Earticle

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

The dynamic evolution process has resulted in the myriad shapes, functions, and systems evident in every living organism. For centuries, people have been harnessing the power of evolution to produce new varieties of plants and animals, such as producing tomatoes from berries and Chihuahuas from wolves. Now scientists are using it to produce better molecules, ranging from drugs to industrial chemicals, and doing it in days or weeks rather than eons. The ingenious process, which creates genetic diversity and selects those with desired features in the laboratory, is called directed evolution or test tube evolution. In this paper, concepts of directed molecular evolution and some examples will be discussed.

Cellobiohydrolase (CBH) I 유전자의 확보는 CBH I 유전자 cellulase 생산 균주인 Trichoderma reesei를 배양, 수거하고 액체 질소를 이용하여 세포를 파쇄 후 RT-PCR kit를 이용하여 CBH I gene을 합성하였다. 그 후 발현벡터인 pYGAL에 cloning하였다. CBH I 유전자 앞부분에는 CBH I 단백질이 cell 외부로 분비할 수 있도록 하는 ppL이 포함되었다. CBHI 단백질 발현은 Protein gel 결과를 통하여 발현을 확인하였다. Cellulase 활성을 증대시키기 위한 분자진화 방법 개발은 error prone PCR과 DNA shuffling을 수행하였다. 얻은 CBH I 유전자를 발현 벡터에 삽입하고 효모에 transformation하여 이것을 다시 screening하였다. 1차 screening 후 confirm test하기 위해 DNS (Dinitrosalicylic Acid) 환원당 측정법을 이용하였으며, 이 결과 121-D8, 228-G2, 389-E3, 412-B4, 456-D2의 cellulase 변이체를 획득할 수 있으며, 456-D2의 경우 original CBH I과 비교하여 약 510%의 활성이 증가된 것을 확인할 수 있었다. 분자 진화 된 cellulase sequence 분석결과 CBH I wild type과 비교하였을 때 121-D8의 경우 변경된 9개의 염기 중 아미노산의 변화에 영향을 준 염기는 6개, 228-G2의 경우 변경된 7개의 바뀐 염기 중 아미노산의 변화에 영향을 준 염기는 4개, 389-E3의 경우 변경된 13개의 바뀐 염기 중 아미노산의 변화에 영향을 준 염기는 9개, 412-B4의 경우 변경된 9개의 바뀐 염기 중 아미노산의 변화에 영향을 준 염기는 6개, 456-D2의 경우 변경된 10개의 바뀐 염기 중 아 미노산의 변화에 영향을 준 염기는 7개이었다. Cellulase 생산 공정 최적화는 5 L 발효기를 이용하여 고 농도 배양을 실험한 결과 최종 O.D. 120까지 진행할 수 있었으며, 약 1.2 g/L의 cellulase를 얻을 수 있었다. Cellulase 생산 공정 scale-up 5 L 규모에서 확립된 최적 fed-batch 발효 공정을 300 L로 scale-up하여 실험하였다. 최종 O.D.는 약 280 정도이며 cellulase의 발현양은 약 3.2 g/L 수준임을 확인하였다. Cellulase 정제 공정 최적화 결과 80%의 수율과 95%의 순도를 확보하였다.

Although Energy demands of modern society increase rapidly, curent energy would be exhausted shortly. Therefore development of bio-ethanol production process from cellulose containing materials was extrmly demanded. Therefore development of highly functional cellulase is requisite for this purpose. In this study cellobio-hydrolase (CBH1) gene from Trichorderma reesei was used to increase cellulase activity by directed evolution and highly functional cellobio-hydrolase was obtained and characterized.

Twin-Arginine translocation (Tat) pathway has been known with a unique cellular protein folding quality control mechanism in gram negative bacteria, which exports only correctly folded proteins across cytoplasmic membrane. Recently, we identified for the first time that a certain type of translocation intermediate of the recombinant Tat substrate is stably displayed on the surface of Escherichia coli. Furthermore, it was revealed that this display is dependent on a functional signal sequence and a folding efficiency of substrate. Taking advantage of the features of the display, a new protein engineering technology permitting simultaneous engineering of in vivo folding and affinity of proteins was exploited. This display technology should accelerate screening and engineering processes for the development of therapeutically valuable proteins such as single-chain variable fragment (scFv) and alternative binding scaffolds readily expressed in bacteria.

