Ann Microbiol (2016) 66:1049–1055 DOI 10.1007/s13213-015-1190-2 ORIGINAL ARTICLE L-phenylacetylcarbinol production by yeast petite mutants Mohsen Doostmohammadi1 & Mohammad Ali Asadollahi1 & Iraj Nahvi2 & Davoud Biria1 & Gholam Reza Ghezelbash3 & Maryam Kheyrandish1 Received: 19 September 2015 /Accepted: 28 December 2015 /Published online: 20 January 2016 # Springer-Verlag Berlin Heidelberg and the University of Milan 2016 Abstract The yeast Saccharomyces cerevisiae is able to Introduction biotransform benzaldehyde into L-phenylacetylcarbinol (L-PAC), a key intermediate in the production of ephedrine Ephedrine and pseudoephedrine can be extracted and isolated and pseudoephedrine, by the action of pyruvate decarobxylase from numerous plant species of the genus Ephedra. These (PDC) enzyme. This biotransformation can alternatively be alkaloids exhibit anti-asthmatic and decongestant properties performed by acetohydroxyacid synthase (AHAS) which is (Borchardt 2003). Moreover, their potential application in a mitochondrial enzyme. In the yeast petite mutants, AHAS obesity control has been reported (Astrup et al. 1992). Since accumulates in the cytosol. In the current study, wild-type extraction from plants involves tedious, costly, and time- yeast cells and yeast petite mutants were examined for consuming steps, attempts have been made to find alternative L-PAC biosynthesis. The results showed higher L-PAC titers methods for industrial production of these alkaloids. Chemical in the yeast petite mutants. In addition, the effect of cell synthetic routes which involve resolution of racemic mixtures immobilization and carbon source (glucose or molasses) on of alkaloids are also not appropriate for commercial produc- L-PAC production was investigated. It was found that cell tion (Abourashed et al. 2003). The current large-scale process immobilization enhances L-PAC formation. The highest for the production of ephedrine is via a combined fermentation L-PAC concentration (2.4 g/l) was obtained at 2 g/l of and chemical synthesis process (Fig. 1). In this process, benzaldehyde using the immobilized petite mutants grown benzaldehyde is first converted to L-phenylacetylcarbinol on molasses. (L-PAC) as the key intermediate in the production of ephed- rine and pseudoephedrine. Ephedrine is subsequently obtain- ed by reductive amination of the carbinol (Abourashed et al. Keywords L-Phenylacetylcarbinol . Acetohydroxyacid 2003;Doostmohammadietal.2015). synthase . Saccharomyces cerevisiae . Petite mutants . Pyruvate decarboxylase (PDC) is a homotetrameric Immobilization . Benzaldehyde enzyme that catalyzes the non-oxidative decarboxylation of thiamine pyrophosphate (TPP)-bound pyruvate to acetalde- hyde and carbon dioxide. As the second function, PDC can catalyze carboligation reaction of bound decarboxylated * Mohammad Ali Asadollahi pyruvate to yield acetoin via an aldol type condensation. [email protected] This ability is exploited for the commercial production of L-PAC using a combination of benzaldehyde with the acetal- dehyde obtained from pyruvate decarboxylation reaction 1 Department of Biotechnology, Faculty of Advanced Sciences and (Fig. 2; Agarwal et al. 2015). In parallel, alcohol dehydroge- Technologies, University of Isfahan, Isfahan 81746-73441, Iran nase (ADH) converts benzaldehyde to benzyl alcohol as a 2 Department of Biology, Faculty of Science, University of Isfahan, by-product (Neuberg and Hirsch 1921; Netrval and Vojtíšek Isfahan 81746-73441, Iran 1982; Tripathi et al. 1997). 3 Department of Biology, Faculty of Science, Shahid Chamran The efficiency of the process is limited by factors such as University, Ahvaz, Iran by-product formation, benzaldehyde toxicity toward cells, and 1050 Ann Microbiol (2016) 66:1049–1055 Fig. 1 Semi-synthetic process for large-scale production of Unreacted Benzaldehyde ephedrine a yeast fermentation for L-PAC the biotransformation of Ephedrine endogenous pyruvate and exogenously supplied benzaldehyde to L-PAC; b vessel for extraction of L-PAC from the Metal culture medium; c reductive catalyst amination of L-PAC using metal catalyst in the presence of H2 and H2 methylamine Methylamine a) b) c) irreversible inactivation of the enzyme by acetaldehyde decreases significantly. This prevents import of many (Rosche et al. 2001). Many attempts have been made to preproteins into mitochondria. As such, cytosolic accumula- improve the efficiency of the fermentation process. Some tion of AHAS precursor in the yeast petite mutants has been researchers have employed the isolated PDC enzyme instead reported (Dasari and Kölling 2011). of the whole cell as a biocatalyst to prevent by-product In this study, we have compared the ability of wild-type formation (Shin and Rogers 1996; Gunawan et al. 2008). and petite mutants of the yeast S. cerevisiae for L-PAC Acetohydroxyacid synthase (AHAS) is an enzyme present production. Both free and immobilized yeast cells were in plants, bacteria, and fungi which catalyzes the first commit- employed. Molasses and glucose were used as carbon sources. ted step in the