The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past 5 years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes. Here, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made β-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana. The results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as β-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins.

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植物半纤维素的模块化生物合成及其对酵母细胞的影响

几个世纪以来，包裹植物细胞的碳水化合物聚合物造福于人类，并具有宝贵的生物技术用途。在过去的 5 年中，基于多糖的生物材料工程出现了令人兴奋的可能性。尽管基于细菌纤维素的水凝胶取得了令人瞩目的进展，但关于如何在适合生物技术目的的细胞中重组和调节植物半纤维素的知识相对较少。在这里，我们使用优化的毕赤酵母平台组装纤维素合酶样 A (CSLA) 酶，以在酵母中生产可调节的异甘露聚糖 (HM) 多糖。通过交换植物甘露聚糖和葡甘露聚糖合酶的结构域，我们设计了嵌合 CSLA 蛋白，使 β-1,4 连接的甘露聚糖的数量超过了天然酶的数量，同时最大限度地减少了酵母生长的负担。魔芋葡甘露聚糖合酶的长期表达对酵母细胞有毒：减少生物量积累并最终导致细胞活力受损。然而，工程化葡甘露聚糖合酶以及 CSLA 纯甘露聚糖合酶和 CSLC 葡聚糖合酶不抑制生长。有趣的是，根据 CSLA 蛋白质序列的组成，毕赤酵母细胞的大小可以增加或减少。通过将嵌合 CSLA 蛋白与来自拟南芥的甘露聚糖合成相关 (MSR) 辅因子共表达，可以进一步提高 HM 产量和葡萄糖掺入。结果为可持续生物经济所需的多糖生物材料工程提供了新途径。酵母中嵌合纤维素合酶样酶的表征为以可调方式生产植物多糖提供了一条令人兴奋的途径。此外，用无毒植物多糖（如 β-甘露聚糖）修饰的细胞提供了模块化底盘来生产和封装敏感货物，如治疗性蛋白质。