Tøndervik Anne, Aune Randi, Degelmann Adelheid, Piontek Michael, Ertesvåg Helga, Skjåk-Bræk Gudmund, Sletta Håvard
Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway.
ARTES Biotechnology GmbH, Langenfeld, Germany.
Front Plant Sci. 2022 Jun 6;13:837891. doi: 10.3389/fpls.2022.837891. eCollection 2022.
Alginates are linear polysaccharides produced by brown algae and some bacteria and are composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). Alginate has numerous present and potential future applications within industrial, medical and pharmaceutical areas and G rich alginates are traditionally most valuable and frequently used due to their gelling and viscosifying properties. Mannuronan C-5 epimerases are enzymes converting M to G at the polymer level during the biosynthesis of alginate. The epimerases AlgE1-AlgE7 share a common structure, containing one or two catalytic A-modules (A), and one to seven regulatory R-modules (R). Despite the structural similarity of the epimerases, they create different M-G patterns in the alginate; AlgE4 (AR) creates strictly alternating MG structures whereas AlgE1 (ARRRAR) and AlgE6 (ARRR) create predominantly G-blocks. These enzymes are therefore promising tools for producing tailor-made alginates. Efficient epimerization of alginates requires availability of recombinantly produced alginate epimerases, and for this purpose the methylotrophic yeast is an attractive host organism. The present study investigates whether is a suitable expression system for future large-scale production of AlgE1, AlgE4, and AlgE6. expression strains were constructed using synthetic genes with reduced repetitive sequences as well as optimized codon usage. High cell density cultivations revealed that the largest epimerases AlgE1 (147 kDa) and AlgE6 (90 kDa) are subject to proteolytic degradation by proteases secreted by the yeast cells. However, degradation could be controlled to a large extent either by co-expression of chaperones or by adjusting cultivation conditions. The smaller AlgE4 (58 kDa) was stable under all tested conditions. The results obtained thus point toward a future potential for using in industrial production of mannuronan C-5 epimerases for tailoring of alginates.
藻酸盐是由褐藻和一些细菌产生的线性多糖，由β-D-甘露糖醛酸（M）和α-L-古洛糖醛酸（G）组成。藻酸盐在工业、医学和制药领域有众多当前和潜在的未来应用，富含G的藻酸盐由于其胶凝和增粘特性，传统上最有价值且使用频繁。甘露糖醛酸C-5差向异构酶是在藻酸盐生物合成过程中在聚合物水平将M转化为G的酶。差向异构酶AlgE1 - AlgE7具有共同结构，包含一个或两个催化A模块（A）和一到七个调节R模块（R）。尽管差向异构酶结构相似，但它们在藻酸盐中产生不同的M - G模式；AlgE4（AR）产生严格交替的MG结构，而AlgE1（ARRRAR）和AlgE6（ARRR）主要产生G块。因此，这些酶是生产定制藻酸盐的有前途的工具。藻酸盐的高效差向异构化需要重组产生的藻酸盐差向异构酶，为此，甲基营养酵母是一种有吸引力的宿主生物。本研究调查了[具体酵母名称]是否是未来大规模生产AlgE1、AlgE4和AlgE6的合适表达系统。使用具有减少重复序列以及优化密码子使用的合成基因构建了[具体酵母名称]表达菌株。高细胞密度培养表明，最大的差向异构酶AlgE1（147 kDa）和AlgE6（90 kDa）会被酵母细胞分泌的蛋白酶进行蛋白水解降解。然而，通过共表达伴侣蛋白或调整培养条件，降解在很大程度上可以得到控制。较小的AlgE4（58 kDa）在所有测试条件下都稳定。因此，所获得的结果表明[具体酵母名称]在工业生产用于定制藻酸盐的甘露糖醛酸C-5差向异构酶方面具有未来潜力。
