As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.

Bio-based polyurethane (PU) has recently drawn our attention due to the increasing interest in sustainability and the risks involved with petroleum depletion. Herein, bio-based self-healing PU with a novel polyol, i.e., eugenol glycol dimer (EGD), was synthesized and characterized for the first time. EGD was designed to have pairs of primary, secondary, and aromatic alcohols, which all are able to be involved in urethane bond formation and to show self-healing and antioxidant effects. EGD was incorporated into a mixture of the prepolymer of polyol (tetramethylene ether glycol) and 4,4′methylene diphenyl diisocyanate to synthesize PU. EGD-PU showed excellent self-healing properties (99.84%), and it maintained its high self-healing property (84.71%) even after three repeated tests. This dramatic self-healing was induced through transcarbamoylation by the pendant hydroxyl groups of EGD-PU. The excellent antioxidant effect of EGD-PU was confirmed by 2,2-diphenyl-1-picrylhydrazyl analysis. Eugenol-based EGD is a promising polyol chain extender that is required in the production of bio-based, self-healing, and recyclable polyurethane; therefore, EGD-PU can be applied to bio-based self-healable films or coating materials as a substitute for petroleum-based PU.

Several regiospecific enantiomers of hydroxy-(S)-equol (HE) were enzymatically synthesized from daidzein and genistein using consecutive reduction (four daidzein-to-equol–converting reductases) and oxidation (4-hydroxyphenylacetate 3-monooxygenase, HpaBC). Despite the natural occurrence of several HEs, most of them had not been studied owing to the lack of their preparation methods. Herein, the one-pot synthesis pathway of 6-hydroxyequol (6HE) was developed using HpaBC (EcHpaB) from Escherichia coli and (S)-equol-producing E. coli, previously developed by our group. Based on docking analysis of the substrate or products, a potential active site and several key residues for substrate binding were predicted to interpret the (S)-equol hydroxylation regioselectivity of EcHpaB. Through investigating mutations on the key residues, the T292A variant was verified to display specific mono-ortho-hydroxylation activity at C6 without further 3′-hydroxylation. In the consecutive oxidoreductive bioconversion using T292A, 0.95 mM 6HE could be synthesized from 1 mM daidzein, while 5HE and 3′HE were also prepared from genistein and 3′-hydroxydaidzein (3′HD or 3′-ODI), respectively. In the following efficacy tests, 3′HE and 6HE showed about 30∼200-fold higher EC50 than (S)-equol in both ERα and ERβ, and they did not have significant SERM efficacy except 6HE showing 10% lower β/α ratio response than that of 17β-estradiol. In DPPH radical scavenging assay, 3′HE showed the highest antioxidative activity among the examined isoflavone derivatives: more than 40% higher than the well-known 3′HD. In conclusion, we demonstrated that HEs could be produced efficiently and regioselectively through the one-pot bioconversion platform and evaluated estrogenic and antioxidative activities of each HE regio-isomer for the first time.