A Cell‐Free Platform Based on Nisin Biosynthesis for Discovering Novel Lanthipeptides and Guiding their Overproduction In Vivo

Abstract Lanthipeptides have extensive therapeutic and industrial applications. However, because many are bactericidal, traditional in vivo platforms are limited in their capacity to discover and mass produce novel lanthipeptides as bacterial organisms are often critical components in these systems. Herein, the development of a cell‐free protein synthesis (CFPS) platform that enables rapid genome mining, screening, and guided overproduction of lanthipeptides in vivo is described. For proof‐of‐concept studies, a type I lanthipeptide, nisin, is selected. Four novel lanthipeptides with antibacterial activity are identified among all nisin analogs in the National Center for Biotechnology Information (NCBI) database in a single day. Further, the CFPS platform is coupled with a screening assay for anti‐gram‐negative bacteria growth, resulting in the identification of a potent nisin mutant, M5. The titers of nisin and the nisin analog are found to be improved with CFPS platform guidance. Owing to the similarities in biosynthesis, the CFPS platform is broadly applicable to other lanthipeptides, thereby providing a universal method for lanthipeptide discovery and overproduction.

The desired factions were then collected.

LC-MS Analysis of Nisin Z and Targeted Proteomics Analysis:
The original strain, J1-004, and the engineered L. lactis cells collected from the fermentation broth were analyzed by LC-MS for nisin Z and by a targeted proteomics approach according to a previously reported method [2] . Briefly, L. lactis cells collected from the fermentation broth were pelleted by centrifugation 8,000 × g for 10 min at 4°C, and washed thrice with wash buffer (100 mmol L -1 NaCl, 25 mmol L -1 Tris−HCl, pH 7.5). The per 10 mL of buffer. The suspended sample was vortexed for 30 s twice and disrupted by sonication [3] . The supernatant from the lysed cells was collected by centrifugation (13,000 × g for 45 min at 4°C). Proteins from the cell lysates were measured using a noninterference protein assay kit (Sangon Biotech, Shanghai, China) and adjusted to 2 μg/μL using lysis buffer. First, 50 μL of the supernatant (100 μg of total protein) was mixed with an equal volume of 100 mM ammonium bicarbonate buffer (pH 8.0). Next, the sample was reduced at 30°C for 1 h by the addition of 3 mmol L -1 tris (2carboxyethyl)-phosphine (TCEP) and alkylated by the addition of 15 mmol L -1 iodoacetamide (IAA).
The samples were incubated in dark conditions at 30°C for an additional 1 h. The sample was diluted with ammonium bicarbonate buffer to reduce the urea concentration to 1 mol L -1 . Trypsin was added to the mixture (trypsin/total protein 1:50, w/w) and incubated at 37°C for 14 h. The detergent and salt in the digested peptide sample were removed by passage through a Pierce Detergent Removal Spin Column (Thermo Fisher Scientific) and a SepPak C18 cartridge (Waters Corp.), respectively.
The purified peptides were freeze-dried and stored at -80°C for subsequent liquid chromatographytandem mass spectrometry (LC-MS-MS) analysis.
The peptide samples were analyzed using a hybrid quadrupole-time-of-flight (TOF) liquid chromatography (LC) tandem mass (MS/MS) spectrometer (TripleTOF 5600+, AB Sciex, Foster City, CA, USA) equipped with a nanospray ion source. Peptides were first loaded onto a C18 trap column (5 µm, 5 × 0.3 mm; Agilent Technologies, Santa Clara, CA, USA) and then eluted into a C18 analytical column (75 μm × 150 mm, 3 μm particle size, 100 Å pore size; Eksigent, Dublin, CA, USA). Mobile phase A (3% DMSO, 97% H2O, 0.1% formic acid) and mobile phase B (3% DMSO, 97% ACN, 0.1% formic acid) were used to establish a 100-min gradient as follows: 0 min of 5% B, 65 min of 5-23% B, 20 min of 23-52% B, 1 min of 52-80% B, maintenance at 80% B for 4 min, 0.1 min of 80-5% B, and a final step of 5% B for 10 min. A constant flow rate was set at 300 nL/min. MS scans were conducted from 350 amu-1500 amu, with a 250-ms time span. For the MS/MS analysis, each scan cycle consisted of one full-scan mass spectrum (with m/z ranging from 350-1500 and charge states from 2-5) followed by 40 MS/MS events. The threshold count was set to 120 to activate MS/MS accumulation and former target ion exclusion was set to 18 s. Raw data obtained by TripleTOF 5600+ were analyzed using ProteinPilot 5.0 (AB SCIEX) against the designated proteome database.  Biotechrabbit, Berlin, Germany). 100 ng of pJL1-sfGFP was added to 40 μL of the cell-free system and the commercial cell-free synthesis kit, respectively. The cell-free reaction was conducted according to our described method or kit instructions, respectively. The calculation of cost was based on of price for each raw material or kit in this study. After incubation for 6 h, the titer of sfGFP produced by our cell-free system was 64.97 mg L -1 , and that produced by the commercial kit was 802.34 mg L -1 . The commercial cell-free synthesis kit has been demonstrated to result in very high productivity; therefore, the 12-fold lower titer of sfGFP in our system indicated the acceptable efficiency of our system. Moreover, since our cell-free system is about 50-fold cheaper than the commercial kit, a scale-up study is feasible. Overall, this comparison demonstrated that our system can meet the needs of subsequent research.  pET28a-nisB, pET28a-nisC, and pET28a-nisP. Cell-free nisin Z: pJL1-nisZ (0.13 nmol L -1 ), NisB (800 nmol L -1 ), NisC (500 nmol L -1 ), and pET28-nisP (0.1 nmol L -1 ) were added to the cell-free system. After 6 h of incubation, 2 μL of the reaction mixture was used in a bioassay with M. luteus as the indicator strain. (B) Production of sfGFP in different cell-free systems (n=3). Control: blank cell-free system incubated for 6 h; added pJL1-sfGFP at 6 h: followed by the addition of pJL1-nisZ (0.13 nmol L -1 ), NisB (800 nmol L -1 ), NisC (500 nmol L -1 ), and pET28a-nisP (0.1 nmol L -1 ) and incubation for 6 h, and then 100 ng pJL1-sfGFP was added to the system and incubated for another 6 h. added pJL1-sfGFP at 0 h: 100 ng of pJL1-sfGFP was added into cell-free system at 0 h and incubated for 6 h. The data represented mean ± SD.     Purified modified precursor peptides were digested with trypsin to form mature RL6, RL8, RL13, RL14, M4, M5, and S29A and treated with NEM. Commercialized nisin A and nisin Z were also treated with NEM. The thioether crosslinks are formed via a Michael-type addition by reducing cysteine thiols (-SH) in Cys residues to dehydro-amino acids. This step does not cause any change in molecular weight between substrates and products. However, if there is a Cys in the protein that does not form a thioether crosslink, there will be a complete -SH on the peptide. If the -SH undergoes an alkylation reaction with NEM, the product will have a larger molecular mass (125 Da) than the substrate. The eight-fold dehydrated core peptides are highlighted in yellow. Theoretical alkylated core peptides with 1-5 NEM adducts are highlighted in red. The results showed all five thioester rings were formed in mature lanthipeptides.

