Earticle

Deoxynucleoside kinases exist as heterodimeric pairs specific for deoxyadenosine/deoxyguanosine kinase (dAK/dGK) and deoxyadenosine/deoxycytidine kinase (dAK/dCK). The aspartic acid-84 in dGK was mutated to alanine, asparagine and glutamic acid by site-directed mutagenesis. The mutation resulted in a drastic decease in dGK activity compared to the unmodified cloned enzyme while it increased production of dAK activity. The mutated dak/dgk genes, which synthesize tandem deoxyadenosine/deoxyguanosine kinase, were inserted back to the Lactobacillus acidophilus and Lactococcus lactis by electroporation to determine the effect of site-directed mutation of he enzymes on the microbial growth. However, no significant change was observed in cell growth and lactic acid production between wild type and mutant lactic acid bacteria.

Pentose phosphate pathway generates reducing equivalents, NADPH, and 5-carbon sugars. The ribose 5-phosphate produced by this pathway can be recycled into glucose 6-phosphate (G6P) by transketolase, transaldolase, and some of the enzymes of the gluconeogenic pathway. Consequently, complete oxidization of 1 mole of G6P to CO2 can generate 12 moles of NADPH. However, in most cases, glycolysis is much more activated than gluconeogenesis to generate energy sufficient for cell survival. Moreover, glycolysis and gluconeogenesis are reciprocally regulated so that they do not take place simultaneously in the same cell to a significant extent. Therefore, it is important to deregulate gluconeogenic pathway to fully oxidize G6P for generation of NADPH. Fructose-1,6-bisphosphatase (FBPase) is a key enzyme that participate in the regulation of gluconeogenesis. It is allosterically inhibited by both AMP and G6P. Here, we engineered FBPase to be deregulated by AMP and G6P based on the 3D-structure. Variants of FBPase purified by Ni-NTA column were characterized in terms of kinetic parameters.

Green fluorescent protein (GFP) Nobel Prize protein from jellyfish is a versatile reporter protein for monitoring gene expression and protein localization in variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral prosperities, including several blue emission variants, all of which contains the single point mutation in tyrosine analogue in green fluorescent protein chromophore. Generally, GFP variants will be generated through replacement of naturally occurring amino acids by site directed mutation or directed evolution methodology and disadvantages of this methodology is limited with 20 naturally occurring side chains. In other hand unnatural amino acid mutagenesis has been used to selectively substitute target amino acids with our interesting side chains in our objective protein. Current studies have explored incorporation of unnatural amino acid analogues into green fluorescent protein and alter the spectral emission and excitation properties. We have selected different tyrosine analogues and successfully incorporated into GFP to alter the specific characters. We have selected the tyrosine analogues due to the important role in the chromophore and structure formation. Current study not only demonstrated the importance of unnatural amino acid mutagenesis and also demonstrated new approach to generate tailormade proteins with interesting and useful spectral properties.

Polylactic acid (PLA) is an increasingly attractive polymer that can be used in many industries. It is currently synthesized by two-step processes composed of fermentative production of lactic acid followed by rather complicated chemical polymerization step. In this lecture, I will report on the results of combined metabolic and enzyme engineering for the establishment of new pathways in E. coli for the biosynthesis of PLA and lactate containing polyester copolymers. [This work was supported by the Korean Systems Biology Research Project from the Ministry of Education, Science and Technology. Further supports by LG Chem and WCU (R32-2008-000-10142-0) program are appreciated.]

Glycodiversification and glycorandomization, invaluable tools for generating biochemical diversity, can be catalyzed by flexible glycosyltransferases (GTs), which attach various activated sugar “donors” onto different type of “acceptor” molecules. Among discovered and successfully developed protein engineering techniques, creation of hybrid protein have been evaluated as very powerful approach to produce enzyme with new activity. Theoretically, hybrid protein was built up by different fragments from various parent molecules. Particularly, engineered GTs have been used as wonderful catalysts for making new glycosylated compound, for example, the generation of new hybrid glycopeptides or glycosylation of scopoletin by GT from Arabidopsisthalianaorproducing novel urdamycin derivatives, etc. Applying protein modeling, we have constructed a hybrid protein HT1 that is composed of DnrS-derived N-terminal and DesVII-originated C-terminal. The enzyme activity was tested using various activated sugar produced Escherichia coli system (such as TDP-rhamnose, UDP-xylose and UDP-6-deoxy-allose) in combination with the transformation of phenolic substrate (flavonoid, stilbene, quinone, etc). The initial result showed that hybrid protein significant change in regio-specific activity in comparison with parent molecule (DnrSandDesVII), particularly, enabling produce flavonol and flavanone-O-rhamnoside such as quercetin, kaempferol, myricetin, andnaringenin-O-rhamnoside. The experiment of glyco-diversification is continuously carrying out to determine the level of enzyme’s promiscuity and which type of the most favorite substrate was recruited. Therefore, the chimeric protein HT1 is apromising tool to divert the natural products. Furthermore it is possible to improve or alter enzyme activity by domain swapping from far related glycosyltransferases.

