Modification of C20 oxidase in tanshinone biosynthesis pathway / 中国中药杂志

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Advances of high-throughput screening system in reengineering of biological entities / 生物工程学报

Enzymes and cell factories are the core of industrial biotechnology. They play important roles in various fields such as medicine, chemical industry, food, agriculture, and energy. Usually, natural enzymes and cells need to be engineered to improve the catalytic efficiency, stability and enantioselectivity. Directed evolution makes it possible to rapidly improve the properties of enzymes and cell factories. Sensitive and reliable high-throughput screening approaches are the key for successful and efficient engineering of enzymes and cell factories. In this review, we first summarize the advantages and disadvantages of different screening methods and signal generation strategies as well as their application scope; we then describe the latest advances of ultra-high throughput screening technology applied in the directed evolution of enzymes and cell factories in the past three years. On this basis, we discuss the limiting factors that need to be further improved for high-throughput screening systems and forecast the future development trends of high-throughput screening methods, hoping that researchers in various fields including biotechnology and instrument development can cooperate closely to enhance the reliability and applicability of the high-throughput screening techniques.

Strategies for engineering the thermo-stability of glycosidase / 生物工程学报

Glycosidases are widely used in food and pharmaceutical industries due to its ability to hydrolyze the glycosidic bonds of various sugar-containing compounds including glycosides, oligosaccharides and polysaccharides to generate derivatives with important physiological and pharmacological activity. While glycosidases often need to be used under high temperature to improve reaction efficiency and reduce contamination, most glycosidases are mesophilic enzymes with low activity under industrial production conditions. It is therefore critical to improve the thermo-stability of glycosidases. This review summarizes the recent advances achieved in engineering the thermo-stability of glycosidases using strategies such as directed evolution, rational design and semi-rational design. We also compared the pros and cons of various techniques and discussed the future prospects in this area.

Methanol dehydrogenase, a key enzyme of one-carbon metabolism: a review / 生物工程学报

One-carbon compounds such as methanol and methane are cheap and readily available feedstocks for biomanufacturing. Oxidation of methanol to formaldehyde catalyzed by methanol dehydrogenase (MDH) is a key step of microbial one-carbon metabolism. A variety of MDHs that depend on different co-factors and possess different enzymatic properties have been discovered from native methylotrophs. Nicotinamide adenine dinucleotide (NAD)-dependent MDHs are widely used in constructing synthetic methylotrophs, whereas this type of MDH usually suffers from low methanol oxidation activity and low affinity to methanol. Consequently, methanol oxidation is considered as a rate-limiting step of methanol metabolism in synthetic methylotrophs. To accelerate methanol oxidation, thereby improving the methanol utilization efficiency of synthetic methylotrophs, massive researches have focused on discovery and engineering of MDHs. In this review, we summarize the ongoing efforts to discover, characterize, and engineer various types of MDHs as well as the applications of MDHs in synthetic methylotrophs. Directed evolution of MDH and construction of multi-enzyme complexes are described in detail. In the future prospective part, we discuss the potential strategies of growth-coupled protein evolution and rational protein design for acquisition of superior MDHs.

Progress in the construction and screening of random mutation library / 生物工程学报

Directed evolution is a cyclic process that alternates between constructing different genes and screening functional gene variants. It has been widely used in optimization and analysis of DNA sequence, gene function and protein structure. It includes random gene libraries construction, gene expression in suitable hosts and mutant libraries screening. The key to construct gene library is the storage capacity and mutation diversity, to screen is high sensitivity and high throughput. This review discusses the latest advances in directed evolution. These new technologies greatly accelerate and simplify the traditional directional evolution process and promote the development of directed evolution.

Enzyme ancestral sequence reconstruction and directed evolution / 生物工程学报

The amino acid sequence of ancestral enzymes from extinct organisms can be deduced through in silico approach termed ancestral sequence reconstruction (ASR). ASR usually has six steps, which are the collection of nucleic acid/amino acid sequences of modern enzymes, multiple sequence alignment, phylogenetic tree construction, computational deduction of ancestral enzyme sequence, gene cloning, and characterization of enzyme properties. This method is widely used to study the adaptation and evolution mechanism of molecules to the changing environmental conditions on planetary time scale. As enzymes play key roles in biocatalysis, this method has become a powerful method for studying the relationship among the sequence, structure, and function of enzymes. Notably, most of the ancestral enzymes show better temperature stability and mutation stability, making them ideal protein scaffolds for further directed evolution. This article summarizes the computer algorithms, applications, and commonly used computer software of ASR, and discusses the potential application in directed evolution of enzymes.

Directed evolution of tyrosine ammonia-lyase to improve the production of p-coumaric acid in Escherichia coli / 生物工程学报

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.