Stem cells have significant promise for regenerative medicine, but they first require an understanding of molecular or environmental cues that can regulate their proliferation and differentiation, as well as means to manipulate these signals to control cell behavior. Delivery of genes encoding molecules capable of regulating stem cell function can serve as an effective means to both investigate stem cell biology and to control cell fate for therapeutic applications. Additionally, gene delivery coupled with gene targeting has the potential to introduce mutations, and thereby generate disease models, as well as to correct deleterious genetic mutations in stem cell populations. However, a major obstacle to such applications continues to be the development of efficient and safe gene delivery vectors. Adenoassociated viral (AAV) vectors, which are being broadly explored in clinical trials, have significant promise as therapeutic vectors due to their safety and delivery efficiency, as well as their potential for gene targeting. Unfortunately, no natural AAV variants have been found with optimal properties for infecting stem cells. Due to the significant advantages of the vector, however, engineering AAV vectors to overcome rate limiting steps (i.e., cellular binding, intracellular trafficking, viral unpackaging, etc.) in stem cell transduction may have a high impact for stem cell investigations. Current approaches to design custom AAV vectors are limited to rational peptide insertion into or chemical modifications of the viral capsid structure. However, since the structure-function relationships of the complex AAV capsids are not fully understood, rationally designing and modifying the AAV capsid to meet specific needs are still challenges. In this presentation, a powerful tool (i.e. directed evolution) to create de novo bio-inspired nanoparticles (i.e., AAV vectors) that can significantly enhance the capabilities of gene delivery as well as gene targeting within stem cells will be introduced, and a variety of potential applications using gene-targeted stem cells will be discussed.

Stationary phase expression system has promising potential for production of toxic proteins such as antimicrobial peptide in Escherichia coli. Here we characterized and evoluted the promoter of ptsL, a gene identified by 2-D gel to be expressed from the stationary phase. The promoter region of ptsL is fused with the gene encoding enhanced green fluorescence protein (EGFP) to check the characteristic of stationary phase expression of this system by analyzing the fluorescence generated by cells. Furthermore, mlc which is encoding a repressor of ptsL and led by pBAD promoter to control its expression by different dose of arabinose, is cloned into the same plasmid. The purpose of introducing mlc gene here is to inhibit possible leak expression of EGFP prior to stationary phase, which is privotal when this system is used to produce toxic proteins. Finally, directed evolution was applied to enhance the expression level of ptsL promoter. [This work was supported by the Korean Systems Biology Research Program (M10309020000-03B5002-00000) of the Ministry of Science and Technology]

In bacteria and fungi, the process of carotenoid desaturation step is derived by a single enzyme, the phytoene desaturase (CrtI). Here we constructed beneficial mutants, which produce only neurosporene or lycopene up to 99% as a major product through random mutagenesis and site-directed mutagenesis. Three generations of random mutagenesis was performed and nine positive mutants were isolated and sequenced. Then we determined the effect of each amino acid changes using by site-directed mutagenesis. Through site-directed mutagenesis, we found two critical mutation points, L163P and G511C. To compare the carotenoid biosynthetic pathway of R.spheroides and P.agglomerans, each gene CrtE (GGPP synthase), CrtB (phytoene synthase), was modified to a self-expression module and then reconstructed as a synthetic operon (pACM_ERS_BRS and pACM_EPA_BPA) in Escherichia coli. With constructed two types of synthetic operon, we compared the different effect when expressed with mutant CrtIRS. The comparison result showed that nuerosporene expression rate was better when mutant CrtIRS was introduced into E.coli that harboring pACM_ERS_BRS, while lycopene expression rate was better when it was introduced into E.coli that harboring pACM_EPA_BPA. For better understanding in functional effect of each mutated point, predicted crystal structure modeling of phytoene desaturase from R.spheariodes was performed and analyzed.

The N-linked glycan on the Fc (fragment crystallizable) domain of IgG antibody is indispensable for binding to effector Fc gamma receptors (FcγRs) expressed on various immune cells and hence for the clearance of abnormal target cells. In IgG molecules, removal of the invariant glycan at Asn297 abolishes binding to FcγRs and effector functions. Recently, we have engineered aglycosylated IgG Fc variants that showed binding to FcγRI with affinity nearly identical to that of glycosylated antibodies. Interestingly, the engineered aglycosylated antibodies were able to potentiate tumor cell killing with dendritic cells (DCs) as effectors, while the glycosylated antibodies did not induce the DC-mediated ADCC (antibody dependent cell-mediated cytotoxicity). Additionally, we have isolated an aglycosylated antibody with selectivity towards activating FcγRIIa over inhibitory FcγRIIb and with superior macrophage-mediated ADCP (antibody dependent cell-mediated phagocytosis) when compared with a clinical grade glycosylated antibody. A set of Fc engineered versions of aglycosylated antibody with various receptor selectivities and unique effector functions have been generated and will be discussed.

