Process Biochemistry 50 (2015) 1581–1590 Contents lists available at ScienceDirect Process Biochemistry jo ur nal home p age: www.elsevier.com/locate/procbio Development of a novel Aspergillus uracil deficient expression system and its application in expressing a cold-adapted -amylase gene from Antarctic fungi Geomyces pannorum Youzhi Mao a , Yanchen Yin a , Lujia Zhang a , Siti Aisyah Alias b , Bei Gao a,∗ , Dongzhi Wei a a State Key Lab of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China b Institute of Ocean and Earth Sciences, C308 Institute of Postgraduate Studies, University Of Malaya, Kuala Lumpur 50603, Malaysia a r t i c l e i n f o Article history: Received 4 February 2015 Received in revised form 15 June 2015 Accepted 16 June 2015 Available online 21 June 2015 Keywords: Cold-adapted -amylase pyrF Aspergillus oryzae Geomyces pannorum Heterologous expression a b s t r a c t Filamentous fungi have increasingly been used as hosts for heterologous protein production because of their high secretion capability and ability to add eukaryotic post-translational modifications. In this study, a novel uracil-deficient Aspergillus transformation system, which was based on an orotate phosphoribo- syltransferase (pyrF) nutritional selection marker, was discovered. Additionally, a universal, purify-able vector that directed genes into the Aspergillus host strain was engineered. A genomic DNA segment encod- ing a novel -amylase was isolated from the psychrotolerant fungus Geomyces pannorum and the open reading frame was determined, deduced 497 amino acids. G. pannorum -amylase was then expressed in the newly constructed Aspergillus oryzae system, with an amylase activity reaching 958 U/ml. It was purified to electrophoretic homogeneity and has a molecular mass of approximately 54 kDa. The enzyme exhibited an optimal activity at pH 5.0 and 40 ◦ C and retained over 20% of maximal activity over the temperature range 0–20 ◦ C. To our knowledge, this report is the first of the heterologous expression of a cold-adapted enzyme in filamentous fungi. G. pannorum -amylase is an economical amylase with many potential applications. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction The filamentous fungus Aspergillus oryzae, for which the com- plete genome sequence is known [1], is used extensively in the manufacture of fermented foods because of its proven safety record in the food industry and being generally recognized as safe (GRAS). A. oryzae has also been considered a favourable host for enzyme production (e.g. amylases, proteases, phytases, lipases, etc.) because of its ability to secrete proteins into the extracellular medium and introduce eukaryotic post-translational modifications [2]. Indeed, for many years, classical genetic strategies and recom- binant DNA techniques have been applied to the construction of excellent host strains, providing for a high level of gene expres- sion and protein secretion [3]. However, A. oryzae is not used as a host to express cold-adapted enzymes thus far. Like most ∗ Corresponding author at: East China University of Science and Technology, P.O.B. 311, 130 Meilong Road, Shanghai 200237, China. E-mail addresses: maoyz08@yeah.net (Y. Mao), tjuirisyin@163.com (Y. Yin), ljzhang@ecust.edu.cn (L. Zhang), saa@um.edu.my (S.A. Alias), gaobei@ecust.edu.cn (B. Gao), dzhwei@ecust.edu.cn (D. Wei). fungi, the Aspergilli lack natural extra-chromosomally replicating DNA elements. Accordingly, researchers have attempted to con- struct artificial plasmids and expression systems for Aspergillus [4]. Since the initial report by Boeke [5], genetic methods requiring the loss of function of orotidine 5 ′ -monophosphate decarboxylase (URA3) or its orthologue (pyrG) by selection on a 5-fluoroorotic acid (5-FOA) agar plate have been used with a number of mycelial fungus, including Aspergillus niger, Aspergillus oryzae, Aspergillus parasiticus, Trichoderma reesei and Monascus aurantia- cus. By the mid-1990s, a range of Aspergilli transformation systems and expression vectors had been developed, besides the pyrG described above, most based on nutritional selection markers: niaD (encoding nitrate reductase), sC (encoding ATP sulfurylase), adeA (encoding N-succinyl-5-aminoimidazole-4-carboxamide ribotide synthetase), argB (encoding ornithine carbamoyltransferase), etc. Recently, the polyauxotroph mutants have become popular in genetic engineering [6]. Nevertheless, the further development of new transformation selection markers is still required so that increased amounts of more important proteins can be produced in A. oryzae. In the pyrimidine biosynthetic pathway, the final two steps in the de novo biosynthesis of uridine 5 ′ -monophosphate (UMP) http://dx.doi.org/10.1016/j.procbio.2015.06.016 1359-5113/© 2015 Elsevier Ltd. All rights reserved.