Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Production of Astaxanthin Using CBFD1/HFBD1 from Adonis aestivalis and the Isopentenol Utilization Pathway in Escherichia coli.

Astaxanthin is a powerful antioxidant and is used extensively as an animal feed additive and nutraceutical product. Here, we report the use of the ß-carotene hydroxylase (CBFD1) and the ß-carotene ketolase (HBFD1) from Adonis aestivalis, a flowering plant, to produce astaxanthin in E. coli equipped with the P. agglomerans ß-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%).

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

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.

Fast media optimization for mixotrophic cultivation of Chlorella vulgaris.

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.

Cell free biosynthesis of isoprenoids from isopentenol.

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.

Metabolic engineering of Escherichia coli for the production of isoprenoids.

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.