Brocca S, Schmidt-Dannert C, Lotti M, Alberghina L, Schmid R D
Institut für Technische Biochemie, Universität Stuttgart, Germany.
Protein Sci. 1998 Jun;7(6):1415-22. doi: 10.1002/pro.5560070618.
The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site-directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pastoris cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lipl of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.
二态性酵母皱褶假丝酵母具有不寻常的密码子使用情况，这会妨碍源自该酵母的基因在传统异源宿主中的功能表达。皱褶假丝酵母脂肪酶（CRL）的商业样品在工业中广泛使用，但包含由脂肪酶基因家族编码的几种不同同工型，其中由lip1基因编码的同工型最为突出。在首次艰苦尝试中，通过定点诱变对lip1基因进行了系统修饰，以使其在酿酒酵母中实现功能表达。作为替代方法，根据酵母中的异源表达和简化的基因操作，用优化的核苷酸序列完全合成了该基因（1647 bp）。合成基因在酿酒酵母和巴斯德毕赤酵母这两种宿主中都实现了功能表达，并研究了异源前导序列对表达和分泌的影响。特别是，使用巴斯德毕赤酵母细胞，合成基因实现了功能过表达，首次能够在150 U/mL培养基的水平上生产高纯度的重组Lip1。将重组脂肪酶的物理化学和催化特性与含有脂肪酶同工型的商业非重组皱褶假丝酵母脂肪酶制剂的特性进行了比较。
