*4.5. Everything Looks Great, Right?*

There are still many challenges facing the widespread adoption of plastid transformation technologies. These include: low transformation efficiency; lack of efficient screening methods for homoplasmy in transgenic plants outside of tobacco, especially in important crops such as rice, corn and other monocotyledonous plants, due to the lack of suitable selection markers and regulatory elements [167]; lack of appropriate tissue-specific regulatory sequences [168]; degradation of foreign proteins [169]; and foreign protein expression sometimes causing male sterility, yellow leaves and stunting. Inducible systems such as the ethanol-induced T7 promoter system, IPTG-lac system, and theophylline-inducible riboswitch system were developed because of the damage caused by constitutive foreign protein expression in host plants, but even these systems suffer from drawbacks: they are complex, toxic and costly. Although plastid transformation has been applied to various fields, it will also be critical to raise awareness among the general public of the usefulness of marker-free transplastomic plants.

#### **5. Conclusions and Prospects**

There is no doubt that plastid transformation has afforded a new direction for plant genetic engineering and constitutes a research hotspot because of its many advantages over nuclear transformation. Indeed, high transgene expression and engineering of polygenic traits are not amenable to classical nuclear transformation. However, plastid transformation can meet human needs, it is cost-effective, environmentally friendly, safe and efficient. It can be used to modify agronomic traits, and for phytoremediation and biofuels production. More importantly for human health, antigen vaccines and protein-based drugs can be produced in chloroplasts. Although plastid transformation has been achieved in many crops, many others still remain recalcitrant to plastid transformation [137]. The protocols of plastid transformation in crops would offer significant advantages for the production of edible vaccines and medical proteins, biofuels and industrial enzymes, as well as enhanced agronomic traits. In this review, we have illustrated the potential advantages from new studies and technologies like CRISPR-Cas9 for introducing double-strand DNA breaks for HR and create new varieties/mutants (*acc2*) to increase selection efficiency during plastid transformation. No longer limited to biolistic methods, nanotubes can be an alternative material for DNA delivery to increase the donor DNA into chloroplasts. Although they have not yet been applied to plastid transformation, the morphogenic regulators (*BBM*, *WUS2*, and cytokinin biosynthesis genes) and the new chemicals/technology for tissue culture and regeneration can increase the regeneration of transformed cells. These new approaches should also entice researchers to reconsider protoplast-based strategies for plastid transformation. Combined with nuclear transformation and other methods, plastid transformation may allow the production of important proteins.

**Author Contributions:** C.S.L. conceived and designed the review. C.-S.L., P.-C.Y. and W.-J.C. prepared the plastid transformation section. Y.Y., P.-C.Y., W.-J.C., and K.Y. prepared the plastid transformation application section. P.-C.Y., W.-J.C. and C.-S.L. wrote the manuscript with input from all co-authors. All authors read and approved the final manuscript.

**Funding:** This research was supported by Academia Sinica, Innovative Translational Agricultural Research Administrative Office (AS-KPQ-107-ITAR-10, AS-KPQ-108-ITAR-10, AS-KPQ-109-ITAR-10), and the Ministry of Science and Technology (105-2313-B-001-007 -MY3; 108-2313-B-001 -011 -), Taiwan.

**Acknowledgments:** We thank Plant Editors for English editing.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **Abbreviations**


**1. Introduction**

The first natural isoquinolinequinone isolated from bacteria were reported by Fukum et al. in 1977 [1] and then by Kubo et al. in 1988 [2]. A few others were isolated from porifera, including cribrostatins (produced by the blue marine sponge *Cribrochalina* sp.) [3], renierones (from *Reniera*, *Petrosia*, and *Haliclona* spp.) [4,5], and caulibugulones (found in the marine bryozoon *Caulibugula inermis)* [6]. These isoquinolinequinones showed a potent antimicrobial activity against Gram positive bacteria and yeast (*Candida albicans*) and a pronounced cytotoxicity against L1210 and other cell lines with IC50 values as low as 30 ngmL−1 [7,8]. In 1998, we isolated mansouramycin A (**5**) as a trace component from the marine derived *Streptomyces* sp. B3497 [9]. Mansouramycin A (**5**) and the synthetic analogue 3-methyl-7-(methylamino)-5,8-isoquinolinedione (**4a**) were re-isolated from the marine-derived *Streptomyces* sp. isolate Mei37, together with three new mansouramycins B–D (**1a**–**3a**) [10].  Recently, mansouramycin A (**5**) was also obtained from the marine-derived *Streptomyces albus* J1074 and found to be a potent inhibitor of the methicillin-resistant *Staphylococcus aureus* ATCC 43300 with an MIC of 8 μgmL−1. *S. albus* J1074 produced additionally the novel isoindoloquinone albumycin [11].

