Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds

The yeast Saccharomyces cerevisiae has been modified successfully for production of numerous metabolites and therapeutic proteins through metabolic engineering, but has not been utilized to date for the production of lipid-derived compounds. We developed a lipid metabolic engineering strategy in S. cerevisiae based upon culturing techniques that are typically employed for studies of peroxisomal biogenesis; cells were grown in media containing fatty acids as a sole carbon source, which promotes peroxisomal proliferation and induction of enzymes associated with fatty acid beta-oxidation. Our results indicate that growth of yeast on fatty acids such as oleate results in extensive uptake of these fatty acids from the media and a subsequent increase in total cellular lipid content from 2% to 15% dry cell weight. We also show that co-expression of plant fatty acid desaturases 2 and 3 ( FAD2 and FAD3), using a fatty acid-inducible peroxisomal gene promoter, coupled the processes of fatty acid uptake with the induction of a new metabolic pathway leading from oleic acid (18:1) to linolenic acid (18:3). Finally, we show that cultivation of yeast cells in the presence of triacylglycerols and exogenously supplied lipase promotes extensive incorporation of triglyceride fatty acids into yeast cells. Collectively, these results provide a framework for bioconversion of low-cost oils into value-added lipid products.     

Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides

Ginsenosides are the primary bioactive components of ginseng, which is a popular medicinal herb and exhibits diverse pharmacological activities. Protopanaxadiol is the aglycon of several dammarane-type ginsenosides, which also has anticancer activity. For microbial production of protopanaxadiol, dammarenediol-II synthase and protopanaxadiol synthase genes of Panax ginseng, together with a NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana, were introduced into Saccharomyces cerevisiae, resulting in production of 0.05 mg/g DCW protopanaxadiol. Increasing squalene and 2,3-oxidosqualene supplies through overexpressing truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, farnesyl diphosphate synthase, squalene synthase and 2,3-oxidosqualene synthase genes, together with increasing protopanaxadiol synthase activity through codon optimization, led to 262-fold increase of protopanaxadiol production. Finally, using two-phase extractive fermentation resulted in production of 8.40 mg/g DCW protopanaxadiol (1189 mg/L), together with 10.94 mg/g DCW dammarenediol-II (1548 mg/L). The yeast strains engineered in this work can serve as the basis for creating an alternative way for production of ginsenosides in place of extraction from plant sources.     

Metabolic engineering of Saccharomyces cerevisiae for itaconic acid production

Renewable alternatives for petroleum-derived chemicals are achievable through biosynthetic production. Here, we utilize Saccharomyces cerevisiae to enable the synthesis of itaconic acid, a molecule with diverse applications as a petrochemical replacement. We first optimize pathway expression within S. cerevisiae through the use of a hybrid promoter. Next, we utilize sequential, in silico computational genome-scanning to identify beneficial genetic perturbations that are metabolically distant from the itaconic acid synthesis pathway. In this manner, we successfully identify three non-obvious genetic targets (?ade3 ?bna2 ?tes1) that successively improve itaconic acid titer. We establish that focused manipulations of upstream pathway enzymes (localized refactoring) and enzyme re-localization to both mitochondria and cytosol fail to improve itaconic acid titers. Finally, we establish a higher cell density fermentation that ultimately achieves itaconic acid titer of 168 mg/L, a sevenfold improvement over initial conditions. This work represents an attempt to increase itaconic acid production in yeast and demonstrates the successful utilization of computationally guided genetic manipulation to increase metabolic capacity.     

Metabolic engineering of Saccharomyces cerevisiae for 2,3-butanediol production

Applied Microbiology and Biotechnology - Saccharomyces cerevisiae is a work horse for production of valuable biofuels and biochemicals including 2,3-butanediol (2,3-BDO), a platform chemical with...     

Metabolic engineering of glycerol production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1delta nde1delta nde2delta gut2delta quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h(-1) at 100 g of glucose. liter(-1)). In aerated batch cultures grown on 400 g of glucose. liter(-1), this engineered S. cerevisiae strain produced over 200 g of glycerol. liter(-1), corresponding to a molar yield of glycerol on glucose close to unity.     

Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers

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Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

Background

The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved.

Results

Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the K_M for NADPH in the mutated xylose reductase (K341 → R N343 → D), while K_M for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)^-1 as compared to 4.2 mg × (L × h)^-1 for the wild type strain.

Conclusion

Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.     

Metabolic engineering of muconic acid production in Saccharomyces cerevisiae

The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141 mg/L muconic acid in a shake-flask culture, a value nearly 24-fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts.     

The temporal program of chromosome replication: genomewide replication in clb5{Delta} Saccharomyces cerevisiae

Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Delta strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins ("late" origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.     

Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production

Fatty alcohols are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We have engineered Saccharomyces cerevisiae to produce 1-hexadecanol by expressing a fatty acyl-CoA reductase (FAR) from barn owl (Tyto alba). In order to improve fatty alcohol production, we have manipulated both the structural genes and the regulatory genes in yeast lipid metabolism. The acetyl-CoA carboxylase gene (ACC1) was over-expressed, which improved 1-hexadecanol production by 56% (from 45 mg/L to 71 mg/L). Knocking out the negative regulator of the INO1 gene in phospholipid metabolism, RPD3, further enhanced 1-hexadecanol production by 98% (from 71 mg/L to 140 mg/L). The cytosolic acetyl-CoA supply was next engineered by expressing a heterologous ATP-dependent citrate lyase, which increased the production of 1-hexadecanol by an additional 136% (from 140 mg/L to 330 mg/L). Through fed-batch fermentation using resting cells, over 1.1 g/L 1-hexadecanol can be produced in glucose minimal medium, which represents the highest titer reported in yeast to date.     

