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. 2021 Dec 17;26(24):7649.
doi: 10.3390/molecules26247649.

Accessing Nystatin through Mariculture

James J La Clair  1
Affiliations

Accessing Nystatin through Mariculture

James J La Clair. Molecules. .

Abstract

Understanding our oceans and their marine ecosystems has enabled the development of sustainable systems for mariculture. While the bulk of studies to date have focused on the production of food, its remarkable expanse has inspired the translation of other markets towards aquatic environments. This manuscript outlines an approach to pharmaceutical mariculture, by demonstrating a benchmark for future prototyping. Here, design, field evaluation and natural product chemistry are united to successfully produce nystatin at sea. This study begins by evaluating new designs for culture flasks, illustrating a next step towards developing self-contained bioreactors for culturing in marine environments. Through pilot studies, an underwater system was developed to cost effectively produce cultures that yielded 200 mg of nystatin per deployment. Overall, this study demonstrates the potential for the practical culturing of microbes in a marine environment and provides an important next step for the fledgling field of molecular mariculture.

Keywords: drug development; mariculture; marine biotechnology; natural product production.

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Conflict of interest statement

The author declares no conflict of interest.

Figures

Figure 1
Figure 1
Structure of the antifungal agent, nystatin. Like amphotericin B and natamycin, nystatin can act as an ionophore. While the exact mode and mechanisms of its antifungal action remain contested, nystatin has been shown to bind to sterols within fungal cell membranes. When present in sufficient concentrations, it forms pores in the membrane that lead to K+ leakage, acidification, and cell death.
Figure 2
Figure 2
Flasks. Three different flask types were explored: (a) a LPDE spherical flask; (b) a LPDE bottle (comparable to Nalgene narrow mouth LDPE bottle 2202-0005); and (c) a sterile bag (Flexboy FFB102704). The spherical flasks and bottles was custom manufactured with a 16 cm diameter and 1.5 mm thickness and contained 2 eyelets for attachment. An identical capping system was used for the bottles and spherical flasks (see specifications for Nalgene 2202-0005). Each unit was deployed by attachment to a 5/16” galvanized eye and eye swivel (Pro-LifT 3478). Attachments were made using 1/8′ (3.2 mm) type 316 stainless steel 1 × 19 wire (Alps, Chicago, IL, USA). The flasks were mounted using a Stainless Steel Standard S-Bail Snap Shackle (RONSTAN–2 11/16” L) to ease in deployment.
Figure 3
Figure 3
Arial (top view) of a six-flask manifold. Twelve 8 mm holes were drilled into a 60 cm stainless steel mast hoop (24” hoop, Sailrite 11730). The first 6 were drilled vertically (top to the bottom) at 60° intervals. The second set of 6 were drilled horizontally at 60° intervals offset 30° from the vertical holes. Here, alternating horizontal and vertical holes allowed attachment of the flasks (vertical) and mooring chain (horizontal). Using stainless steel wire rope (1/8′ or 3.2 mm type 316 stainless steel 1 × 19 wire, Alps), 6 snap shackles were mounted 30 cm from the hoop and attached by a marine-grade Brazier head rivet. The system was deployed by passing each wire through a single link of mooring chain, and then riveting them when taunt with a riveting clamp (commonly used for sail rigging). This process was either conducted on a boat (prior to installation of a mooring rig) or underwater (to an existing mooring rig), typically ≤10 min. A snap clamp was used to allow practical attachment and harvesting of the flasks (see Figure 4).
Figure 4
Figure 4
A plug-and-mariculture system. A system was designed to culture six 2 L spherical flasks. The manifold shown in Figure 3 was mounted on a mooring through a single chain link. Six flasks could be attached though shackles. Sizes are provided for the manifold and flask. Culturing was conducted at 1.3 L per flask with the remaining volume (0.7 L) charged with air.
Figure 5
Figure 5
NMR analyses of maricultures. 1H NMR spectra (600 MHz) in CD3OD of: (a) deployment A; (b) deployment B; (c) media that was deployed under the same protocols as (a,b) without inoculation (negative control); or (d) a sample of purified nystatin. NMR spectra in (a–c) was taken on crude extracts prior to purification (fractionation and recrystallization). The negative control (c) identifies peaks in (a,b) that were derived from the media. NMR spectra are provided from 0.5 to 6.5 ppm. Additional spectral data, including further expansions of the 1H NMR spectra in (a–c) as well as gCOSY, NOESY, HSQC and HMBC data on purified nystatin in (d) have been provided in the Supplementary Material.
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