scholarly journals Engineering of CYP153A33 With Enhanced Ratio of Hydroxylation to Overoxidation Activity in Whole-Cell Biotransformation of Medium-Chain 1-Alkanols

Author(s):  
Hyuna Park ◽  
Doyeong Bak ◽  
Wooyoung Jeon ◽  
Minjung Jang ◽  
Jung-Oh Ahn ◽  
...  

α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.

2015 ◽  
Vol 5 (5) ◽  
pp. 2622-2629 ◽  
Author(s):  
Zheng-Jiao Luan ◽  
Fu-Long Li ◽  
Shuai Dou ◽  
Qi Chen ◽  
Xu-Dong Kong ◽  
...  

Error-prone PCR and site-directed mutagenesis around substrate channel were employed for improving an esterase (RhEst1) activity towards Cilastatin building block. RhEst1A147I/V148F/G254A showed 20 times higher activity than the native enzyme in whole cell biotransformation.


2017 ◽  
Vol 84 (4) ◽  
Author(s):  
Shan-Chi Hsieh ◽  
Jung-Hao Wang ◽  
Yu-Chen Lai ◽  
Ching-Yeuh Su ◽  
Kung-Ta Lee

ABSTRACTMedium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A fromMarinobacter aquaeoleiVT8 (CYP153AM.aq; the strain is also known asMarinobacter hydrocarbonoclasticusVT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153AM.aqis limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkRM.aqis functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153AM.aqoperon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) fromPseudomonas putidaGPo1. Using a recombinantEscherichia colistrain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane–1-dodecanol substrate mixture. This study reports a very efficient method of producing C12/C14alkanols and C121,12-alkanediol by whole-cell biotransformation.IMPORTANCETo produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.


2012 ◽  
Vol 2 (8) ◽  
pp. 1602 ◽  
Author(s):  
Manpreet Singh ◽  
Sawraj Singh ◽  
Sateesh Deshaboina ◽  
Hare Krishnen ◽  
Richard Lloyd ◽  
...  

2009 ◽  
Vol 75 (12) ◽  
pp. 4202-4205 ◽  
Author(s):  
Wei Wang ◽  
Feng-Qing Wang ◽  
Dong-Zhi Wei

ABSTRACT A new cytochrome P450 monooxygenase, FcpC, from Streptomyces virginiae IBL-14 has been identified. This enzyme is found to be responsible for the bioconversion of a pyrano-spiro steroid (diosgenone) to a rare nuatigenin-type spiro steroid (isonuatigenone), which is a novel C-25-hydroxylated diosgenone derivative. A whole-cell P450 system was developed for the production of isonuatigenone via the expression of the complete three-component electron transfer chain in an Escherichia coli strain.


2021 ◽  
Author(s):  
Hye-Jin Jo ◽  
Jun-Hong Kim ◽  
Ye-Na Kim ◽  
Pil-Won Seo ◽  
Chae-Yun Kim ◽  
...  

A novel biocatalytic system for the synthesis of the industrially relevant C2 chemicals (e.g., ethylene glycol (3)) from formaldehyde (1) was established. The biocatalytic system consisted of a newly discovered...


2013 ◽  
Vol 468 ◽  
pp. 88-94 ◽  
Author(s):  
Fátima M. Nunes ◽  
Gabriel F. dos Santos ◽  
Natália N. Saraiva ◽  
Marília A. Trapp ◽  
Marcos C. de Mattos ◽  
...  

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