The tyrocidines and analogues are cationic cyclodecapeptides [cyclo (D-Phe¹-L-Pro²-L-(Phe³/Trp³)-D-(Phe⁴/Trp⁴)-L-Asn⁵-L-Gln⁶-L-(Tyr⁷/Phe⁷/Trp⁷)-L-Val⁸-L-(Orn⁹/Lys⁹)-L-Leu¹⁰], produced together with the neutral linear pentadecapeptide gramicidins, in the antibiotic tyrothricin complex by Brevibacillus parabrevis. Despite discovery 80 years ago, it was still uncertain whether these peptides are secreted or sequestered intracellularly. We resolved this by utilising high resolution electrospray mass spectrometry to confirm the predominantly intracellular sequestration of the peptides in the tyrothricin complex. A “peptidomics” approach allowed us to map the intracellular production of 16 cyclodecapeptides and 6 gramicidins over 16 days of culturing. Gramicidin production remained relatively constant, with Val-gramicidin A the predominant analogue produced throughout the 16 day fermentation period. The tyrothricin cyclodecapeptides have four variable positions and there was a culturing time related shift from the Phe-rich A analogues, containing a L-Phe³-D-Phe⁴ aromatic dipeptide unit, to the Trp-rich C analogues with L-Trp³-D-Trp⁴. For the other variable aromatic residue position, Tyr⁷ was preferentially incorporated above Trp⁷, with a minor incorporation of Phe⁷ over the whole culturing period. For the variable basic amino acid residue, there was time-sensitive shift from Orn⁹ to Lys⁹ incorporation. Modulation of the cyclodecapeptide profile over time does not correlate with the reported non-ribosomal peptide synthetase affinity, specifically for Trp in the variable aromatic residue positions, indicating additional supply-demand control in the cyclodecapeptides production by B. parabrevis. These novel observations are not only of importance for production and purification of selected peptide analogues from the tyrothricin complex, but also for insight into microbial control of non-ribosomal peptide production that extends beyond the peptide synthetase machinery.

的短杆菌酪肽和类似物是阳离子cyclodecapeptides [环（d-PHE ¹ -L-Pro的² -L-丙氨酸（Phe ³ /色氨酸³）-D-丙氨酸（Phe ⁴ /色氨酸⁴）-L-天冬酰胺⁵ -L-GLN ⁶ - L-（Tyr ⁷ / Phe ⁷ / Trp ⁷）-L-Val ⁸ -L-（Orn ⁹ / Lys ⁹）-L-Leu ¹⁰ ]，与中性线性五肽葡糖苷配基一起，由短杆菌属在酪氨酸酪氨酸复合物中产生短伞。尽管在80年前被发现，但仍不确定这些肽是在细胞内分泌还是被隔离。我们通过利用高分辨率电喷雾质谱法确认酪氨酸蛋白复合物中肽的主要细胞内螯合解决了这一问题。“肽组学”方法使我们能够在培养的16天中绘制出16种环十肽和6种短杆菌肽的细胞内产量图。格拉米霉素的产量保持相对稳定，Val-gramicidin A是整个16天发酵期生产的主要类似物。酪氨酸环十二肽具有四个可变位置，并且与含有L-Phe ³ -D-Phe ⁴的富Phe A类似物存在培养时间相关的变化。芳香族二肽单元，与L-Trp ³ -D-Trp ⁴结合为富含Trp的C类似物。对于其他可变的芳族残基位置，在整个培养期间，优先将Tyr ⁷掺入Trp ⁷之上，并少量掺入Phe ⁷。对于可变的碱性氨基酸残基，存在从Orn ⁹到Lys ⁹掺入的时间敏感性转变。随时间变化的环十肽谱的调节与所报道的非核糖体肽合成酶的亲和力不相关，特别是对于可变芳香族残基位置上的Trp，表明副短双歧杆菌生产环十肽的额外供需控制。这些新颖的观察结果不仅对于从酪氨酸复合物生产和纯化所选肽类似物具有重要意义，而且对于深入了解超出肽合成酶机制的非核糖体肽生产的微生物控制具有重要意义。