Detection of Penicillin G Produced by Penicillium Chrysogenum KF 425 In
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bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984930; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Detection of Penicillin G Produced by Penicillium chrysogenum KF 425 in Vivo with Raman Microspectroscopy and Multivariate Curve Resolution-Alternating Least Squares Methods Shumpei Horii†,‡, Masahiro Ando§,⊥, Ashok Z. Samuel§, Akira Take║, §,, Takuji Nakashima║, Atsuko Matsumoto║, Yōko Takahashi║, and Haruko Takeyama‡ §,∇,O,* †Department of Advanced Science Engineering , and OConsolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169- 8555, Japan ‡Computational Bio Big-Data Open Innovation Laboratory, AIST-Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan §Research Organization for Nano & Life Innovation,Waseda University, 513 Wasedatsurumaki- cho, Shinjuku-ku, Tokyo 162-0041, Japan ⊥PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332- 0012, Japan ║ Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984930; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 108-8641, Japan ∇Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984930; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT Raman microspectroscopy is a minimally invasive technique that can identify molecular structure without labeling. In this study, we demonstrate in vivo detection of the bioactive compound penicillin G inside Penicillium chrysogenum KF425 fungus cells. Highly overlapped spectroscopic signatures acquired using Raman microspectroscopic imaging are analyzed using a multivariate curve resolution-alternating least squares (MCR-ALS) method to extract the pure spectra of individual molecular constituents. In addition to detecting multiple constituents such as proteins and lipids, we observe the subcellular localization of penicillin G like granule particle inside the fungus body. To date, there have been no reports of direct visualization of intracellular localization of penicillin G. The methodology we present in this article is expected to be applied as a screening tool for the production of bioactive compounds by microorganisms. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984930; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Antibiotics are indispensable for modern medicine in the treatment of diseases caused by pathogenic bacteria.1 In addition, these drugs have been widely used as antifungal, antiviral, and anticancer agents in humans.2,3 They are often discovered from secondary metabolites produced by microorganisms which are treasure box for antibiotics because they survive adaptively to a wide variety of environments.4 To date, 22,500 antibiotics with various pharmacological effects have been identified from microorganisms.5 A representative antibiotic is penicillin, which inhibits bacterial cell-wall synthesis. Penicillin was discovered from the culture broth of Penicillium chrysogenum by Alexander Fleming in 1928. Ernst Chain and co-workers begun to purify and study the chemistry of penicillin in 1939. Because of the poor penicillin productivity of the mold strain, 500 L of the mold filtrate per a week were necessary for a program of animal experiments and clinical trials of penicillin. This Penicillium strain, however, produced only traces of penicillin when grown in submerged culture. Therefore, various Penicillium strains were screened in a global search, and better penicillin-producing strains were obtained. Furthermore, it was found that upon exposing the culture of P. chrysogenum to X-rays and ultraviolet radiation, its productivity was increased still further.6 This strain was obtained thanks to a great amount of effort: the isolation of many wild Penicillium strains was required, as well as the cell culture to produce penicillin and the extraction of the penicillin, and bioassays to verify the effectiveness of the extracted compound had to be carried out. In 1942, over 10 years after its discovery, penicillin was ready for practical use. If it had been possible to detect a high- producing strain instantly, from its mold colony, without extraction and bioassay, penicillin may have been developed earlier, saving more human lives. Raman spectroscopy can be applied to the in vivo molecular analysis of biological samples. It provides characteristic information on the molecular structure of metabolites and does not 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984930; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. require any sample pretreatment such as dye labeling or genetic manipulation. In addition, it allows rapid and minimally invasive observation, and can be used for detection of the proteins and lipids constituting living cells.7,8 Moreover, Raman microspectroscopy, the combination of Raman spectroscopy with optical microscopy, allows the creation of high-spatial-resolution (~300 nm), molecular distribution images at the single-cell level.9 Therefore, our research group has been investigating the application of Raman spectroscopy as a primary screening method in the search for new antibiotics, for the rapid and facile execution of investigative steps, up to and including the identification of new antibiotics. We reported the successful Raman imaging of Streptomyces species using confocal Raman microspectroscopy.10 The Raman spectroscopic images of these bacteria reveal clear and distinct spatial distributions of proteins, lipids, cytochromes, and so on. At the same time, we successfully visualized the distribution of amphotericin B (Amph-B) as a secondary metabolite. Thus, we were able to confirm that Amph- B is locally accumulated in mycelia. However, when Raman imaging technology is applied practically to various types of microorganisms, a difficulty is encountered. Raman spectra obtained from biological systems are typically superpositions of spectra of multiple molecular species. The Raman spectra are derived from multiple constituent biomolecules, and background spectra originating from substrates and autofluorescence also contribute. If Raman bands of the specific molecule of interest do not overlap with spectra originating from other constituent molecules, it is straightforward to identify the existence of the target molecule, as already demonstrated.10 In contrast, if Raman bands of the target molecule overlap with those of other cellular components, which is more commonly the case, the analysis becomes much more difficult. Specifically, the overlap hinders the accurate estimation of Raman scattering intensities that originate purely from the target molecule. 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984930; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Therefore, a suitable analytical method is required for the separation of the spectral components of interest from those of the other cellular constituents. Hence, to overcome this problem, we have applied a multivariate curve resolution-alternating least squares (MCR-ALS) method to decompose complicated Raman spectra into spectral components that are assignable to specific molecules.11 In the MCR method, the measured data is approximated as a linear combination of several spectral components. The decomposition of the spectral data is carried out via an ALS calculation. As this calculation is carried out under the constraint of all the spectral profiles and intensities being non-negative, physically and chemically interpretable solutions are preferably obtained.11 It should be emphasized that, rather than focusing on a few Raman bands, MCR-ALS analysis can extract components as patterns spanning the entire spectrum, so that even if some bands overlap with those of other molecular components, the spectral contribution of a particular molecule can be accurately determined. To date, MCR-ALS-based methods have been applied to the analysis of various biomaterials, as well as for the successful analysis of ingredients in pharmaceutical tablets, for the detection of marker molecules for cancer diagnosis, and for the imaging of multiple molecular components during cell division.12–15 In this article, we show that MCR-ALS analysis of Raman microspectroscopy results allows the detection and visualization of the production of spectroscopically overlapping secondary metabolites. A study on Penicillium chrysogenum is reported, and Raman mapping measurements of the fungal cells are presented. P. ch ry s og enu