Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine

Abstract

Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l−1 with a yield of 67 mg (g glucose)−1 in shake-flask cultures and a titre of 5.1 g l−1 in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals.

The sole introduction of two single-point mutations establishes glycerol utilization in Saccharomyces cerevisiae CEN.PK derivatives

“…In particular, strains of the CEN.PK family have become a popular platform in both fundamental research and industrial applications as they offer a good compromise between physiological properties (e.g., the growth characteristics in shake-flask cultures) and genetic properties (e.g., transformation efficiency) [7]. CEN.PK strains have been applied in numerous metabolic and evolutionary engineering studies, such as for the production of lactate and pyruvate [8, 9], isoprenoids [10, 11], C 4 -dicarboxylic acids [12], ornithine [13], n-butanol [14], and 3-hydroxypropionic acid [15], and for the fermentation of pentose sugars [16–18]. Although some of these processes could profit from utilizing glycerol as a carbon source due to its high reducing power, wild-type strains of the CEN.PK family cannot utilize glycerol at all in synthetic medium, particularly when complex medium supplements such as yeast extract or peptone are omitted [19, 20].…”

Technology development for natural product biosynthesis in Saccharomyces cerevisiae

“…Qin and coworkers carried out a comprehensive, modular engineering of yeast metabolism for high-level production of the amino acid intermediate l -ornithine. Pathway rewiring of the urea cycle and TCA cycle in combination with other metabolic engineering strategies enabled a 23-fold improvement in l -ornithine titers [47]. …”

“…implemented pathway re-localization as a part of their modular pathway rewiring strategy for production of the amino acid intermediate l -ornithine. When a portion of the l -ornithine biosynthetic pathway was localized to the mitochondria, titers dropped significantly, whereas re-localization of the complete pathway to the cytosol resulted in increased l -ornithine production [47]. …”

A Review Article: Electrokinetic Bioremediation Current Knowledge and New Prospects

“…QS is well studied for bacterial pathogens as QS regulate bacterial genes and function at the community level so that bacteria collectively act or infect host together, so QS coordinates infection. In addition, bacterial QS can be interfered by chemicals produced by host organisms or already available in nature [21] [22]. In nature, QS may help bacteria for better and enhanced bioremediation.…”

“…1) Exploring the indigenous bacteria metabolic pathways and using genetic engineering to change or rewire bacterial metabolic pathways to strengthen microorganisms' ability for bioremediation [21] [22]. Also, modifying bacterial genes and regulatory networks to make them tougher to survive and tolerate high contaminant concentration in soils [23].…”