Journal of Biotechnology 163 (2013) 184–193 Contents lists available at SciVerse ScienceDirect Journal of Biotechnology j ourna l ho me pag e: www.elsevier.com/locate/jbiotec Production of aromatics in Saccharomyces cerevisiae—A feasibility study Jens O. Krömer a,b,∗ , Dariela Nunez-Bernal a , Nils J.H. Averesch a , Jennifer Hampe a , Javier Varela c,1 , Cristian Varela c a Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, Brisbane, Queensland, Australia b Centre for Microbial Electrosynthesis (CEMES), Advanced Water Management Centre, Gehrmann labs (Bldg 60-620), The University of Queensland, Queensland 4072, Australia c Australian Wine Research Institute (AWRI), Adelaide, South Australia, Australia a r t i c l e i n f o Article history: Received 2 March 2012 Received in revised form 18 April 2012 Accepted 25 April 2012 Available online 3 May 2012 Keywords: p-Hydroxybenzoic acid p-Aminobenzoic acid S. cerevisiae Toxicity Yields Production a b s t r a c t Aromatics are amongst the most important bulk feedstocks for the chemical industry, however, no viable bioprocess exists today and production is still dependent on petro-chemistry. In this article the produc- tion of aromatic precursors such as p-hydroxybenzoic acid (PHBA) and p-amino benzoic acid (PABA) in Saccharomyces cerevisiae was evaluated using metabolic network analysis. Theoretical mass yields for PHBA and for PABA obtained by metabolic network analysis were 0.58 and 0.53 g g glucose -1 , respectively. A major setback for microbial production of aromatics is the high toxicity of the products. Therefore, PHBA and PABA toxicity was evaluated in S. cerevisiae. Minimal inhibitory concentrations of 38.3 g L -1 for PHBA and 0.62 g L -1 for PABA were observed. However, PABA toxicity could be alleviated in adaptation experiments. Finally, metabolic engineering was used to create proof of principle first generation strains of S. cerevisiae. Overall accumulation of 650 M PHBA and 250 M PABA could be achieved. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Aromatic chemicals are essential feedstocks for the global chemical industry. The majority of the aromatic compounds are produced from BTX (benzene, toluene and xylene), which are obtained from both petroleum refining and natural gas. It is widely accepted that long term increase in demand and decreasing avail- ability of these feedstocks as well as environmental compliance costs will drive prices up. Therefore there is a worldwide push in the chemical industry to move from fossil fuel derived feedstocks to renewable bio-derived feedstocks. It is estimated that the current 3–4% share of bio-feedstocks could increase to 17% of the global chemical business, equivalent to 425 billion $US, by 2025 (Reisch, Abbreviations: PEP, phosphoenolpyruvate; PYR, pyruvate; aKG, -ketoglutarate; AC-CoA, acetyl-coenzymeA; H-CoA, coenzymeA; CO2, carbondioxide; ATP, adeno- sine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; NADH/NAD, nicotinamide adenine dinucleotide (reduced/oxidized); NADPH/NADP, nicotinamide adenine dinucleotide phosphate (reduced/oxidized); PHBA, p- hydroxybenzoic acid; NH3, ammonia; TYR, tyrosine; GLU, glutamate; CHOR, chorismate. ∗ Corresponding author at: University of Queensland, Advanced Water Manage- ment Centre, Centre for Microbial Electrosynthesis (CEMES), Gehrmann labs (Bldg 60-620), Queensland 4072, Australia. Fax: +61 7 3365 4726. E-mail address: j.kromer@uq.edu.au (J.O. Krömer). 1 Current address: Escuela de Bioquimica, Universidad Nacional Andres Bello, Santiago, Chile. 2009). Over the last years the first biological diamines for nylon production (Kind and Wittmann, 2011), dialcohols (Nakamura and Whited, 2003) and some dicarboxylic acids (Lee et al., 2008) for polyesters are being developed as bio-replacement chemicals, but to date only very few bio-replacement compounds are cost com- petitive. For many important compound classes there is either no bio-process available, or the discovered biological routes of man- ufacturing are not economically feasible. One class of compounds falling into this group are aromatics. Aromatic compounds are among the most important build- ing blocks in the chemical polymer industry. It is estimated that the aromatic molecule terephthalic acid will exceed a market vol- ume of 50 million t by 2012. Based on the current price of US$ 1300/t this would equal a market of US$ 65 billion in 2012. The problem is that most commercial aromatics have no biological counterparts in biochemical pathways but are at best substrates in bio-remediation pathways of a few specialized microbes. The challenge is to find suitable precursor molecules that could be produced biotechnologically and then chemically converted to the desired compounds. One such candidate is p-amino benzoic acid (PABA), which is a potential precursor molecule for tereph- thalic acid. PABA could be chemically converted to terephthalic acid mononitrile using a Sandmeyer reaction and a subsequent reduc- tion. PABA is an intermediate in folate biosynthesis in microbes but to date no over producing strains have been described. PABA is currently chemically synthesized and is used as a moiety in the pharmaceutical industry (Kluczyk et al., 2002) and as a 0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2012.04.014