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Ruizhao Wang,Xiaoli Gu,Mingdong Yao,Caihui Pan,Hong Liu,Wenhai Xiao,Ying Wang,Yingjin Yuan

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 89-99 doi: 10.1007/s11705-017-1628-0

摘要： The conversion of -carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps of ketolation are catalyzed by -carotene hydroxylase (CrtZ) and -carotene ketolase (CrtW) respectively. Here, astaxanthin biosynthesis pathway was constructed in by introducing heterologous CrtZ and CrtW into an existing high -carotene producing strain. Both genes and were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE_Sc118060 with CrtW from DC263 and CrtZ from sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of in strain SyBE_Sc118060 was replaced from FBA1p to TEF1p, the astaxanthin yield was increased by 30.4% (from 3.4 to 4.5 mg/g DCW). In the meanwhile, 33.5-fold increase on transcription level and 39.1-fold enhancement on the transcriptional ratio of to were observed at early exponential phase in medium with 4% (w/v) glucose. Otherwise, although the ratio of to were increased at mid-, late-exponential phases in medium with 2% (w/v) glucose, the transcription level of both and were actually decreased during the whole time course, consequently leading to no significant improvement on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance desired compounds accumulation by employing the key enzyme(s) in microbes.

关键词： synthetic biology astaxanthin β-carotene hydroxylase β-carotene ketolase Saccharomyces cerevisiae

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Multistep conversion of cresols by phenol hydroxylase and 2,3-dihydroxy-biphenyl 1,2-dioxygenase

Shengnan SHI,Fang MA,Tieheng SUN,Ang LI,Jiti ZHOU,Yuanyuan QU

《环境科学与工程前沿（英文）》 2014年第8卷第4期页码 539-546 doi: 10.1007/s11783-013-0616-y

摘要： A multistep conversion system composed of phenol hydroxylase (PH ) and 2,3-dihydroxy-biphenyl 1,2-dioxygenase (BphC ) was used to synthesize methylcatechols and semialdehydes from - and -cresol for the first time. Docking studies displayed by PyMOL predicted that cresols and methylcatechols could be theoretically transformed by this multistep conversion system. High performance liquid chromatography mass spectrometry (HPLC-MS) analysis also indicated that the products formed from multistep conversion were the corresponding 3-methylcatechol, 4-methylcatechol, 2-hydroxy-3-methyl-6-oxohexa-2,4-dienoic acid (2-hydroxy-3-methyl-ODA) and 2-hydroxy-5-methyl-6-oxohexa-2,4-dienoic acid (2-hydroxy-5-methyl-ODA). The optimal cell concentrations of the recombinant strain BL21 (DE3) expressing phenol hydroxylase (PH ) and 2,3-dihydroxy-biphenyl 1,2-dioxygenase (BphC ) and pH for the multistep conversion of - and -cresol were 4.0 (g·L cell dry weight) and pH 8.0, respectively. For the first step conversion, the formation rate of 3-methylcatechol (0.29 μmol·L ·min ·mg cell dry weight) from -cresol was similarly with that of methylcatechols (0.28 μmol·L ·min ·mg cell dry weight) from -cresol by strain PH . For the second step conversion, strain BphC showed higher formation rate (0.83 μmol·L ·min ·mg cell dry weight) for 2-hydroxy-3-methyl-ODA and 2-hydroxy-5-methyl-ODA from -cresol, which was 1.1-fold higher than that for 2-hydroxy-3-methyl-ODA (0.77 μmol·L ·min ·mg cell dry weight) from -cresol. The present study suggested the potential application of the multistep conversion system for the production of chemical synthons and high-value products.

关键词： multistep conversion cresols phenol hydroxylase 2 3-dihydroxybiphenyl 1 2-dioxygenase methylcatechols