Metabolic engineering probiotic yeast produces 3S, 3'S-astaxanthin to inhibit B16F10 metastasis.

3S, 3'S-Astaxanthin is the most powerful antioxidant to scavenge free radicals in the world. In this study, a 3S, 3'S-astaxanthin biosynthesis pathway was constructed in a probiotic yeast, Kluveromyces marxianus, denoted YEAST, and its bioactive metabolites were extracted for biofunctional assessments. The bio-safety examination was achieved by two animal models as following: First, no significant toxic effects on YEAST groups were found in zebrafish; Second, after feeding YEAST for 4 weeks, the rat-groups showed no visible abnormality, and no significant change of the body weight and blood biochemistry tests. The inhibition of lung metastasis of melanoma cells and the increment of the survival rate were demonstrated by feeding YEAST and injecting the intravenous commercial astaxanthin in vivo rodent model. Based on in vitro assays of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging analysis, ferrous ion chelating ability, reducing power assessment, and mushroom tyrosinase inhibition evaluation, YEAST-astaxanthin showed anti-oxidative and tyrosinase suppressive properties. Taken together, the 3S, 3'S-astaxanthin producing probiotic yeast is safe to be used in the bio-synthesis of functional and pharmaceutical compounds, which have broad industrial applications on cosmetic, food and feed additive and healthcare.

Characterizing an engineered carotenoid-producing yeast as an anti-stress chassis for building cell factories.

BACKGROUND: A microorganism engineered for non-native tasks may suffer stresses it never met before. Therefore, we examined whether a Kluyveromyces marxianus strain engineered with a carotenoid biosynthesis pathway can serve as an anti-stress chassis for building cell factories. RESULTS: Carotenoids, a family of antioxidants, are valuable natural products with high commercial potential. We showed that the free radical removal ability of carotenoids can confer the engineered host with a higher tolerance to ethanol, so that it can produce more bio-ethanol than the wild type. Moreover, we found that this engineered strain has improved tolerance to other toxic effects including furfurals, heavy metals such as arsenate (biomass contaminant) and isobutanol (end product). Furthermore, the enhanced ethanol tolerance of the host can be applied to bioconversion of a natural medicine that needs to use ethanol as the delivery solvent of hydrophobic precursors. The result suggested that the engineered yeast showed enhanced tolerance to ethanol-dissolved hydrophobic 10-deacetylbaccatin III, which is considered a sustainable precursor for paclitaxel (taxol) bioconversion. CONCLUSIONS: The stress tolerances of the engineered yeast strain showed tolerance to several toxins, so it may serve as a chassis for cell factories to produce target products, and the co-production of carotenoids may make the biorefinary more cost-effective.

Constructing a cellulosic yeast host with an efficient cellulase cocktail.

Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host-engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei, and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with eight cellulase gene-cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel.

Metabolic engineering a yeast to produce astaxanthin.

In this study, an astaxanthin-biosynthesis Kluyveromyces marxianus strain Sm23 was first constructed, which could produce 31µg/g DCW astaxanthin. Then, repeated genome integration of the key astaxanthin biosynthesis genes Hpchyb and bkt was done to increase gene copy number and astaxanthin yield. Four improved strains were obtained and the yield of astaxanthin and the total yield of carotenoids in a strain increased with the copy numbers of Hpchyb and bkt. To improve the yield further, the gene Hpchyb from Haematococcus pluvialis was modified by site-directed mutagenesis to increase the enzyme efficiency or/and to prevent the heterologous protein degradation by ubiquitination. Using repeated-integration approach of bkt and the mutated Hpchyb into Sm23, the S3-2 strain was obtained and shown to produce the 3S, 3'S-astaxanthin at 9972µg/g DCW in a 5L fermentor.

Integrating an algal ß-carotene hydroxylase gene into a designed carotenoid-biosynthesis pathway increases carotenoid production in yeast.

The algal ß-carotene hydroxylase gene Crchyb from Chlamydomonas reinhardtii, Czchyb from Chlorella zofingiensis, or Hpchyb from Haematococcus pluvialis and six other carotenoid-synthesis pathway genes were co-integrated into the genome of a yeast host. Each of these three algal genes showed a higher efficiency to convert ß-carotene to downstream carotenoids than the fungal genes from Phaffia rhodozyma. Furthermore, the strain with Hpchyb displayed a higher carotenoid productivity than the strains integrated with Crchyb or Czchyb, indicating that Hpchyb is more efficient than Crchyb and Czchyb. These results suggest that ß-carotene hydroxylase plays a crucial role in the biosynthesis of carotenoids.