Achieving a high product titer through pathway optimization often requires screening many combinations of enzymes and genetic parts. Typically, a library is screened in a single chassis that is a model or production organism. Here, we present a technique where the library is first introduced into B. subtilis XPORT, which has the ability to transfer the DNA to many Gram-positive species using an inducible integrated conjugated element (ICE). This approach is demonstrated using a two-gene pathway that converts tyrosine to melanin, a pigment biopolymer that can serve as a protective coating. A library of 18 pathway variants is conjugated by XPORT into 18 species, including those isolated from soil and industrial contaminants. The resulting 324 strains are screened and the highest titer is 1.2 g/L in B. amyloliquefaciens BT16. The strains were evaluated as co-cultures in an industrial process to make mycelia-grown bulk materials, where the bacteria need to be productive in a stressful, spatially non-uniform and dynamic environment. B. subtilis 3A35 is found to perform well under these conditions and make melanin in the material, which can be seen visually. This approach enables the simultaneous screening of genetic designs and chassis during the build step of metabolic engineering.

通过通路优化实现高产品滴度通常需要筛选酶和遗传部分的多种组合。通常，在作为模型或生产生物体的单个底盘中筛选文库。在这里，我们提出了一种技术，其中首先将库引入枯草芽孢杆菌XPORT，该技术能够使用诱导型集成共轭元件 (ICE) 将 DNA 转移到许多革兰氏阳性物种。使用将酪氨酸转化为黑色素的双基因途径证明了这种方法，黑色素是一种可用作保护涂层的色素生物聚合物。一个包含 18 个途径变体的文库被 XPORT 共轭成 18 个物种，包括从土壤和工业污染物中分离出来的物种。筛选出 324 株菌株，最高滴度为 1.2 g/L解淀粉芽孢杆菌BT16。这些菌株被评估为工业过程中的共培养物，以制造菌丝体生长的散装材料，其中细菌需要在压力大、空间不均匀和动态的环境中生产。B. subtilis 3A35 被发现在这些条件下表现良好，并在材料中产生黑色素，这可以用肉眼看到。这种方法可以在代谢工程的构建步骤中同时筛选基因设计和底盘。