Metabolic engineering of Saccharomyces cerevisiae for the production of triacetic acid lactone

Biobased chemicals have become attractive replacements for their fossil-fuel counterparts. Recent studies have shown triacetic acid lactone (TAL) to be a promising candidate, capable of undergoing chemical conversion to sorbic acid and other valuable intermediates. In this study, Saccharomyces cerevisiae was engineered for the high-level production of TAL by overexpression of the Gerbera hybrida 2-pyrone synthase (2-PS) and systematic engineering of the yeast metabolic pathways. Pathway analysis and a computational approach were employed to target increases in cofactor and precursor pools to improve TAL synthesis. The pathways engineered include those for energy storage and generation, pentose biosynthesis, gluconeogenesis, lipid biosynthesis and regulation, cofactor transport, and fermentative capacity. Seventeen genes were selected for disruption and independently screened for their effect on TAL production; combinations of knockouts were then evaluated. A combination of the pathway engineering and optimal culture parameters led to a 37-fold increase in titer to 2.2 g/L and a 50-fold increase in yield to 0.13 (g/g glucose). These values are the highest reported in the literature, and provide a 3-fold improvement in yield over previous reports using S. cerevisiae. Identification of these metabolic bottlenecks provides a strategy for overproduction of other acetyl-CoA-dependent products in yeast.     

Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid

The production of bio-based succinic acid is receiving great attention, and several predominantly prokaryotic organisms have been evaluated for this purpose. In this study we report on the suitability of the highly acid- and osmotolerant yeast Saccharomyces cerevisiae as a succinic acid production host. We implemented a metabolic engineering strategy for the oxidative production of succinic acid in yeast by deletion of the genes SDH1, SDH2, IDH1 and IDP1. The engineered strains harbor a TCA cycle that is completely interrupted after the intermediates isocitrate and succinate. The strains show no serious growth constraints on glucose. In glucose-grown shake flask cultures, the quadruple deletion strain Δsdh1Δsdh2Δidh1Δidp1 produces succinic acid at a titer of 3.62 g L^?1 (factor 4.8 compared to wild-type) at a yield of 0.11 mol (mol glucose)^?1. Succinic acid is not accumulated intracellularly. This makes the yeast S. cerevisiae a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale.     

Metabolic engineering of Saccharomyces cerevisiae for hydroxytyrosol overproduction directly from glucose

Hydroxytyrosol (HT) is one of the most powerful dietary antioxidants with numerous applications in different areas, including cosmetics, nutraceuticals and food. In the present work, heterologous hydroxylase complex HpaBC from Escherichia coli was integrated into the Saccharomyces cerevisiae genome in multiple copies. HT productivity was increased by redirecting the metabolic flux towards tyrosol synthesis to avoid exogenous tyrosol or tyrosine supplementation. After evaluating the potential of our selected strain as an HT producer from glucose, we adjusted the medium composition for HT production. The combination of the selected modifications in our engineered strain, combined with culture conditions optimization, resulted in a titre of approximately 375 mg l⁻¹ of HT obtained from shake-flask fermentation using a minimal synthetic-defined medium with 160 g l⁻¹ glucose as the sole carbon source. To the best of our knowledge, this is the highest HT concentration produced by an engineered S. cerevisiae strain.     

Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol(-1), mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.     

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid–derived hydrocarbons

Fatty acid–derived hydrocarbons attract increasing attention as biofuels due to their immiscibility with water, high‐energy content, low freezing point, and high compatibility with existing refineries and end‐user infrastructures. Yeast Saccharomyces cerevisiae has advantages for production of fatty acid–derived hydrocarbons as its native routes toward fatty acid synthesis involve only a few reactions that allow more efficient conversion of carbon substrates. Here we describe major biosynthetic pathways of fatty acid–derived hydrocarbons in yeast, and summarize key metabolic engineering strategies, including enhancing precursor supply, eliminating competing pathways, and expressing heterologous pathways. With recent advances in yeast production of fatty acid–derived hydrocarbons, our review identifies key research challenges and opportunities for future optimization, and concludes with perspectives and outlooks for further research directions.     

Metabolic engineering of Saccharomyces cerevisiae producing nicotianamine: potential for industrial biosynthesis of a novel antihypertensive substrate

Nicotianamine (NA), a metal chelator, is ubiquitous in higher plants. In humans, NA inhibits angiotensin I-converting enzyme (ACE), and consequently reduces high blood pressure. Nicotianamine is synthesized from the trimerization of S-adenosylmethionine (SAM) by NA synthase (NAS). Here, we aimed to produce large amounts of NA fermentatively by introducing the Arabidopsis AtNAS2 gene into Saccharomyces cerevisiae strain SCY4. This strain can accumulate up to 100 times the usual amount of SAM, and this is considered desirable for overproduction of NA. Nicotianamine was produced in the engineered yeast, and the NA level increased with incubation time until the stationary phase. The maximum concentration of intracellular NA obtained was 766+/-33 microg/g wet weight. Successful production of NA in S. cerevisiae should pave the way for industrial production of this novel antihypertensive substrate.