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Development of cell-surface display expression system
The power of biotechnology, especially genetic engineering, enables us to develop expertise on protein expression system. Taking advantage of Thailand’s vast biodiversity, several native enzymes from fungi have been isolated and characterized, some of which exhibited excellent properties with potential industrial applications. However, in general, the yield of endogenous enzyme is low. Thus, heterologous expression for more efficient production of these target enzymes in appropriate hosts is necessary. Several expression systems, mostly E. coli or yeasts, have been utilized worldwide. Pichia pastoris is a yeast strain which grows quickly in defined medium and can grow to very high cell densities. It is especially useful for the large scale production of target proteins. In addition, the target proteins are glycosylated, which make P. pastoris yeast a favorable host for expressing fungal, plant or human proteins. However, the need for further downstream process such as purification and separation make the cost for large scale production prohibitive. We therefore exploit cell surface display technology to express secreted target proteins on the yeast cell surface as anchored proteins. Production of enzymes that are immobilized on the cell surface thus obviates tedious purification processes. The yeast cells with anchored proteins can then be used further as a whole-cell biocatalyst.

Improvement of protein production
Multi-copy expression of target enzymes has been proven to be an useful tool to increase the level of protein production. This technique is also employed in our laboratory to improve the level of protein expression both extracellularly and intracellularly. In addition, the viral cis-acting hydrolase element is being studied for its potential to improve the level of enzyme production. Alternative promoters from other organisms are also being investigated for strong and efficient expression of enzymes in yeast.

Development of Thai-isolated new yeast strains as alternative host
The methylotrophic yeast P. pastoris has become a widely studied host for heterologous protein expression, which has several advantages over E. coli including high density of the cultivation, gene stability, and high level of extracellular protein production under a controllable induction system. P. pastoris has been successfully exploited in our laboratory for heterologous expression of several enzymes including cellulase, xylanase and phytase. However, the current P. pastoris strains used are commercial strains under patent, which might cause further complications and additional cost when large scale production of target enzymes aimed for industrial applications is involved. Therefore, we aim to develop new alternative yeast strains with properties suitable and efficient for heterologous protein expression.

Expression of fungal enzymes in protease-deficient strain, Aspergillus oryzae
Filamentous fungi are hosts that have been exploited for expression of target proteins, as they have a high capacity to produce large amounts of target protein and large scale production is relatively cheap compared with other hosts. The filamentous fungal host system is also an attractive host to express proteins originally from other fungi. Thus, our lab utilizes one of the most widely known fungi, Aspergillus oryzae for the production of heterologous proteins, especially fungal enzymes.

Development of novel plasmid vectors for gene expression in Lactobacillus and Bacillus
Lactobacillus and Bacillus have been used as GRAS starter cultures in broad applications of food, functional food, agriculture and bioremediation. Hence, they are attractive hosts as cell factories. Genetic engineering technology has been used to improve those strains as desired properties for expansion their utilization. Plasmid vectors play an important role in the genetically modified process. In our laboratory, we have constructed and developed the novel plasmid vectors by modifying the native indigenous plasmid of Lactobacillus plantarum. The native plasmid has been isolated and modified to generate 4 shuttle vectors, pPR1, pPR2, pPR3 and pPR4. All plasmids were able to replicate in L. plantarum, Escherichia coli and Bacillus subtilis. The pPR1 contains erythromycin and ampicillin resistance genes whereas pPR2 harbors only erythromycin resistance gene. Derivatives of both plasmids which are pPR3 and pPR4, respectively, also contained gfp gene. Recently, pPR3 has been used to monitor the specific bacterial strains in various studies. These plasmids will be further developed to be the efficient expression vectors for heterologous gene expression in Lactobacillus and Bacillus.

The recombinant B.subtilis-pPR3 and E.coli-pPR3 packed cells on UV box

National Center for Genetic Engineering and Biotechnology (BIOTEC)
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