field of recombinant protein production in microalgae, and developments toward the use of microalgae as a vehicle for drug delivery.

Production of recombinant and therapeutic proteins in microalgae.

Astaxanthin is a powerful antioxidant and is used extensively as an animal feed additive and nutraceutical product. Here, we report the use of the β-carotene hydroxylase (CBFD1) and the β-carotene ketolase (HBFD1) from , a flowering plant, to produce astaxanthin in  equipped with the  β-carotene pathway and an over-expressed 4-methylerythritol-phosphate (MEP) pathway or the isopentenol utilization pathway (IUP). Introduction of the over-expressed MEP pathway and the IUP resulted in a 3.2-fold higher carotenoid content in LB media at 36 h post-induction compared to the strain containing only the endogenous MEP. However, in M9 minimal media, the IUP pathway dramatically outperformed the over-expressed MEP pathway with an 11-fold increase in total carotenoids produced. The final construct split the large operon into two smaller operons, both with a T7 promoter. This resulted in slightly lower productivity (70.0 ± 8.1 µg/g·h vs. 53.5 ± 3.8 µg/g·h) compared to the original constructs but resulted in the highest proportion of astaxanthin in the extracted carotenoids (73.5 ± 0.2%).

Article	Year
Metabolic engineering of Escherichia coli for the production of isoprenoids. FEMS microbiology letters\| PubMed ID: 29718190	2018
Efficient Extraction of a Docosahexaenoic Acid (DHA)-Rich Lipid Fraction from sp. Using Ionic Liquids. Materials (Basel, Switzerland)\| PubMed ID: 30326602	2018
Cell free biosynthesis of isoprenoids from isopentenol. Biotechnology and bioengineering\| PubMed ID: 31429926	2019
Fast media optimization for mixotrophic cultivation of Chlorella vulgaris. Scientific reports\| PubMed ID: 31848403	2019
Strategies for production of hydrophobic compounds. Current opinion in biotechnology\| PubMed ID: 35074666	2022
Kinetic characterization and modeling of sequentially entrapped enzymes in 3D-printed PMMA microfluidic reactors for the synthesis of amorphadiene via the isopentenol utilization pathway. Biotechnology and bioengineering\| PubMed ID: 35099806	2022
Editorial overview: Chemical Biotechnology. Current opinion in biotechnology\| PubMed ID: 35570175	2022
Production of recombinant and therapeutic proteins in microalgae. Current opinion in biotechnology\| PubMed ID: 36095993	2022
Production of Astaxanthin Using CBFD1/HFBD1 from and the Isopentenol Utilization Pathway in . Bioengineering (Basel, Switzerland)\| PubMed ID: 37760135	2023

Valerie C. A. Ward

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