Although pepticinnamin E has been discovered 30 years ago, the pcm BGC has only been described recently in two different producers, S. mirablilis OK006 and Streptomyces sp. PKU-MA01144, in 2019 and 2020, respectively, through key gene disruption and heterologous expression. The two microorganisms while encoding closely similar BGC, produces new types of pepticinnamins, pepticinnamins G-I, with difference in chirality of first NRPS amino acid, L-instead of D-tyrosine, and N-methyl-L-alanine instead of N-methyl-L-phenylalanine. In 2019, Santa Maria et al. took advantage of the presence of the N-terminal cinnamoyl moiety of pepticinnamin E in other natural products with characterized BGCs, such as skyllamycin A. By using Sky28, an enzyme responsible for the formation of the benzene ring in the cinnamoyl moiety of skyllamycin A as a biosynthetic "probe", the pepticinnamin E biosynthetic gene cluster (pcm BGC) was discovered in S. mirabilis OK006 with estimated size of 45 kbp. The pcm biosynthetic gene cluster consisted of two NRPSs, Pcm2 and Pcm30. The entire NRPS consisted of five modules, which is consistent with the uptake of five amino acid residues composing pepticinnamin E. The modular architecture of NRPS domains suggested that the biosynthesis uses L-amino acids, however this was not the case for pepticinnamin E, as they were composed of D-tyrosine and D-serine. In the study by Ge et al. in 2020, the new pepticinnamin analogues, pepticinnamins G-M, were discovered and isolated from the marine Streptomyces sp. PKU-MA01144. They confirmed the biosynthetic gene cluster responsible for production of these pepticinnamins, pep, through heterologous cloning, knock out studies and feeding experiments. There are two important differences between the two BGCs. Firstly, the pepticinnamins encoded by pep BGC contained an L-tyrosine residue compared to a D-tyrosine in pcm BGC. Secondly, the two strains are significantly different, sharing whole genome average nucleotide identity of only 81 %. The pepticinnamin BGC from S. mirabilis P8-A2 is close to identical to the pcm BGC of S. mirabilis OK006, share 99.1% nucleotide identity and average protein identity of 98%. As expected, the S. sp. PKU-MA01144 pep BGC is significantly different, sharing 64.3% nucleotide identity and protein identities between 45% and 91%. The differences between pcm and pep BGC likely explain the differences in chirality of two BGC products. We performed BGC alignment to highlight similarities in the architecture and sequence similarity between the three BGCs using clinker and antiSMASH (Figure ). The pcm BGC was earlier analyzed by Santa Maria et al. for presence of NRPS domains that might accept D-tyrosine or have epimerase activity, however bioinformatic analysis indicated L-tyrosine. We came to the same conclusion after comparative genomics and analysis of NRPS domains using the latest antiSMASH 7.0 HMMER models, all indicating that L-amino acid is used. The Dtyrosine and D-serine in pepticinnamin E biosynthesis remains a mystery. Within the NRPS, there are two N-methyltransferases domains which are functional as we detect methylation of tyrosine (M2) and phenylalanine (M3). Other modifications are performed by the halogenase, Pep1 18 , the methyltransferase, Pep9 18 and oxygenase, Pep10 which have previously been characterized through knock out studies in S. sp. PKU-MA01144 and in vitro feeding experiments. These genes are shared across all three pcm/pep BGCs. The polyketide/fatty acid sidechain in the pepticinnamin biosynthesis is likely synthesized by eight genes encoding acyl carrier and/or beta-ketoacyl proteins, a 3-oxoacyl-ACP reductase and a beta-hydroxyacyl-ACP dehydratase based on the protein function predictions, however there is no experimental evidence of which specific genes are involved. Pepticinnamins O and N with a shorter peptide chain are likely shunt products derived from pepticinamin biosynthesis. Pepticinnamin E and its analogues were detected in four fractions (1-4). Fraction 1 was further fractionated using a linear gradient from 50% to 80% MeCN in Milli-Q water over 20 min by an Agilent Infinity 1290 HPLC-DAD (Agilent Technologies) system, with a flow rate of 4 mL/min, UV monitoring at 220, 254, and 282 nm, and a column temperature at 40 °C using a Luna ® 5 µm Phenyl-Hexyl 100 Å, LC Column (250 x 10 mm) to yield two subfractions (subfraction 1A and 1B corresponding to pepticinnamin N and O respectively). Fraction 2 was further fractionated using a linear gradient from 65% to 80% MeCN in Milli-Q water over 20 min to yield three subfractions (subfraction 2A, 2B and 2C corresponding to pepticinnamin N, E and O respectively). Fraction 3 was further fractionated using a linear gradient from 60% to 80% MeCN in Milli-Q water over 20 min to yield two subfractions (subfraction 3A and 3B corresponding to pepticinnamin O and P respectively). Fraction 4 was further fractionated using a linear gradient from 60% to 80% MeCN in Milli-Q water over 20 min to yield one subfraction (subfraction 4A corresponding to pepticinnamin P). Subfraction 1A and 2A were mixed to yield pepticinnamin N (2, 2 mg). Subfraction 1B and 2B were mixed to yield of pepticinnamin E (1, 5 mg). Subfraction 2C and 3A were mixed to yield pepticinnamin O (3, 2.5 mg), and finally subfraction 3B and 4A were mixed to yield pepticinnamin P (4, 3 mg).