Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered <i>Saccharomyces cerevisiae</i>

Biomass, the most abundant carbon source on the planet, may in the future become the primary feedstock for production of fuels and chemicals, replacing fossil feedstocks. This will, however, require development of cell factories that can convert both C6 and C5 sugars present in lignocellulosic biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP), a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or β-alanine intermediates into a xylose-consuming yeast. Using controlled fed-batch cultivation, we obtained 7.37±0.17g 3HPL-1 in 120hours with an overall yield of 29±1%Cmol 3HPCmol-1 xylose. This study is the first demonstration of the potential of using S. cerevisiae for production of 3HP from the biomass sugar xylose

Engineering of Yarrowia lipolytica for production of astaxanthin

Astaxanthin is a red-colored carotenoid, used as food and feed additive. Astaxanthin is mainly produced by chemical synthesis, however, the process is expensive and synthetic astaxanthin is not approved for human consumption. In this study, we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation. First, we screened 12 different Y. lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis: bi-functional phytoene synthase/lycopene cyclase (crtYB) and phytoene desaturase (crtI) from the red yeast Xanthophyllomyces dendrorhous. The best strain produced 31.1 ± 0.5 mg/L β-carotene. Next, we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG1) and geranylgeranyl diphosphate synthase (GGS1/crtE) in the best producing strain and obtained 453.9 ± 20.2 mg/L β-carotene. Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1 ± 57.2 mg/L. Then we introduced β-carotene ketolase (crtW) from Paracoccus sp. N81106 and hydroxylase (crtZ) from Pantoea ananatis to convert β-carotene into astaxanthin. The constructed strain accumulated 10.4 ± 0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates, 5.7 ± 0.5 mg/L canthaxanthin, and 35.3 ± 1.8 mg/L echinenone. Finally, we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW (54.6 mg/L) of astaxanthin in a microtiter plate cultivation. Our study for the first time reports engineering of Y. lipolytica for the production of astaxanthin. The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y. lipolytica-based fermentation process for astaxanthin production

De novo production of resveratrol from glucose or ethanol by engineered <i>Saccharomyces cerevisiae</i>

Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.73±0.05mgL-1 resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85±0.31mgL-1 resveratrol from glucose as the sole carbon source. Next we improved the supply of the precursor malonyl-CoA by over-expressing a post-translational de-regulated version of the acetyl-CoA carboxylase encoding gene ACC1; this strategy further increased resveratrol production to 6.39±0.03mgL-1. Subsequently, we improved the strain by performing multiple-integration of pathway genes resulting in resveratrol production of 235.57±7.00mgL-1. Finally, fed-batch fermentation of the final strain with glucose or ethanol as carbon source resulted in a resveratrol titer of 415.65 and 531.41mgL-1, respectively

Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance

Biologically produced 3-hydroxypropionic acid (3HP) is a potential source for sustainable acrylates and can also find direct use as monomer in the production of biodegradable polymers. For industrial scale production there is a need for robust cell factories tolerant to high concentration of 3HP, preferably at low pH. Through adaptive laboratory evolution we selected S. cerevisiae strains with improved tolerance to 3HP at pH 3.5. Genome sequencing followed by functional analysis identified the causal mutation in SFA1 gene encoding S-(hyclroxymerhyl)glutathione dehydrogenase. Based on our findings, we propose that 3HP toxicity is mediated by 3-hydroxypropionic aldehyde (reuterin ) and that glutathione-dependent reactions are used for reuterin detoxification. The identified molecular response to 3HP and reuterin may well be a general mechanism for handling resistance to organic acid and aldehydes by living cells. (C) 2014 International Metabolic Engineering Society Published by Elsevier Inc. On behalf of International Metabolic Engineering Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/