In this work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes.. Aliivibrio wodanis as production host: development of genetic tools and expression of cold-active enzymes

Background: Heterologous expression of cold-adapted proteins represent today one of the biggest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from cold environment, e.g., the cold oceans that represents largely untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes typically form inactive aggregates. It is therefore a need to develop new cold-temperature expression systems, including identifying new host organisms and genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanctis, Schewanella and Rhodococcus erythropolis have all been explored for such use, but none of them are commercially available, or are available for common use as efficient expression systems. In this work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes.Results: We have tested twelve bacterial strains, available vectors and promoters, reporter systems, used RNA-sequencing to identify the most highly expressed genes in A. wodanis (and thus their intrinsic promoters), explored a novel 5’-fusion to stimulate protein expression and solubility, and finally tested expression of a set of “difficult-to-express” enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be expressed in soluble and active form, also when expression fails in E. coli due to formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5’-end of the Awod_I1781 gene that stimulates expression of the Green Fluorescent Protein and improves expression of cold-active enzymes when used as a 5’-fusion. Conclusions: Our results support that A. wodanis and associated genetic tools are well suited for low-temperature expression, and that A. wodanis represent an interesting platform for further developments of an expression systems that can promote further cold-enzyme discoveries.

背景：冷适应蛋白的异源表达，是当前从寒冷环境（如尚未得到充分开发的寒冷海洋）中挖掘新型酶类的生物勘探工作面临的核心瓶颈之一。在嗜温表达宿主如大肠杆菌（Escherichia coli）中，冷适应酶通常会形成无活性的聚集物。因此，亟需开发新型低温表达系统，包括筛选新型宿主生物与配套遗传工具。此前已有研究尝试将嗜冷细菌——包括海假交替单胞菌（Pseudoalteromonas haloplanctis）、希瓦氏菌（Schewanella）以及红球菌（Rhodococcus erythropolis）——作为此类表达宿主，但相关菌株均未实现商业化，亦未作为高效表达系统得到广泛应用。本研究探索了亚北极细菌沃氏弧菌（Aliivibrio wodanis）作为冷活性酶异源表达潜在宿主的可行性。
结果：本研究测试了12株细菌菌株、商用载体与启动子，并采用了报告基因系统；通过RNA测序（RNA-sequencing）鉴定了沃氏弧菌中表达量最高的基因及其天然启动子；开发了一种新型5'端融合策略以促进蛋白表达与可溶性表达；最终测试了一组源自多种细菌及1种古菌的“难表达”酶的表达情况。结果表明，即便在大肠杆菌中因包涵体形成而无法正常表达的冷适应酶，也可在本系统中以可溶性活性形式获得表达。此外，我们鉴定出一段来自Awod_I1781基因5'端的60 bp/20个氨基酸的片段，将其作为5'融合标签时，可增强绿色荧光蛋白（Green Fluorescent Protein）的表达，并提升冷适应酶的表达效果。
结论：本研究结果证实，沃氏弧菌及其配套遗传工具非常适用于低温表达体系，其有望成为进一步开发助力低温酶挖掘的表达系统的优质平台。