Nowadays, many enzymes are engineered by using directed evolution for application in the industrial processes. There exists a functional gap between natural conditions for the activity of enzymes and industrial conditions that we require for using enzymes under industry. Among them, xylanase A is one of the important enzymes which catalyzes the hydrolysis of xylan, a major constituent of hemicelluloses. The goal of this research is to improve alkaline pH stability of xylanase A from Bacillus subtilis 168, which has optimum activity in pH 5, using directed evolution. At first, strain and expression vector for this research, which can produce xylanase A, were selected to make mutant libraries. Congo red dying method was used in order to check the expression and the secretion of xylanase A from a variety of hosts. E. coli w3110 and Pucp19A were selected for this study. Then, error-prone PCR was used to make mutant libraries easily. Congo red method was also used to screen the mutant which has xylanase A with the improved alkaline pH stability.

Glycolysis is the most important metabolic pathway for growth and maintenance of living organisms, and it is designed highly robust by nature. However, relevant modification of glycolytic activity is significant in metabolic engineering for the product accumulation and it is considered as one of main technical challenges for the strain improvement. gapA is a one of the key glycolytic genes and encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which mediates oxidative phosphorylation of glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate and has vital role in glycolysis. Wild-type GAPDH has strong activity and it is not easy to control its expression level by the conventional controllable promoters. Therefore, it is required to modify GAPDH for the sensitive regulation of glycolysis in E. coli. In this study, we developed a gapA mutant for the regulationsensitive glycolysis in E. coli by directed evolution approach using the gapAdeleted mutant obtained by Red recombination system and temperature sensitive promoter system. Directed evolution of GAPDH was conducted by error-prone PCR combined with growth rate based screening system. By several round of evolution processes, several mutants with the different levels of regulation sensitivities could be obtained. From the sequence analysis of the mutants, activity and sequence relationship was also examined.

Cellulosic materials are abundant natural resources, however, it is very hard to hydrolyze them due to their structural recalcitrance. Therefore, cellulose engineering has been extensively studied in recent years. We tried to characterize and improve cellulases from Cellulomonas fimi in terms of catalytic activity, substrate specificity, and protein solubility. First, we tested the solubility and functional expression of recombinant cellulases in E. coli. Out of seven catalytic domains of cellulases, four proteins were expressed as inclusion bodies in E. coli. Only Cex (encoding an exo-cellulase) among the four proteins showed a detectable catalytic activity, which was measured using a colorimetric method with p-nitrophenyl-β-D-celloglycoside. Solubility and activity of Cex will be improved by random mutagenesis. Furthermore, based on three-demensional structural analysis, plasticity residues in Cex were rationally selected and engineered using site-specific saturation mutagenesis for the improvement of catalytic activity and modification of substrate specificity. In this poster, we report a rational strategy for enhancement of catalytic efficiency and substrate specificity of cellulases.

Carotenoids are a structurally diverse group of natural pigmented chemicals of emerging importance as food supplements or colorants and in nutraceutical and pharmaceutical applications1). Carotenoids are structurally classified based on the number of backbone carbon molecules, usually C30, C40 or C50. Carotenoid biosynthesis occurs via a head to head condensation reaction of isoprenoid precursors followed by a desaturation reaction to increase the number of conjugated double bonds generating the distinctive carotenoid chromophore. Non-carotenogenic Escherichia coli cells can be transformed with heterologous carotenoid biosynthetic pathway genes of microbial or plant sources for the production of structurally diverse of carotenoids1). A new carotenoid desaturase homologue from S. aureus (CrtOx) was identified2). When expressed in engineered E. coli cells synthesizing linear C30 carotenoids3), novel polar carotenoid products were generated, identified as aldehyde and carboxylic acid C30 carotenoid derivatives. The major product in this engineered pathway is the fully desaturated C30 dialdehyde carotenoid 4,4’-diapolycopen-4,4’-dial. Very low carotenoid yields were observed when CrtOx was complemented with the C40 carotenoid lycopene pathway. But extension of an in vitro evolved pathway of the fully desaturated 2,4,2’,4’-tetradehydrolycopene produced the structurally novel fully desaturated C40 dialdehyde carotenoid 2,4,2’,4’-tetradehydrolycopendial. Directed evolution of CrtOx by error-prone PCR resulted in a number of variants with higher activity on C40 carotenoid substrates and improved product profiles. These findings may provide new biosynthetic routes to highly polar carotenoids with unique spectral properties desirable for a number of industrial and pharmaceutical applications. These results also demonstrate the utility of extending an in vitro evolved central metabolic pathway with catalytically promiscuous downstream enzymes in order to generate structurally novel compounds that are inaccessible without directed evolution4).

