高通透红色红球菌细胞封皮催化剂创制及其性能探究

生物催化剂工业化开发是我国生物制造产业重大需求。微生物全细胞催化剂 “最有效、最稳定、最方便应用”，但受细胞膜包被限制，通常不能用于大分子底物。基于红色红球菌特有的高强度细胞封皮结构、高有机溶剂耐受性且可使用廉价尿素高效诱导表达目标酶特点，提出通过噬菌体穴蛋白基因可控表达实现细胞膜按需穿孔、创制高通透细胞封皮型红色红球菌细胞催化剂新思路，突破狭窄底物谱制约。经λ噬菌体、分枝杆菌和红球菌噬菌体穴蛋白基因比选，以四环素诱导、升温诱导及细胞群体感应反馈调节为对照，重点采用磷酸盐饥饿诱导新方法实现优选穴蛋白基因在发酵末期可控表达，成功创制高通透细胞封皮。设计荧光标记寡肽库，研究高通透细胞封皮的底物谱规律；以转氨酶、醛缩酶、CPC酰化酶等为模式酶，揭示实际底物催化转化规律。进一步探究细胞封皮内双酶协同催化合成医药大分子适用性规律。发表SCI论文4-7篇，申请专利3-6项，实现基础研究成果转化。

Biocatalyst industrialization development is the major demand of the industrial bio-manufacturing. “The most effective, stable and convenient form for the biocatalyst is a whole organism”, but it is always limited by the encapsulation of cell membrane, therefore can’t be used for the system with large substrate molecules. Based on a series of unique properties of Rhodococcus ruber, e.g. high intensity cell-envelope structure, high organic solvent tolerance and overexpression of target enzyme with cheap urea as inducer, we propose here a novel idea to creatively construct a special R. ruber cell-envelope biocatalyst with high permeability through holin effect, thereby breakthrough the constraints of the limited substrate spectrum. Different holin genes from phage λ, phage mycobacteria and phage Rhodococcus will be designed and synthesized according to the R. ruber codon preference, and the optimal one will be used for the subsequent inducible expression with phosphate salt starvation induction, tetracycline induction, temperature-shift induction, and also Quorum Sensing (QS) regulation. The rare phosphate salt starvation induction is particularly highlighted as the most preferred approach with high industrial feasibility to efficiently initiate the holin expression in the late stage of fermentation and then generate the expected cell membrane holes. For the harvested high substrate-permeable R. ruber cell-envelope type biocatalyst, a library containing fluorescence labeling oligo-peptides with 2-6 amino acids will be designed and synthesized, and used for visualization investigation on the changes of the substrate spectrum. Selecting transaminase, aldolase and cephalosporin C acylase, etc. as the model enzymes, the catalysis and biotransformation rules of the single enzyme in the novel R. ruber cell-envelope will be characterized. At last, an intracellular dual-enzyme concerted-catalysis system, also in the type of high permeable R. ruber cell-envelope, is further proposed to produce high-value medicine intermediates, expanding the application fileds of the novel method developed in this study.