Westman Johan O, Nyman Jonas, Manara Richard M A, Mapelli Valeria, Franzén Carl Johan
Chalmers University of Technology, Department of Biology and Biological Engineering, Division of Industrial Biotechnology, 412 96 Göteborg, Sweden.
University of Southampton, Chemistry, University Road, Southampton SO17 1BJ, UK.
Metab Eng Commun. 2018 Apr 9;6:49-55. doi: 10.1016/j.meteno.2018.04.001. eCollection 2018 Jun.
Yeast flocculation is the reversible formation of multicellular complexes mediated by lectin-like cell wall proteins binding to neighbouring cells. Strong flocculation can improve the inhibitor tolerance and fermentation performance of yeast cells in second generation bioethanol production. The strength of flocculation increases with the size of the flocculation protein and is strain dependent. However, the large number of internal repeats in the sequence of from S288c makes it difficult to recombinantly express the gene to its full length. In the search for novel flocculation genes resulting in strong flocculation, we discovered a DNA sequence, , that gives NewFlo phenotype flocculation in CEN.PK 113-7D. The nucleotide sequence of the internal repeats of differed from those of . We hypothesized that a chimaeric flocculation gene made up of a variant derived from S288c and additional repeats from from CCUG 53310 would be more stable and easier to amplify by PCR. The constructed gene, , had 22 internal repeats compared to 18 in . Expression of in otherwise non-flocculating strains led to strong flocculation. Despite the length of the gene, the cassette containing could be easily amplified and transformed into yeast strains of different genetic background, leading to strong flocculation in all cases tested. The developed gene can be used as a self-immobilization technique or to obtain rapidly sedimenting cells for application in sequential batches without need for centrifugation.
酵母絮凝是由类凝集素细胞壁蛋白与相邻细胞结合介导的多细胞复合物的可逆形成。在第二代生物乙醇生产中，强絮凝可提高酵母细胞对抑制剂的耐受性和发酵性能。絮凝强度随絮凝蛋白大小的增加而增强，且具有菌株依赖性。然而，S288c菌株的絮凝蛋白序列中存在大量内部重复序列，这使得该基因难以全长重组表达。在寻找导致强絮凝的新型絮凝基因的过程中，我们发现了一个DNA序列，它能使CEN.PK 113-7D菌株呈现NewFlo表型絮凝。该序列内部重复序列的核苷酸序列与S288c的不同。我们推测，由源自S288c的絮凝蛋白变体和来自CCUG 53310的额外重复序列组成的嵌合絮凝基因会更稳定，且更易于通过PCR扩增。构建的基因有22个内部重复序列，而S288c只有18个。在原本不絮凝的菌株中表达该基因会导致强絮凝。尽管该基因长度较长，但包含该基因的盒式结构易于扩增，并可转化到不同遗传背景的酵母菌株中，在所有测试情况下均能导致强絮凝。所开发的基因可作为一种自我固定化技术，或用于获得快速沉降的细胞，以便在连续批次中应用而无需离心。
