Institute for Biology and Biotechnology of Plants, University of Münster, Schlossplatz 8, 48143, Münster, Germany.
Institute for Molecular Microbiology and Biotechnology, University of Münster, Corrensstr. 3, 48149, Münster, Germany.
Appl Microbiol Biotechnol. 2021 Feb;105(4):1547-1561. doi: 10.1007/s00253-021-11112-5. Epub 2021 Feb 1.
Chitin is an abundant waste product from shrimp and mushroom industries and as such, an appropriate secondary feedstock for biotechnological processes. However, chitin is a crystalline substrate embedded in complex biological matrices, and, therefore, difficult to utilize, requiring an equally complex chitinolytic machinery. Following a bottom-up approach, we here describe the step-wise development of a mutualistic, non-competitive consortium in which a lysine-auxotrophic Escherichia coli substrate converter cleaves the chitin monomer N-acetylglucosamine (GlcNAc) into glucosamine (GlcN) and acetate, but uses only acetate while leaving GlcN for growth of the lysine-secreting Corynebacterium glutamicum producer strain. We first engineered the substrate converter strain for growth on acetate but not GlcN, and the producer strain for growth on GlcN but not acetate. Growth of the two strains in co-culture in the presence of a mixture of GlcN and acetate was stabilized through lysine cross-feeding. Addition of recombinant chitinase to cleave chitin into GlcNAc, chitin deacetylase to convert GlcNAc into GlcN and acetate, and glucosaminidase to cleave GlcN into GlcN supported growth of the two strains in co-culture in the presence of colloidal chitin as sole carbon source. Substrate converter strains secreting a chitinase or a β-1,4-glucosaminidase degraded chitin to GlcNAc or GlcN to GlcN, respectively, but required glucose for growth. In contrast, by cleaving GlcNAc into GlcN and acetate, a chitin deacetylase-expressing substrate converter enabled growth of the producer strain in co-culture with GlcNAc as sole carbon source, providing proof-of-principle for a fully integrated co-culture for the biotechnological utilization of chitin. Key Points• A bacterial consortium was developed to use chitin as feedstock for the bioeconomy.• Substrate converter and producer strain use different chitin hydrolysis products.• Substrate converter and producer strain are mutually dependent on each other.
壳聚糖是虾和蘑菇产业的丰富废弃物，因此是生物技术过程的合适的二次原料。然而，壳聚糖是一种嵌入在复杂生物基质中的结晶基质，因此难以利用，需要同样复杂的壳聚糖酶机制。我们采用自下而上的方法，在这里描述了一个互利的、非竞争性的联合体的逐步发展，其中一个赖氨酸缺陷型大肠杆菌底物转化器将壳聚糖单体 N-乙酰葡萄糖胺（GlcNAc）裂解为葡萄糖胺（GlcN）和醋酸盐，但只使用醋酸盐，而将 GlcN 留给赖氨酸分泌的谷氨酸棒杆菌生产菌株生长。我们首先设计了底物转化器菌株以在醋酸盐上生长而不是 GlcN 上，以及生产菌株以在 GlcN 上而不是醋酸盐上生长。通过赖氨酸交叉喂养稳定了两种菌株在含有 GlcN 和醋酸盐混合物的共培养物中的生长。添加重组壳聚糖酶将壳聚糖裂解为 GlcNAc，壳聚糖脱乙酰酶将 GlcNAc 转化为 GlcN 和醋酸盐，以及葡萄糖胺酶将 GlcN 裂解为 GlcN，支持了两种菌株在胶体壳聚糖作为唯一碳源的共培养物中的生长。分泌壳聚糖酶或β-1,4-葡萄糖胺酶的底物转化器菌株将壳聚糖降解为 GlcNAc 或 GlcN 分别为 GlcN，但需要葡萄糖生长。相比之下，通过将 GlcNAc 裂解为 GlcN 和醋酸盐，表达壳聚糖脱乙酰酶的底物转化器使生产菌株能够在 GlcNAc 作为唯一碳源的共培养物中生长，为壳聚糖的生物技术利用提供了一个完全集成的共培养物的原理证明。要点•开发了一种细菌联合体，以将壳聚糖用作生物经济的原料。•底物转化器和生产菌株使用不同的壳聚糖水解产物。•底物转化器和生产菌株相互依赖。
