Improved Efficiency of the Desulfurization of Oil Sulfur Compounds in Escherichia coli Using a Combination of Desensitization Engineering and DszC Overexpression
Lu LiYi-Bo LiaoYifan LuoGuangming ZhangXihao LiaoWei ZhangSuiping ZhengShuangyan HanYing LinShuli Liang
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The 4S pathway of biodesulfurization, which can specifically desulfurize aromatic S-heterocyclic compounds without destroying their combustion value, is a low-cost and environmentally friendly technology that is complementary to hydrodesulfurization. The four Dsz enzymes convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). Of these four enzymes, DszC, the first enzyme in the 4S pathway, is the most severely affected by the feedback inhibition caused by HBP. This study is the first attempt to directly modify DszC to decrease its inhibition by HBP, with the results showing that the modified protein is insensitive to HBP. On the basis of the principle that the final HBP product could show a blue color with Gibbs reagent, a high-throughput screening method for its rapid detection was established. The screening method and the combinatorial mutagenesis generated the mutant AKWC (A101K/W327C) of DszC. After the IC50 was calculated, the feedback inhibition of the AKWC mutant was observed to have been substantially reduced. Interestingly, the substrate inhibition of DszC had also been reduced as a result of directed evolution. Finally, the recombinant BL21(DE3)/BADC*+C* (C* represents AKWC) strain exhibited a specific conversion rate of 214.84 μmolHBP/gDCW/h, which was 13.8-fold greater than that of the wild-type strain. Desensitization engineering and the overexpression of the desensitized DszC protein resulted in the elimination of the feedback inhibition bottleneck in the 4S pathway, which is practical and effective progress toward the production of sulfur-free fuel oil. The results of this study demonstrate the utility of desensitization of feedback inhibition regulation in metabolic pathways by protein engineering.Keywords:
Dibenzothiophene
Protein Engineering
Biocatalysis
Saturated mutagenesis
Protein Engineering
Thermostability
Site-directed mutagenesis
Protein design
Directed mutagenesis
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Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering—guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.
Protein Engineering
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Protein engineering has been the most attractive strategy for biologists to redesign enzymes. As the simplest technique of protein engineering, directed evolution has been applied to many fields, such as industry, agriculture and medicine. An experiment of directed evolution comprises mutant libraries creation and screening or selection for enzyme variants with desired properties. Therefore, a successful application of directed evolution depends on whether or not one can generate a quality library and perform effective screening to find the desired properties. Directed evolution is already increasingly used in many laboratories to improve protein stability and activity, alter enzyme substrate specificity, or design new activities. Meanwhile, many more effective novel strategies of mutant library generation and screening or selection have emerged in recent years, and will continue to be developed. Combining computational/rational design with directed evolution has been developed as more available means to redesign enzymes. Keywords: Protein engineering, directed evolution, sequence diversity creation, novel strategy, computational design, rational design
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Molecular evolution based on mutagenesis is widely used in protein engineering. However, optimal proteins are often difficult to obtain due to a large sequence space. Here, we propose a novel approach that combines molecular evolution with machine learning. In this approach, we conduct two rounds of mutagenesis where an initial library of protein variants is used to train a machine-learning model to guide mutagenesis for the second-round library. This enables us to prepare a small library suited for screening experiments with high enrichment of functional proteins. We demonstrated a proof-of-concept of our approach by altering the reference green fluorescent protein (GFP) so that its fluorescence is changed into yellow. We successfully obtained a number of proteins showing yellow fluorescence, 12 of which had longer wavelengths than the reference yellow fluorescent protein (YFP). These results show the potential of our approach as a powerful method for directed evolution of fluorescent proteins.
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Abstract Biodesulfurization of fuel oils is a two‐phase (oil/water) process which may offer an interesting alternative to conventional hydrodesulfurization due to the mild operating conditions and reaction specificity afforded by the biocatalyst. For biodesulfurization to realize commercial success, a variety of process considerations must be addressed including reaction rate, emulsion formation and breakage, biocatalyst recovery, and both gas and liquid mass transport. This study evaluates emulsion formation and breakage using two biocatalysts with differing hydrophobic characteristics. A Gram‐positive ( Rhodococcus erythropolis ) biocatalyst, expressing the complete 4S desulfurization pathway, and a Gram‐negative biocatalyst ( Escherichia coli ), expressing only the gene for conversion of dibenzothiophene (DBT) to DBT sulfone, are compared relative to their ability to convert DBT and the ease of phase separation as well as biocatalyst recovery following desulfurization.
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Biocatalysis
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This account provides a personal view on the development of biocatalysis over the last two decades. Examples include the use of commercial enzymes, such as lipases, (recombinant) esterases, transaminases, and Baeyer–Villiger monooxygenases for the synthesis of optically pure compounds. The opportunity provided by modern protein engineering methods to tailor design an enzyme for a given scientific problem (substrate scope, selectivity, stability) is emphasized together with concepts to boost this technology in terms of timelines and success. 1 Introduction 2 Unexpected Discoveries 2.1 To Protect and Serve 2.2 ‘Abnormal’ Access to β-Amino Acids 3 Defined Enzyme Is Better Than Crude Extract 4 New Horizons Opened by Protein Engineering 4.1 Random Mutagenesis Can Give Random Results 5 Exploring Sequence and Structure Databases 5.1 Massive Alignment Identifies Evolutionary Variations 5.2 Fixing Wrong Annotations Can Yield a Toolbox of Novel Enzymes 6 Conclusion
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