Recombinant human growth hormone (hGH) has been used as a therapeutic agent to treat children's growth disorders and adult growth hormone deficiency. hGH has been known to be highly susceptible to rapid proteolysis and has a short half-life of only a few hours in human blood stream. Here we developed an experimental and rational method to select modification sites in hGH polypeptide, where any changes (glycosylation or site-directed mutagenesis) can be applied. First, we selected several putative permissive sites and performed site-specific glycosylations for improved proteolytic stability. hGHs with site-specific mutations were expressed in Saccharomyces cerevisiae, and their glycosylations were confirmed by SDS-PAGE analysis and glycosidase treatment. The activities of glycosylated hGHs were tested using Nb2 cell proliferation bioassay. When compared with the wild-type unglycosylated hGH, the glycosylated hGH did not lose its biological activity. The proteolytic stability of glycosylated hGH was also tested by western blot analysis after treatment of plasmin. As a result, glycosylated hGH showed enhanced stability compared with the wild-type hGH. These results indicate that glycosylation at internal permissive sites was successful to develop protease-resistant hGH.

Streptomyces peucetius ATCC 27952 contains two similar genes (1068 nucleotides), dnmL and rmbA, which encode for glucose-1-phosphate (G-1-P) thymidylyltransferases (69% identity and 86% similarity). When the genes were expressed in E. coli under similar conditions, rmbA was expressed in soluble form, and it showed G-1-P thymidylyltransferase activity. But, dnmL was expressed as inclusion bodies. Thorough comparative analysis of DnmL and RmbA by sequence alignment, hydrophobicity scale and homology modeling inferred that the expression of RmbA in soluble form is contributed to the helical structure formed by the high hydrophobicity content in C-terminal sequence of RmbA. Similarly, the formation of inclusion bodies of DnmL was attributed to a loop conformation attained by its C-terminal sequence. To test the significance of these sequences, the C-terminal sequences of DnmL was substituted with that of RmbA. The fusion DnmL was expressed in soluble form and it had similar G-1-P thymidylyltransferase activity as compared to RmbA. These observations demonstrated that the hydrophobic effect plays a significant role in protein folding or structure, and the solubility of the expressed proteins depends on the protein conformations. In conclusion, this method of protein engineering will be a rational tool for enhancing solubility of proteins expressed in E. coli.

Polyguluronate specific lyase (EC 4.2.2.11) from Streptomyces sp. ALG-5 was cloned in pColdI vector and characterized previously. We named the polyG-specific lyase as ALG5 lyase. Alg5 was engineered to improve the alginate degrading activity based on comparative homology modeling. The homology modeling of ALG5 lyase was constructed by using the 3D crystal structure (1UAI.pdb) of alginate lyase 2 from Corynebacterium sp. Aly1 as a template. ALG5 lyase belongs to polysaccharide lyase family 7 which the fold of the structure is β-jolly sandwich. ALG5 lyase had 8 β-strands (SA1-SA8)at upper layer and 7 β-strands (SB1-SB7) at lower layer. Active sites were formed with Arg72 of SA3, Gln117 of SA5, His119 of SA5, Tyr188 of SA4 and Tyr194 of SA5. In the case of water soluble protein, protein surface was known to be composed with hydrophilic amino acid residues whereas protein core was composed with hydrophobic amino acid residues. Stability of mutants was calculated with ProSa2003. Glu101 on SB4 was mutated to hydrophobic amino acid, Phe, Leu, Met, Ile and Val. When Glu101 was mutated to Leucine, the alginate degrading activity was increased about 5 fold than that of original ALG5 lyase.

