ACIB-Austrian Centre of Industrial Biotechnology, Krenngasse 37, 8010, Graz, Austria.
Institute of Biotechnology and Biochemical Engineering, NAWI Graz, Graz University of Technology, Petersgasse 12, 8010, Graz, Austria.
Microb Cell Fact. 2022 Apr 9;21(1):61. doi: 10.1186/s12934-022-01781-w.
Soluble cello-oligosaccharides (COS, β-1,4-D-gluco-oligosaccharides with degree of polymerization DP 2-6) have been receiving increased attention in different industrial sectors, from food and feed to cosmetics. Development of large-scale COS applications requires cost-effective technologies for their production. Cascade biocatalysis by the three enzymes sucrose-, cellobiose- and cellodextrin phosphorylase is promising because it enables bottom-up synthesis of COS from expedient substrates such as sucrose and glucose. A whole-cell-derived catalyst that incorporates the required enzyme activities from suitable co-expression would represent an important step towards making the cascade reaction fit for production. Multi-enzyme co-expression to reach distinct activity ratios is challenging in general, but it requires special emphasis for the synthesis of COS. Only a finely tuned balance between formation and elongation of the oligosaccharide precursor cellobiose results in the desired COS.
Here, we show the integration of cellodextrin phosphorylase into a cellobiose-producing whole-cell catalyst. We arranged the co-expression cassettes such that their expression levels were upregulated. The most effective strategy involved a custom vector design that placed the coding sequences for cellobiose phosphorylase (CbP), cellodextrin phosphorylase (CdP) and sucrose phosphorylase (ScP) in a tricistron in the given order. The expression of the tricistron was controlled by the strong T7 promoter and strong ribosome binding sites (RBS) for each open reading frame. The resulting whole-cell catalyst achieved a recombinant protein yield of 46% of total intracellular protein in an optimal ScP:CbP:CdP activity ratio of 10:2.9:0.6, yielding an overall activity of 315 U/g dry cell mass. We demonstrated that bioconversion catalyzed by a semi-permeabilized whole-cell catalyst achieved an industrial relevant COS product titer of 125 g/L and a space-time yield of 20 g/L/h. With CbP as the cellobiose providing enzyme, flux into higher oligosaccharides (DP ≥ 6) was prevented and no insoluble products were formed after 6 h of conversion.
A whole-cell catalyst for COS biosynthesis was developed. The coordinated co-expression of the three biosynthesis enzymes balanced the activities of the individual enzymes such that COS production was maximized. With the flux control set to minimize the share of insolubles in the product, the whole-cell synthesis shows a performance with respect to yield, productivity, product concentration and quality that is promising for industrial production.
可溶的纤维二糖寡糖（COS，聚合度 DP 为 2-6 的β-1,4-D-葡糖寡糖）在食品和饲料、化妆品等不同工业领域越来越受到关注。开发大规模 COS 应用需要具有成本效益的生产技术。蔗糖酶、纤维二糖酶和纤维二糖磷酸化酶的级联生物催化具有广阔的前景，因为它可以从蔗糖和葡萄糖等便利的底物出发，自下而上地合成 COS。包含合适共表达所需酶活性的全细胞衍生催化剂将是使级联反应适用于生产的重要一步。一般来说，多酶共表达以达到不同的活性比是具有挑战性的，但对于 COS 的合成需要特别强调。只有精细地平衡寡糖前体纤维二糖的形成和延伸，才能得到所需的 COS。
在这里，我们将纤维二糖磷酸化酶整合到一种生产纤维二糖的全细胞催化剂中。我们设计了共表达盒，使它们的表达水平上调。最有效的策略涉及定制载体设计，将纤维二糖磷酸化酶（CbP）、纤维二糖磷酸化酶（CdP）和蔗糖磷酸化酶（ScP）的编码序列按顺序排列在一个三顺反子中。三顺反子的表达受强 T7 启动子和每个开放阅读框的强核糖体结合位点（RBS）控制。在最佳 ScP：CbP：CdP 活性比为 10:2.9:0.6 时，整个细胞催化剂的重组蛋白产量达到总细胞内蛋白的 46%，总活性为 315 U/g 干细胞质量。我们证明，半透性全细胞催化剂的生物转化可实现 125 g/L 的工业相关 COS 产物浓度和 20 g/L/h 的时空产率。使用 CbP 作为提供细胞二糖的酶，可防止通量进入更高的寡糖（DP≥6），并且在转化 6 小时后不会形成不溶性产物。
开发了用于 COS 生物合成的全细胞催化剂。三种生物合成酶的协调共表达平衡了各酶的活性，从而使 COS 的产量最大化。通过将通量控制设置为将产品中不溶性物质的比例降至最低，整个细胞的合成在产率、生产率、产物浓度和质量方面表现出了有希望用于工业生产的性能。
