Stereoselective Synthesis of Chiral δ-Lactones via an Engineered Carbonyl Reductase

2021 ◽  
Author(s):  
Tao Wang ◽  
Xiaoyan Zhang ◽  
Yucong Zheng ◽  
Yunpeng Bai
Keyword(s):  
Serratia Marcescens ◽  
Directed Evolution ◽  
Stereoselective Synthesis ◽  
Specific Activity ◽  
Carbonyl Reductase

A carbonyl reductase variant, SmCRM5, from Serratia marcescens was obtained through structure-guided directed evolution. The variant showed improved specific activity (U mg-1) toward most of 16 tested substrates and gave...

scholarly journals Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Min Li ◽  
Zhi-Jun Zhang ◽  
Xu-Dong Kong ◽  
Hui-Lei Yu ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Engineering ◽  
Directed Evolution ◽  
Specific Activity ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Carbonyl Reductase ◽  
Triple Mutant ◽  
Engineering Strategy ◽  
Substrate Tolerance

ABSTRACT Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T 50 15, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C 50 15, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter−1 day−1. These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor. IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

2001 ◽  
Vol 47 (12) ◽  
pp. 1088-1094 ◽  
Author(s):  
Yew-Loom Chen ◽  
Tsung-Yin Tang ◽  
Kuo-Joan Cheng
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Synergistic Effects ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min–1·(mg protein)–1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

NON-LETHAL ASSIMILATION AND DISTRIBUTION OF RADIOACTIVE PHOSPHORUS IN SERRATIA MARCESCENS

1964 ◽  
Vol 10 (3) ◽  
pp. 317-322 ◽  
Author(s):  
G. E. Myers ◽  
R. G. L. McCready
Keyword(s):  
Serratia Marcescens ◽  
Culture Medium ◽  
Specific Activity ◽  
Test Organism ◽  
High Specific Activity ◽  
Lethal Effect ◽  
Optimal Conditions ◽  
Radioactive Phosphorus ◽  
Lipid Fractions ◽  
Cell Fractions

Optimal conditions for non-lethal assimilation of radioactive phosphorus by Serratia marcescens have been studied. Ten microcuries or less of P32per 300 ml of medium has no significant lethal effect on the test organism. The presence of 1% w/v glucose in the culture medium stimulates phosphorus assimilation causing an increase of approximately 34%. Phosphorus assimilation over a 24 hour period increases by approximately 50% when cultures are incubated at 30 °C rather than 37 °C.The concentration of P32in the various cellular constituents has been determined at various intervals during 24 hour incubation. Incorporation of P32in the acid-soluble and lipid fractions of the cell begins almost immediately whereas there is a slight delay prior to incorporation into the R.N.A. and longer delay before incorporation into D.N.A. Sixty to 65% of the P32assimilated by the bacterial cell is incorporated into the nucleic acids. Although R.N.A. has the highest specific activity of the cell fractions studied, the phospholipid fraction also has high specific activity.

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

2007 ◽  
Vol 388 (12) ◽  
pp. 1291-1300 ◽  
Author(s):  
Ai-Sheng Xiong ◽  
Ri-He Peng ◽  
Jing Zhuang ◽  
Jin-Ge Liu ◽  
Feng Gao ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Dna Sequences ◽  
Active Sites ◽  
Rational Design ◽  
Specific Activity ◽  
Dna Shuffling ◽  
Gene Encoding ◽  
Library Construction ◽  
Key Sites

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.

Efficient reductive desymmetrization of bulky 1,3-cyclodiketones enabled by structure-guided directed evolution of a carbonyl reductase

2019 ◽  
Vol 2 (10) ◽  
pp. 931-941 ◽  
Author(s):  
Xi Chen ◽  
Hongliu Zhang ◽  
Miguel A. Maria-Solano ◽  
Weidong Liu ◽  
Juan Li ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Carbonyl Reductase

scholarly journals Improving the specific activity and pH stability of xylanase XynHBN188A by directed evolution

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
La Xiang ◽  
Yihong Lu ◽  
Hui Wang ◽  
Meixing Wang ◽  
Guimin Zhang
Keyword(s):  
Directed Evolution ◽  
Specific Activity ◽  
Ph Stability
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