Production of zosteric acid and other sulfated phenolic biochemicals in microbial cell factories

Abstract

Biological production and application of a range of organic compounds is hindered by their limited solubility and toxicity. This work describes a process for functionalization of phenolic compounds that increases solubility and decreases toxicity. We achieve this by screening a wide range of sulfotransferases for their activity towards a range of compounds, including the antioxidant resveratrol. We demonstrate how to engineer cell factories for efficiently creating sulfate esters of phenolic compounds through the use of sulfotransferases and by optimization of sulfate uptake and sulfate nucleotide pathways leading to the 3'-phosphoadenosine 5'-phosphosulfate precursor (PAPS). As an example we produce the antifouling agent zosteric acid, which is the sulfate ester of p-coumaric acid, reaching a titer of 5 g L^-1 in fed-batch fermentation. The described approach enables production of sulfate esters that are expected to provide new properties and functionalities to a wide range of application areas.

Fig. 1

The zosteric acid microbial cell factory. a The optimized cell factory for producing sulfated biochemicals is exemplified by the production of zosteric acid, a plant biochemical from eelgrass (right side). The cell factory expresses a tyrosine ammonia-lyase (TAL) for deamination of tyrosine, a phenol sulftransferase (PST) for O-sulfonation of p-coumaric acid, and overexpresses uptake mechanisms for sulfate (here either CysZ or CysPUWA) as well as enzymes for activation of sulfate into 3′-phosphoadenosine 5′-phosphosulfate by CysDN and CysQ, and hydrolysis of 3′-phosphoadenosine 5′-phosphate (CysQ). b Sulfation as a general process for modifying a drug or a biologically produced compound for enhanced properties

Fig. 2

Activity and relationship between screened sulfotransferases. The phylogenetic relationship a of the chosen sulfotransferases shown together with b the consumption of substrates (pHCA (p-coumaric acid), resveratrol, kaempferol, and vanillic acid) and appearance (c) of product peaks. d The substrate specificity of selected phenol sulfotransferases when the production organisms were challenged with selected compounds (p-coumaric acid, orange; resveratrol, light gray; kaemphferol, yellow; vanillic acid, blue; ferulic acid, light green; 3-hydroxy-4-methoxycinnamic acid, navy blue; naringenin, rust red; 4-vinylphenol, dark gray; 4-nitrophenol, golden brown; 4-methylumbelliferone, dark blue; 4-acetamidophenol, dark green; 4-ethylphenol, light blue; 4-ethylguaiacol, light brown). The height of the bars show the relative remaining amount of compounds relative to a strain that harbors an empty expression plasmid shown (as averages of individual points plotted in circles). Source data of Fig. 2d are provided as a source data file

Fig. 3

Production of zosteric acid directly from glucose in minimal medium in recombinant E. coli. Selected strains expressing SULT1A1_Rno in combination with either no tyrosine ammonia-lyase (TAL) or one of the tyrosine ammonia-lyases from Rhodobacter sphaeroides (TAL_Rsp), Rhodobacter capsulatus (TAL_Rca), or Flavobacterium johnsoniae (TAL_Fjo), respectively, were grown in M9 medium with 0.2% glucose for 24 h. Resulting production titers (µM) of p-coumaric acid (pHCA) or zosteric acid (ZA) and standard deviations (± for n = 3) are shown. Source data are provided as a Source Data file

Fig. 4

Improving the supply of activated sulfate promotes production of sulfated products. Strains expressing SULT1A1_Rno from pETDuet-1 alone or with in combination with either an empty pRSFDuet-1 plasmid, a derived plasmid harboring the cysDNC operon, cysQ, a synthetic cysDNCQ operon, various sulfate transport genes or combinations thereof were grown in M9 medium with either low or high concentration of p-coumaric acid (pHCA) and potassium sulfate for 24 h in 96-well plates. Titers (µM) of produced zosteric acid (ZA) are shown together with standard deviations (± for n = 3). No production of ZA occurred in the absence of SULT1A1_Rno. Source data are provided as a Source Data file

Fig. 5

Production of zosteric acid in fed-batch bioreactor cultivation. Growth and production in a 1-L fermenter under fed-batch conditions in minimal salts media without tyrosine (a) or with supplementation of exogenous tyrosine (b)