Improvement of the alkali stability of Penicillium cyclopium lipase by error-prone PCR

BACKGROUND: Lipases are extensively exploited in lots of industrial fields; cold-adapted lipases with alkali-resistance are especially desired in detergent industry. Penicillium cyclopium lipase I (PCL) might be suitable for applications of detergent industry due to its high catalytic efficiency at low temperature and relatively good alkali stability. In this study, to better meet the requirements, the alkali stability of PCL was further improved via directed evolution with error-prone PCR. RESULTS: The mutant PCL (N157F) with an improved alkali stability was selected based on a high-throughput activity assay. After incubating at pH 11.0 for 120 min, N157F retained 70% of its initial activity, which was 23% higher than that of wild type PCL. Combined with the three-dimensional structure analysis, N157F exhibited an improved alkali stability under the high pH condition due to the interactions of hydrophilicity and ß-strand propensity. Conclusions: This work provided the theoretical foundation and preliminary data for improving alkali stability of PCL to meet the industrial requirements, which is also beneficial to improving alkali-tolerance ability of other industrial enzymes via molecular modification.

Directed evolution of chitinase Chisb and biosynthesis of chitooligosaccharides / 生物工程学报

Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.

Recent advances in directed evolution / 生物工程学报

Screening is the bottleneck of directed evolution. In order to address this problem, a series of novel semi-rational designed strategies have been developed based on combinatorial active-site saturation test and iterative saturation mutagenesis, including single code saturation mutagenesis, double code saturation mutagenesis and triple code saturation mutagenesis. By creation of "small and smart" high qualified mutant libraries and combinatorial mutagenesis of specific sites, these new strategies have been successfully applied in multiparameter optimization, e.g. stereo/regioselectivity and activity. This review summarized recent advances in directed evolution and its applications in biocatalysis field.

Preface for special issue on enzyme engineering (2018) / 生物工程学报

Enzyme engineering combines enzymology and engineering, and is one of the major fields of modern biotechnology. To promote enzyme engineering research in China, we present in this special issue with reviews and original articles focusing on recent relevant advances reported by Chinese scientists.

Effect of mutation on aggregation propensity in homology model structures of syntaxin-3 from Homo sapiens.

Perception of molecular mechanism would provide potent additional knowledge on mammalian membrane proteins involved in causing diseases. In human, syntaxin-3 (STX3) is a significant apical targeting protein in the epithelial membrane and in exocytosis process; it also acts as a vesicle transporter by cellular receptor in neutrophils, which is crucial for protein trafficking event. Structurally, syntaxin-3 has hydrophobic domain at carboxyl terminus that directs itself to intra-cellular compartments. In addition, the experimental structure of STX3 is not available and no mutational study has been carried out with natural variants of proteins. Moreover, there is no evidence so far for the natural variant Val286 of STX3 causing any diseases. Hence, in the present study, analyses of residue-based properties of the homology model STX3 were carried out along with mutations at carboxyl terminus of STX3 by implementing protein engineering and in silico approaches. The model structure of STX3 was constructed adopting Modeller v9.11 and the aggregation propensity was analyzed with BioLuminate tool. The results showed that there was reduction in aggregation propensity with point mutation at Val286, instead of Ile, resulting into increasing the structural stability of STX3. In conclusion, the Ccap exposed residue would be a suitable position for further mutational studies, particularly with Val286 of STX3 in human. This approach could gainfully be applied to STX3 for efficient drug designing which would be a valuable target in the cancer treatment.

A roadmap to directed enzyme evolution and screening systems for biotechnological applications

Enzymes have been long used in man-made biochemical processes, from brewing and fermentation to current industrial production of fine chemicals. The ever-growing demand for enzymes in increasingly specific applications requires tailoring naturally occurring enzymes to the non-natural conditions found in industrial processes. Relationships between enzyme sequence, structure and activity are far from understood, thus hindering the capacity to design tailored biocatalysts. In the field of protein engineering, directed enzyme evolution is a powerful algorithm to generate and identify novel and improved enzymes through iterative rounds of mutagenesis and screening applying a specific evolutive pressure. In practice, critical checkpoints in directed evolution are: selection of the starting point, generation of the mutant library, development of the screening assay and analysis of the output of the screening campaign. Each step in directed evolution can be performed using conceptually and technically different approaches, all having inherent advantages and challenges. In this article, we present and discuss in a general overview, challenges of designing and performing a directed enzyme evolution campaign, current advances in methods, as well as highlighting some examples of its applications in industrially relevant enzymes.

Advances in Molecular Modification of Microbial Enzymes / 中国生物工程杂志

In recent years,more and more of the enzyme proteins have been carried out using recombinant microorganism bioreactor for large scale production.For reasons of improved the catalytic capability and environmental suitability,or enhanced expression level of the protein,a variety of genetic engineering technology according to protein molecule modification have been applied extensively.Major strategies and achievements of molecular modification for microbial enzyme,such as site-directed mutagenesis,error-prone PCR,DNA shuffling and optimum codon design were reviewed.