title: Microbial synthesis of triacetic acid lactone.
abstract: Native g2ps1-encoded 2-pyrone synthase (2-PS) from Gerbera hybrida, a mutant Brevibacterium ammoniagenes fatty acid synthase B (FAS-B) and two different mutants of Penicillium patulum 6-methylsalycilic acid synthase (6-MSAS) are examined to identify the best enzyme to recruit for the microbial synthesis of triacetic acid lactone (TAL). To identify the best microbial host for these evaluations, the native TAL-synthesizing activity of g2ps1-encoded 2-PS is expressed in recombinant Escherichia coli and Saccharomyces cerevisiae constructs. Five-fold higher expression levels of 2-PS are observed in S. cerevisiae. Consequently, microbial synthesis of TAL focuses on S. cerevisiae constructs. Comparison of different promoters for the expression of g2ps1 in S. cerevisiae indicates that the alcohol dehydrogenase II promoter (P(ADH2)) affords the highest expression levels of 2-PS. As a result, the genes encoding the various TAL-synthesizing enzyme activities are expressed in S. cerevisiae from a P(ADH2) promoter. To extend TAL-synthesizing activity beyond g2ps1-encoded 2-PS, the ketoreductase domains of fasB-encoded FAS-B and 6-MSAS-encoded 6-MSAS are modified using a single mutation. Modification of the nicotinamide cofactor-binding site of 6-MSAS with a triple mutation is also examined. Separate S. cerevisiae constructs expressing native g2ps1, mutant Y2226F fasB, mutant Y1572F 6-MSAS, and mutant G1419A-G1421P-G1424A 6-MSAS are cultured under the same fermentor-controlled conditions. The highest concentration (1.8 g/L) and yield (6%) of TAL are synthesized from glucose by S. cerevisiae expressing the Y1572F mutant of 6-MSAS.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Dongming
authors.initials: D
authors.lastname: Xie
authors.forename: Zengyi
authors.initials: Z
authors.lastname: Shao
authors.forename: Jihane
authors.initials: J
authors.lastname: Achkar
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Zha
authors.forename: John W
authors.initials: JW
authors.lastname: Frost
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D019659:Asteraceae / Q000201:enzymology*; D019149:Bioreactors; D001951:Brevibacterium / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology; D011753:Pyrones / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D011753:Pyrones; C030720:triacetic acid lactone; D010088:Oxidoreductases; D000217:Acyltransferases; D064429:Fatty Acid Synthases; C021587:6-methylsalicylic acid synthetase; D008025:Ligases; D005947:Glucose