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

Abstract

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.ResultsWe 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0667-z) contains supplementary material, which is available to authorized users.

Nitrogen metabolism in Pseudomonas putida: functional analysis using random barcode transposon sequencing

“…) is an unnatural amino acid that is used in the production of pharmaceuticals, and has been a target molecule for biological production in E. coli (136,137).…”

Biocatalysis in the Swiss Manufacturing Environment

“…Similarly targeting the production of bio-based fine chemicals, the company Evolva SA developed yeast as a whole cell biocatalyst for the production of (S)-2-aminobutyric acid and the hitherto purely synthetic compound (S)-2-aminobutanol. The molecules are the enantiomeric precursors for ethambutol, bivaracetam, and levetiraceram, which are anti-tuberculosis and anti-epilepsy drugs, respectively [32]. By designing a two-step heterologous pathway consisting of a Bacillus subtilis threonine deaminase and a mutated Escherichia coli glutamate dehydrogenase, the industrial scientists could produce enantiopure (S)-2-aminobutyric acid at levels up to 1.7 mg/L.…”

Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids

“…•h −1 (Tao et al, 2014). This MEC module has also been used to generate a heterologous biosynthetic pathway leading to the production of L-ABA in Saccharomyces cerevisiae (Weber et al, 2017). Metabolic engineering allowed the expansion of the latter system for the production of S-2-aminobutanol.…”

“…A scale-up of the process (30 L of reaction in a 50-L fermenter) allowed the production of 29.2 mol L -ABA (97.3% theoretical yield), with a productivity of 6.9 g⋅L –1 ⋅h –1 ( Tao et al, 2014 ). This MEC module has also been used to generate a heterologous biosynthetic pathway leading to the production of L -ABA in Saccharomyces cerevisiae ( Weber et al, 2017 ). Metabolic engineering allowed the expansion of the latter system for the production of S -2-aminobutanol.…”