利用组合生物合成与体外酶催化的方法对必特螺旋霉素进行糖基化改造

Bitespiramycin (Bitespiramycin, BT), the new macrolide developed by genetic engineering, has been completed Phase III clinical trials, and now applying for a national new drug certificate. The sugar moieties of macrolide antibiotics are closely related to antibacterial activity, and it is an effective way to obtain new drugs by exchanging diverse types of sugar. In order to create novel glycosylated derivatives of BT to deal with bacterial resistance in the future, the forosamine at 9-position of BT was replaced by other sugars. The specific biosynthetic genes and glycosyltransferase (GT) genes responsible for forosamine biosynthesis were firstly identified from BT biosynthesis gene clustered by gene disruption and recovery experiments. The domain-swapped forosamine GT genes or other substrate-broad GT genes were introduced into the forosamine-deficient mutants with expressing various other sugar biosynthetic genes, and identifying the novel glycosylated BT derivatives from the resultant strains. As carbomycin has similar chemical structure with the forosamine-deprived BT, it can also be used as a precursor of glycosylation at 9-position by introducing different GTs in its host strain, or by in vitro enzymatic methods. The all glycosylation BT analogs were determinated for their antimicrobial and antitumor activities to screen out more potent compounds for further study.

必特螺旋霉素（bitespiramycin, BT）是采用基因工程技术研制的新型大环内酯类抗生素，已完成III期临床试验，正在申报新药证书。大环内酯类抗生素的糖基与抗菌活性密切相关，对这些糖基进行改造是获得新型药物的有效途径。为应对将来细菌对BT产生耐药性，拟对BT的9位福洛氨糖进行替换，获得新糖基修饰的BT类似物。通过基因阻断和回复实验鉴定出BT生物合成基因簇中负责福洛氨糖的关键生物合成基因和糖基转移酶(GT)基因；在福洛氨糖生物合成缺陷株中导入改造后的GT基因或对底物具有宽容性的异源GT基因，再导入其它糖基的生物合成基因，鉴定出转化子中产生的新糖基化的BT类似物。carbomycin与脱福洛氨糖的BT结构相似，在其宿主菌中导入不同的GT在9位形成糖基修饰，或者利用体外酶学的方法进行改造来获得新的9位糖基化产物，测定所有新糖基化产物的抗菌和抗肿瘤活性，筛选出活性好的化合物做进一步研究。

必特螺旋霉素（bitespiramycin, BT）现改名为可利霉素，在2019年获得国家一类新药证书，它的主组分包括异戊酰螺旋霉素（isovalerylspiramycin, ISP）Ⅰ、Ⅱ、Ⅲ。本项目主要对BT的C9糖基进行改造以获得新的衍生物。首先通过基因阻断、回复验证和异源表达的方法，鉴定出BT在C9位福洛氨糖（Forosamine）的生物合成基因包括bsm18-20、bsm29、bsm26、bsm21、bsm22基因，特异性糖基转移酶(GT)基因为bsm31及辅助蛋白基因bsm30。在bsm22基因失活变株中鉴定出分子量比螺旋霉素Ⅱ和Ⅲ分别小27的化合物，通过化学结构解析证实是Forosamine上缺少2个甲基的4‴-N,N-demethyl-spiramycin Ⅱ和Ⅲ；在Δbsm22中导入异戊酰基转移酶基因后获得4‴-N,N-demethyl-ISPⅡ和4‴-N,N-demethyl-ISPⅢ。卡波霉素B（Carbomycin B）与9位脱Forosamine的ISPⅡ结构相似，因此在Forosamine生物合成缺陷株或Carbomycin产生菌中导入多种NDP-脱氧糖基生物合成元件和GT元件，从转化子中鉴定出ISPⅡ和C9位为4-酮基-6-脱甲氧基-D-葡萄糖修饰的ISPⅡ。在体外表达和纯化出OleD、DesVII、SpnP、Bsm31/Bsm30共4种GT，以C9是羟基的Midecamycin A1为受体，建立体外糖基化反应体系。实验证实OleD可以利用UDP-葡萄糖对Midecamycin A1、螺旋霉素和BT进行葡萄糖基化。对Midecamycin A1糖基化产物进行结构解析发现OleD可以识别UDP-葡萄糖等5种糖基底物，将这些不同糖基催化至2'-OH上。采用蛋白质工程的方法对OleD进行定点突变来提高OleD利用NDP-糖的能力，筛选出催化UDP-GlcNAc转化率提高了7倍的突变体Q327F。但抗菌活性研究发现这些麦迪霉素的糖苷衍生物都完全丧失了抗菌活性，说明在麦迪霉素2'位的糖基化会导致抗生素的失活，这也是首次在麦迪霉素中发现的糖基化失活机制。BT是一种多组分的创新药物，通过已建立的体内外改造BT糖基的体系可以获得大量BT类似物，为筛选出活性更高的单体化合物奠定基础，更好地应对将来BT长期应用后带来的细菌耐药性问题。