Figure S8. OD600 of E. coli DH5α in LB Medium with Different Concentrations of EDTA and
Nisin after 18 h of Co-culture (n=3) ΔOD600 indicates the difference between readings with different concentrations of blank media. Previous studies have reported that nisin has a significant inhibitory effect on E. coli [4] , however, uncommon E. coli strains were used and they are available in our laboratory. In the reported 20-fold nisin concentration condition, DH5α did not exhibit complete inhibition. To enable comparisons with previous studies, EDTA was added to the E. coli culture to increase the sensitivity of E. coli to nisin. After 18 h, E. coli DH5α growth was only slightly inhibited following addition of 320 μM EDTA, however, the inhibitory effects of various concentrations of nisin on E. coli were obvious. The data represented mean ± SD.   Note: * The test lanthipeptide is a mixture of different dehydration molecules. Quantification of lanthipeptides was performed using the eight-fold dehydrated molecules (n=3). The data represented mean ± SD.
Primers of plasmids for mLanA overexpression in E. coli Note: For plasmid construction via restriction enzyme digestion and ligation, complementary sequences were designed using the Primer Premier 5 software. Suitable restriction sites and protective bases were introduced. For plasmid construction via the Gibson cloning method, complementary sequences were designed using the Primer Premier 5 software and were flanked by the homologous sequence. The restriction sites used for cloning are underlined.