Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae (baker's yeast) has great potential as a whole-cell biocatalyst for multistep synthesis of various organic molecules. To date, however, few examples exist in the literature of the successful biosynthetic production of chemical compounds, in yeast, that do not exist in nature. Considering that more than 30% of all drugs on the market are purely chemical compounds, often produced by harsh synthetic chemistry or with very low yields, novel and environmentally sound production routes are highly desirable. Here, we explore the biosynthetic production of enantiomeric precursors of the anti-tuberculosis and anti-epilepsy drugs ethambutol, brivaracetam, and levetiracetam. To this end, we have generated heterologous biosynthetic pathways leading to the production of (S)-2-aminobutyric acid (ABA) and (S)-2-aminobutanol in baker's yeast. RESULTS: We first designed a two-step heterologous pathway, starting with the endogenous amino acid L-threonine and leading to the production of enantiopure (S)-2-aminobutyric acid. The combination of Bacillus subtilis threonine deaminase and a mutated Escherichia coli glutamate dehydrogenase resulted in the intracellular accumulation of 0.40 mg/L of (S)-2-aminobutyric acid. The combination of a threonine deaminase from Solanum lycopersicum (tomato) with two copies of mutated glutamate dehydrogenase from E. coli resulted in the accumulation of comparable amounts of (S)-2-aminobutyric acid. Additional L-threonine feeding elevated (S)-2-aminobutyric acid production to more than 1.70 mg/L. Removing feedback inhibition of aspartate kinase HOM3, an enzyme involved in threonine biosynthesis in yeast, elevated (S)-2-aminobutyric acid biosynthesis to above 0.49 mg/L in cultures not receiving additional L-threonine. We ultimately extended the pathway from (S)-2-aminobutyric acid to (S)-2-aminobutanol by introducing two reductases and a phosphopantetheinyl transferase. The engineered strains produced up to 1.10 mg/L (S)-2-aminobutanol. CONCLUSIONS: Our results demonstrate the biosynthesis of (S)-2-aminobutyric acid and (S)-2-aminobutanol in yeast. To our knowledge this is the first time that the purely synthetic compound (S)-2-aminobutanol has been produced in vivo. This work paves the way to greener and more sustainable production of chemical entities hitherto inaccessible to synthetic biology.

Discovery and reconstitution of the cycloclavine biosynthetic pathway--enzymatic formation of a cyclopropyl group.

The ergot alkaloids, a class of fungal-derived natural products with important biological activities, are derived from a common intermediate, chanoclavine-I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast-based expression platform along with in vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine-I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500 mg L(-1) , thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.

Discovery and Reconstitution of the Cycloclavine Biosynthetic Pathway-Enzymatic Formation of a Cyclopropyl Group.

The ergot alkaloids, a class of fungal-derived natural products with important biological activities, are derived from a common intermediate, chanoclavine-I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast-based expression platform along with in vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine-I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500 mg L-1, thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.

The important ergot alkaloid intermediate chanoclavine-I produced in the yeast Saccharomyces cerevisiae by the combined action of EasC and EasE from Aspergillus japonicus.

BACKGROUND: Ergot alkaloids are a group of highly bioactive molecules produced by a number of filamentous fungi. These compounds have been intensely studied for decades, mainly due to their deleterious effects in contaminated food and feeds, but also for their beneficial pharmaceutical and agricultural applications. Biosynthesis of ergot alkaloids goes via the common intermediate chanoclavine-I, and studies of the key enzymes, EasE and EasC, involved in chanoclavine-I formation, have relied on gene complementation in fungi, whereas further characterization has been hampered by difficulties of poor EasE protein expression. In order to facilitate the study of ergot alkaloids, and eventually move towards commercial production, the early steps of the biosynthetic pathway were reconstituted in the unicellular yeast Saccharomyces cerevisiae. RESULTS: The genomic sequence from an ergot alkaloid producer, Aspergillus japonicus, was used to predict the protein encoding sequences of the early ergot alkaloid pathway genes. These were cloned and expressed in yeast, resulting in de novo production of the common intermediate chanoclavine-I. This allowed further characterization of EasE and EasC, and we were able to demonstrate how the N-terminal ER targeting signal of EasE is crucial for activity in yeast. A putative, peroxisomal targeting signal found in EasC was shown to be nonessential. Overexpression of host genes pdi1 or ero1, associated with disulphide bond formation and the ER protein folding machinery, was shown to increase chanoclavine-I production in yeast. This was also the case when overexpressing host fad1, known to be involved in co-factor generation. CONCLUSIONS: A thorough understanding of the enzymatic steps involved in ergot alkaloid formation is essential for commercial production and exploitation of this potent compound class. We show here that EasE and EasC are both necessary and sufficient for the production of chanoclavine-I in yeast, and we provide important new information about the involvement of ER and protein folding for proper functional expression of EasE. Moreover, by reconstructing the chanoclavine-I biosynthetic pathway in yeast we demonstrate the advantage and potential of this host, not only as a convenient model system, but also as an alternative cell factory for ergot alkaloid production.

Yeast synthetic biology platform generates novel chemical structures as scaffolds for drug discovery.

Synthetic biology has been heralded as a new bioengineering platform for the production of bulk and specialty chemicals, drugs, and fuels. Here, we report for the first time a series of 74 novel compounds produced using a combinatorial genetics approach in baker's yeast. Based on the concept of "coevolution" with target proteins in an intracellular primary survival assay, the identified, mostly scaffold-sized (200-350 MW) compounds, which displayed excellent biological activity, can be considered as prevalidated hits. Of the molecules found, >75% have not been described previously; 20% of the compounds exhibit novel scaffolds. Their structural and physicochemical properties comply with established rules of drug- and fragment-likeness and exhibit increased structural complexities compared to synthetically produced fragments. In summary, the synthetic biology approach described here represents a completely new, complementary strategy for hit and early lead identification that can be easily integrated into the existing drug discovery process.