In the past decade methods of directed evolution has become a widely accepted and broadly applied method for biocatalyst engineering. A directed evolution experiment comprises two main steps: generating diverse mutant libraries and screening for improved protein variants. Although the vast majority of reported directed evolution experiments use a combination of error-prone PCR and DNA shuffling, methods for constructing diverse molecular libraries continue to accumulate. More important than the motivation to circumvent patent laws, these new approaches aim to generate more comprehensive, less biased libraries, because the quality of a mutant library is decisive for the success of a directed evolution experiment. This emerging focus on library quality is also reflected in new computational methods. In parallel with experimental techniques, computational predictive frameworks have been dramatic advances in design and analysis of directed evolution experiments. Recently, chemical DNA synthesis has been adopted to make various controlled mutant libraries with low bias and high fidelity, because of high efficiency, fidelity, cost reduction and process automation. Advanced mathematical and data-mining tools in computer modeling have been applied to analyze sequence-activity or sequence-stability relationships and to assist in the design of proteins with specified properties. It is unclear now, however, whether it is more efficient to mutate an enzyme randomly or to mutate active sites or key sites specifically designed by computer modeling. We expect that the powerful combination of in silico and in vitro methods based on DNA synthesis and computational analysis for directed evolution will further accelerate the successful development of desired biocatalysts.

With the visual system based on color C40 carotenoids formation, the 12 clones having the altered specificity of C15 FPP to C20 GGPP were isolated from the mutant library of farnesyl diphosphate (FPP, C15) synthase (IspA) of Escherichia coli. Some of the mutant IspA clones are reminiscences of the significance of positions and types of amino acids near or before the conserved first aspartate rich motif (FARM), which is generally known as one of the controlling mechanism for chain elongation reaction of all prenyl synthases. One IspA mutant carries a mutation at 2nd amino acid upstream of the conserved region IV, which recently was found to be important region controlling the chain elongation of Type III GGPP synthase. Another IspA mutant carries mutations near the conserved second aspartate rich motif (SARM). The product formation and profiles of wild-type and mutant IspA were examined by in vitro assay of purified enzymes. The model structures of the IspA mutants indicate that the product specificity may be controlled by alternation of the catalytic site of the enzymes that are spaciously related with the conserved regions.

이 연구의 최종 목표는 방향성 분자진화기술 (directed molecular evolution)을 이용한 음이온에 안정한 papain의 개발이다. 음이온 안정성 papain 생산을 위한 유전자의 방향성 분자진화 방법 개발이 이루어졌으며 분자진화된 음이온 안정성 papain의 스크리닝 방법 개발됐다. 분자진화된 papain의 아미노산 서열 및 특성 분석과 분자진화된 재조합 papain의 생산방법 확립되었다. 분자진화된 재조합 papain의 formulation 및 제품 적용화 기술 개발이다. 연구결과 Papain petidase IV 유전자의 확보 및 발현 균주 개발하였고 음이온 안정성 papain 유전자를 얻기 위한 분자진화의 방법 개발 및 조건 확립하였다. 분자진화 방법으로 error prone PCR 방법 확립, DNA shuffling을 통한 mutagenesis 방법 확립, staggered extension process 방법 확립 및 분자 진화된 음이온성 안정성 papain의 효율적 스크리닝 방법 개발하였다. Skim milk agar plate 이용, 활성 및 안정성이 뛰어난 개량형 papain을 Filter paper방법을 이용 하여 screening 방법을 개발하였다.

When the papain, which is a sort of Cystein protease, is applied to the outer skin, it decomposes the protein which forms the peeled outer skin and speeds up metabolism. Therefore, it is one of the most important cosmetics compositic which keeps the function of skin normal. When the papain is used in cosmetics with surfactant, the activity of papain is reduced rapidly. In this study, the modified papain with extreme stability negative ionic environment was developed by directed evolution

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (Kan^R) should be dependent on FucT2 solubility. Kan^R was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (Kan^R) should be dependent on FucT2 solubility. Kan^R was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli $\beta$-galactosidase and $\beta$-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more $\beta$-glucuronidase activity than wild-type $\beta$-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high $\beta$-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.