Improvement of extracellular bacterial protein production in Pichia pastoris by co-expression of endoplasmic reticulum residing GroELeGroES Pijug Summpunn, 1 , 2, * Juntratip Jomrit, 3 and Watanalai Panbangred 4, 5 Food Technologyand Innovation Research Center of Excellence, School of Agricultural Technology, Walailak University, Nakhon Si Thammarat 80161, Thailand, 1 Center of Excellence for Shrimp, School of Agricultural Technology, Walailak University, Nakhon Si Thammarat 80161, Thailand, 2 Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand, 3 Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand, 4 and Mahidol University-Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU:CRC), Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand 5 Received 11 May 2017; accepted 25 September 2017 Available online xxx Pichia pastoris is an established host system for heterologous protein expression. However, the potential productivity of this system can be limited. In this study, the Escherichia coli chaperones (GroESeGroEL) were expressed from the P GAP promoter and targeted to the secretory pathway through the endoplasmic reticulum (ER). The ability of the ER targeted chaperones to improve production of bacterial protein in P. pastoris was evaluated. The chaperones tagged with a-factor secretion- and ER retention-signal sequences were co-expressed with either extracellularly secreted phytase or intra- cellular D-phenylglycine aminotransferase (D-PhgAT) enzymes. The ER residing GroELeGroES successfully increased the levels of active phytase extracellularly, 1.5e2.3-fold higher than the phytase expression alone, but did not enhance the formation of active, intracellular D-PhgAT. These results indicate that the chaperones have the potential to enhance production of active enzymes when present in the same trafficking pathway. This is the first report on the improvement of extracellular bacterial protein production through co-expression with ER residing bacterial chaperones in the Pichia system. The modified P. pastoris expression system may be beneficial for extracellular expression of other prokaryotic proteins. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Chaperone; Expression system; Pichia; Productivity; Protein folding] Production of recombinant or biopharmaceutical proteins as well as bioactive metabolites has traditionally employed bacterial and eukaryotic organisms as hosts. Bacteria and yeasts share some beneficial features such as single cell growth, easy cultivation on inexpensive media, and availability of genetic manipulation sys- tems. The methylotrophic yeast, Pichia pastoris, has attracted increased attention due to its efficient secretion of heterologous proteins, high biomass concentration, ability to perform post- translational modifications, and potential for metabolic engineer- ing applications (1). However, a number of bottlenecks and stress factors limit the potential of this expression system and reduce the final yield of expressed proteins (2,3). Several approaches, including metabolic and cell engineering, have been utilized to improve the productivity in P. pastoris (4e7). Interestingly, Jar- iyachawalid et al. (4) reported the successful use of bacterial chaperones to co-express and enhance intracellular expression of a functional bacterial enzyme in P. pastoris. It was the first report on the use of Escherichia coli GroELeGroES to enhance the yield of a functional enzyme, D-phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri, through heterologous expression in yeast. Moreover, it was reported that increasing gene copy numbers for groEL and groES resulted in a progressive increase in D-PhgAT activity and yield. This indicated that the bacterial chaperone is an important to maximize functional expression of bacterial enzymes in P. pastoris. We hypothesize that in addition to increasing gene dosage, if the retention of co-expressed functional chaperones can be prolonged or recycled within the endoplasmic reticulum (ER), chaperones would be better able assist with the native folding of heterologous expressed proteins. Thus expression of chaperones in the ER may enhance the yield of functional proteins that are ulti- mately secreted. Specific retention sequence (HDEL, HiseAspeGlueLeu) (8) have been successfully used to retrieve enzymes from the Golgi appa- ratus to the ER through the cytosolic coat protein I (COP I) depen- dent pathway. This can facilitate enhanced concentrations of the enzyme in the ER which is important if activity in the ER is required (9,10). In this study, we focused on the improving the expression of functional proteins through correct protein folding in the Pichia expression system. Target proteins were co-expressed with chap- erones, GroELeGroES, localized to the ER with the use of HDEL retention system. The resulting modified Pichia expression system was evaluated as a production host for heterologous expression of either extracellular (phytase) or intracellular (D-PhgAT) proteins. MATERIALS AND METHODS Stains, plasmid, oligonucleotides, enzymes, media and chemicals E. coli DH5a was purchased from Novagen (Madison, WI, USA) and used as a cloning host * Corresponding author at: School of Agricultural Technology, Walailak University, Nakhon Si Thammarat 80161, Thailand. Tel.: þ66 7 567 2371; fax: þ66 7 567 2302. E-mail address: pijug.su@wu.ac.th (P. Summpunn). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2017 1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2017.09.007 Please cite this article in press as: Summpunn, P., et al., Improvement of extracellular bacterial protein production in Pichia pastoris by co- expression of endoplasmic reticulum residing GroELeGroES, J. Biosci. Bioeng., (2017), https://doi.org/10.1016/j.jbiosc.2017.09.007