biosynthesis of branched-chain amino acids (Calvo et al. 1969). In contrast to PDC, AHAS is an enzyme with intrinsic carboligase activity. AHAS decarboxylates Materials and methods pyruvate and catalyzes the condensation of an active acetal- dehyde moiety derived from pyruvate with another molecule Materials of pyruvate to form acetolactate or with ketobutyrate to form acetohydroxybutyrate (Chipman et al. 1998). All solvents and chemicals used were of reagent grade unless The ability of AHAS to catalyze condensation of pyruvate otherwise indicated. Standard L-PAC was a gift from Embio with benzaldehyde and formation of L-PAC has been reported Limited (Mumbai, India). (Engel et al. 2003). In the yeast Saccharomyces cerevisiae, AHAS is a mitochondrial enzyme encoded by ILV2 (Falco Microorganism, culture conditions and cell et al. 1985). immobilization In the yeast petite mutants, generation of adenosine triphos- phate (ATP) by oxidative phosphorylation ceases and S. cerevisiae (CEN.PK113-7D) was grown on yeast peptone membrane potential across the inner mitochondrial membrane dextrose (YPD) agar plates. The medium used for the Fig. 2 Mechanism of L-PAC formation by PDC enzyme Ann Microbiol (2016) 66:1049–1055 1051 production of L-PAC contained 30-g/l glucose and 6-g/l pep- carried out using standard curves generated after each analysis tone. For cultivation of encapsulated cells, CaCl2 at a concen- run (Doostmohammadi et al. 2015). tration of 5 g/l was added to glucose–peptone medium. The composition of the molasses medium was similar to glucose– Experimental methodology peptone medium except that glucose was replaced by 30-g/l molasses. For cell immobilization, 1000 ml of overnight cul- A two-level factorial design of experiments was conducted to ture at an optical density (OD ) of 18 was centrifuged 600 600 investigate the L-PAC production by both the wild-type and and after removing supernatant, the cells (about 3 g of wet petite cells at two concentrations of benzaldehyde and in cells) were added to 35 ml of 4 % (w/v) sterile sodium alginate immobilized and free cell systems. The rationale for selecting solution. This solution was added drop wise into a 2 % (w/v) the two-level factorial design was to have a reputable method CaCl solution under gentle stirring using a magnet stirrer. 2 to compare L-PAC production by the wild-type and the petite cells in various conditions. All of the experiments (Table 1) Induction of petites with UV light were run in duplicate. Analysis of variance (ANOVA) was the statistical method used in this research. The significance of the To generate petite mutants, cells from an overnight YPD culture data was judged by the p value being closer to zero (0.00). For (OD600 of 18) were diluted 1:50 in YPD and incubated at 30 °C a 95 % confidence level, the p value should be less than or for 3 h. Grown cells were exposed to UV light radiation equal to 0.05, indicating a statistically significant effect. The (CROSSLINKER CL-E508.G) with a 254-nm UV source at individual and interactive effects of the benzaldehyde concen- 2 an intensity of 10 J/cm h for 20 min. Cell suspension was then tration (1 and 2 g/l), state (free or immobilized cell), and strain cultivated on YPD medium containing triphenyltetrazolium (wild-type or petite mutant) on L-PAC production as response chloride. A color detection method was used to measure mito- variables were studied as well. chondrial dysfunction in the yeast cells. Wild-type yeasts reduce triphenyltetrazolium chloride to the insoluble red Formosan pig- ment, whereas strains lacking the ability to carry out respiration (mitochondrial function) turn white (Stotz and Linder 1990). Results and discussion Comparison between wild-type cells and yeast petite Biotransformation using free and immobilized yeast cells mutants Cells harvested from 1000 ml of overnight culture medium at PDC is the major enzyme responsible for the biosynthesis of an OD600 of 18 (about 3 g of wet cells) were added to 100 ml L-PAC. However, this enzyme suffers from some disadvan- of biotransformation medium (glucose or molasses medium) tages such as formation of benzyl alcohol as an unwanted by- and incubated for 1 h on a shaker at 30 °C and 200 rpm for product and sensitivity to benzaldehyde concentration and adaptation of cells to the medium. Benzaldehyde and acetal- temperature (Velmurugan et al. 1997; Engel et al. 2003). dehyde were each added at concentrations of 1 and 2 g/l and Alternatively, AHAS can be used to catalyze condensation flasks were incubated at 30 °C and 200 rpm for 28 h. of benzaldehyde with pyruvate leading to L-PAC formation. AHAS is a mitochondrial enzyme in the yeast S. cerevisiae Analysis of L-PAC concentrations (Falco et al. 1985), but in the yeast petite mutants, this enzyme accumulates in the cytosol (Dasari and Kölling 2011). Concentrations of L-PAC were determined by gas chromatog- Therefore, in petite mutants, two parallel pathways should raphy (GC). Samples were taken after 21 h and extracted with exist for the biosynthesis of L-PAC. We compared the ability dichloromethane.