The Strep 13 encodes as sesquiterpene alcohol (germacredinol) synthaseencoding from Streptomyces peucetius ATCC27952. Complete open reading frame (ORF) of Strep 13 shows the production of germacredinol/geosmin. Here, we have explored the independent function of N-terminal and C-terminal protein of strep 13. Both terminal recombinant proteins were expressed at high level in Escherichia coli and analysis were carried out. N-terminal protein (~58.15 kDa) showed the Mg2+ dependent activity for the conversion of conversion of Farnesyl diphosphate to Germacrene D. In contrast, the expressed C-terminal domain of Strep13 has no farnesyl diphosphate cyclase activity.

The epoxide hydrolase gene from Mugil cephalus (MCEH) was engineered by triple site-directed mutagenesis. A comparative homology modeling based on the 3-D structure of Aspergillus niger epoxide hydrolase was analyzed to predict target amino acid residues. Three amino acid of the wild-type MCEH was engineered for proper spatial orientation of nucleophile, an enhancement of charge relay system, and no overlapping of electron density map in the active site. The engineered MCEH showed up to 40-fold enhanced catalytic efficiency compared to that of the wild type. Acknowledgement: This work was supported by the Marine and Extreme Genome Research Center Program, Ministry of Land, Transportation and Maritime Affairs, Republic of Korea.

Through the processes of natural selection and co-option, nature has crafted an astounding array of protein designs with a remarkable repertoire of functions ranging from catalysis to signaling and regulation. A growing list of biotechnology needs for proteins with altered cofactor/substrate specificities, improved/modified functionalities or activities are creating an ever expanding compilation of protein design challenges. Two separate tracks of computational methods will be described depending on the presence or absence of detailed structural information. The first track of methods will concentrate on the design of a known protein structure to introduce an improved or completely novel function such as affinity for a cofactor, substrate, or small molecule (ligand). This will be accomplished by either modifying the existing active site (i.e., redesign) to bring about an altered functionality or by introducing a completely new active site into the fold when a novel functionality is sought. Computations will be first tested by altering the effector binding specificity of the bacterial transcriptional regulatory protein AraC to molecules other than L-arabinose and by grafting a calcium binding pocket onto a cell adhesion protein. The second track of methods will focus on protein design challenges when detailed structural information is absent and limited information is available regarding residue contacts or function. Instead, only the sequences of protein family members are available. In such instances, protein design efforts typically rely on the high throughput screening of large combinatorial libraries generated by accumulating single point mutations and/or recombination events. Here, scoring functions will be put forth and computations will be used to identify the optimal pattern of point mutations and/or recombination events that are likely to maximize functional enrichment in a combinatorial protein library of a pre-specified size. We will benchmark the efficiency of all computational methods by comparing their performance on P450 and beta-lactamase libraries.

Despite all the technical progress in protein engineering, our ability to rationally manipulate the structures and functions of proteins is quite limited, because the genetic code specifies the same 20 amino acids building blocks. The development of a method that makes possible the systematic expansion of the genetic code can enable the evolution of proteins with new or enhanced properties. Recent advances in engineering of translation machinery have made it possible to add over 60 novel amino acids to the genetic code, but the repertoire of unnatural functional groups is still limited and their application is quite restricted. In an effort to overcome these major obstacles, newly discovered aminoacyl tRNA synthetase and tRNA were characterized in terms of structure and biochemical properties. Based on these studies, the cellular translation system was redesigned and evolved to site-specifically incorporate novel unnatural amino acids. Direct incorporation of unnatural amino acids can make considerable contribution to biotechnology and bioenergy research areas such as protein engineering, click chemistry, and nanobiotechnology, and bioenergy production. Detailed experimental design and results will be presented and application of synthetic biology technology will be discussed.

Molecular biology has started since Schrödinger published the report of “What is life” at 1944. At present, when 68 years has passed since the start point, genomics analysis for various species describes genes as vast resources of functional molecules (proteins) which have not be mimicked yet by organic molecules, and structural biology approach gets to hierarchical structure depiction that functional module units as elements are built up to the proteins as nanodevices. Advance in molecular biology has also developed various experimental technologies to analyze biological events from a molecular viewpoint; consequently, recombinant proteins with a non-native function and structure can be generated by means of molecular evolutional techniques and fusion protein engineering. In this molecular biology background, we are proposing the concept of “smart bio-design” for generating functional biomolecules from various protein modules. In the case of antibody, IgG-type antibodies are usually produced in mammalian cell expression systems, but the construction of a stable and large-scale production process in mammalian systems is laborious, time consuming, and expensive. In contrast, antibody fragments, such as single-chain Fv (scFv) and Fab fragments, can be prepared in bacterial secretory expression systems; however, the downsizing of antibodies has some benefits in protein expression processes, while it results in low affinity for antigen-displayed cell surfaces by the degrease of antigenbinding valence. Here, we propose a new construction of low molecular multivalent/bispecific antibody by conjugation of protein engineering and chemical conjugation engineering.

Antibody IgG has been evolved in natural immune system against a variety of pathogens and widely used for therapeutics, diagnostics, and research reagents due to its high evolvability which adopts new functions at ease with tolerating destabilizing mutations. In IgG molecules, removal of the invariant glycan at Asn297 abolishes binding to FcγRs (Fc gamma receptors) expressed on the surface of various immune cells. Therefore, aglycosylated antibodies produced in bacteria have not been used for therapeutic antibodies requiring effector functions such as clearance of tumor cells. In this lecture, I will present a set of Fc (fragment crystallizable) engineered versions of aglycosylated antibody with various FcγRs selectivities and unique therapeutic effector functions that clinical grade glycosylated antibodies do not display. Additionally, cytochrome P450 enzyme, a biocatalyst transforming a broad scope of substrates, has been evolved for a novel diagnostic tool. Strategies to increase sensitivity of the biomedical agent have been developed and will be discussed.

While the expression and screening of genetic libraries for protein engineering is generally carried out in microbial cells, the use of living cells sets intrinsic limitations in numerous dimensions including the throughput of process and choice of assay substrates. These limitations associated with cell-based expression screening can be effectively overcome by expressing enzyme libraries in the cell-free protein synthesis system. Not being subject to the constraints of maintaining cell viability and intact cell membranes, cell-free protein synthesis offers greater flexibility in designing expression and screening procedures. In this study, we will discuss different approaches to express and screen genetic libraries in a cell-free synthesis system derived from Escherichia coli. In particular, the discussion will be focussed on the development of effective methods to assay the expression of individual genes constituting the targeted library.

Carbonic anhydrase (CA, EC 4.2.1) accelerates to uptake of CO2 from air into intracellular system and phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) catalyzes the conversion of HCO3 - concentrated by carbonic anhydrase (CA) into four-carbon organic acid oxaloacetate (OAA). PEPcase is also the main anaplerotic enzyme providing OAA and/or malate, replenishing the tricarboxylic acid (TCA) cycle intermediates in all photosynthetic organisms [1]. Our research objective is to develop the platform technology for high-reactivity and high-production of marine CA and PEPCase. To achieve this goal, the experts on marine biology and genetic and protein engineering are collaborated to perform high-production of marine CA and PEPCase via search, screening and fabrication of high-expression recombinant hosts. In our laboratory, three types of CA in two strains of marine microalga (Dunaliella sp., Phaeodactylum tricornutum CCMP632) were identified. In Dunaliella sp., two types of CA, namely alpha and gamma-type CA, were isolated and cloned. Two types of PEPCase were also identified from marine green algae Dunaliella sp. and Dunaliella salina. The molecular cloning of the two PEPCase genes in Dunaliella salina (DsPEPC1, DsPEPC2) and one in Dunaliella sp. (DspPEPC1) were isolated, respectively. The recombinant micro-algae and microbe containing the developed CA and PEPCase variants will be used as important CO2-utilizing bioconversion system for production of useful C4 chemicals. This platform biotechnology for high-reactive design and high-production of marine CA and PEPCase will be essentially employed for development of efficient enzyme-based CO2 fixation/sequestration process.

AbstractLactide is cyclic dimer composed by two molecules of lactate and is pivotal monomer for poly lactide (PLA) which was promising environmentally friendly polymer and applied in wide industrial field.1 Candida antarctic lipase B (CALB) catalyze hydrolysis, esterification, and transesterification in mild condition compared to chemical catalyst.1,2 Although wild-type CALB consumed methyl R-lactate as substrate completely, conversion of R-lactide reached only about 60 % and the others were converted to oligomers as by-product. Molecular dynamic simulation predicted that Asp134 residue of CALB seemed interacted with methanol, which was by-product during transesterification of methyl R-lactate and functions to degrade ring structure of R-lactide. Among D134-mutants of CALBs, D134A and D134V were selected as candidates for the improvement of R-lactide production. Interestingly, D134A CALB mutant showed 20 folds higher consumption rate of methyl R-lactate as substrate. As conclusion, D134A mutant CALB has a potential to be used as a better biocatalyst for enzymatic synthesis of R-lactide from alkyl R-lactate directly in mild condition compared to wild type CALB.