While the marine-derived *Streptomyces* sp. isolate B1848 was previously noted as a producer of 6-hydroxy-isatine and several other known compounds [12–14], further fermentations led now to the isolation and characterization of three unusual mansouramycins

**Citation:** Shaaban, M.; Shaaban, K.A.; Kelter, G.; Fiebig, H.H.; Laatsch, H. Mansouramycins E–G, Cytotoxic Isoquinolinequinones from Marine Streptomycetes. *Mar. Drugs* **2021**, *19*, 715. https://doi.org/10.3390/ md19120715

Academic Editor: Ipek Kurtboke

Received: 11 November 2021 Accepted: 8 December 2021 Published: 20 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

E–G (**1a**–**3a**) along with mansouramycins A (**5**), D (**6**) (Figure 1) and 13 known metabolites [12–14]. The chemical structures of **1a**–**3a** were elucidated by NMR (1D, 2D) and HRMS, by comparison with related compounds and by computer-assisted methods. The cytotoxic activity of the isolated isoquinolinequinones was determined.

**Figure 1.** Chemical structures of isoquinolinequinones **1–6** produced by *Streptomyces* sp. B1848, and alternative structures **1b–3b**.

#### **2. Results and Discussion**

With a malt extract medium with 50% synthetic seawater (M2+ medium), the marinederived *Streptomyces* sp. isolate B1848 produced only traces of mansouramycins A (**5**) and D (**6**), along with the zizaene derivative albaflavenol [9], 6-hydroxy-isatine [13,14], 2-deoxythymidine, 2-deoxuridine, 2-deoxyadenonsine, anthranilic acid, tyrosol, indolyl-3- acetic acid, phenyl acetamide, indolyl-3-carboxylic acid, *N*ß-acetyltryptamine, *N*-acetyltyramine, and *p*-hydroxybenzoic acid [12]. Better yields of the mansouramycins and further red pigments were obtained now on a meat extract medium in a fermentation with a 50 L shaker culture. After extraction and chromatographic separation, the strain B1748 afforded under these conditions the mansouramycins A (**5**) and D (**6**) and three new congeners, the mansouramycins E–G (**1a**–**3a**) as dark red solids. The isoquinolinequinones gave brown-red zones on TLC, with UV absorptions in solution similar as of *peri*-hydroxyquinones. Their reversible color change with sodium dithionite from orange to nearly colorless confirmed quinones; *peri*-hydroxyquinones were excluded, however, by the missing bathochromic shift with sodium hydroxide. Unlike the orange-red phenoxazinone chromophore of actinomycins and related pigments, which are becoming red with concentrated sulfuric acid, the isoquinolinequinones turned yellow. Further physicochemical properties of compounds **1a**−**3a** are summarized in Table 1.

Silica gel G/UV254; **1b, 2b, 3b** (CH2Cl2/7% MeOH); sh = shoulder.

## *2.1. Structure Elucidation*

a

Compound **1a** was obtained as red powder of moderate polarity. The molecular formula was determined as C16H11N3O2 by EI-HRMS, indicating 13 double bond equivalents (DBE). The color change to yellow with concentrated sulfuric acid and the characteristic UV curve with a flat absorption at λmax 509 nm as for **5**, **6** pointed to an isoquinolinequinone moiety as well [10] (Table 1). The 13C NMR spectrum (Table 2) showed six aromatic/olefinic methines and one methyl signal. Furthermore, signals of nine non-protonated carbon atoms were observed, of which two at *δ* 182.8 and 181.4 pointed to carbonyl groups of a quinone. In the proton NMR spectrum (Table 2), the CH singlets at *δ* 9.01 (H-1) and 5.71 (H-6), in addition to a broadened NH signal at *δ* 7.83 and a methyl doublet at *δ* 2.85 of the CH3NH fragment were typical for mansouramycins.

The proton H-6 showed HMBC correlations (Figure 2) with C-4 (4*J*), 4a, 5, 7, and C-8; correlations of the N-methyl signal with C-7, and of H-1 with C-3, 4a and 8 resulted in a 3,4-disubstitued 7-methylamino-isoquinolinequinone skeleton as in **4a**–**6**; unfortunately, NH HMBC correlations were not visible for **1a**.

A 1,2-disubstituted benzene ring was deduced from the typical signal pattern of four *o*,*m*-coupled protons at *δ* 8.23 (d), 7.79 (d), 7.61 (td), and 7.31 (td) ppm and from the expected HMBC correlations (Figure 2). A further broadened NH signal was seen at *δ* 11.98, which formed with the remaining atoms an aniline residue. With respect to the two open valencies in both fragments, the isoquinoline and the aniline unit can be merged only in two ways under formation of structures **1a** or **1b**. The more in-depth analysis of the NMR data by means of the structure elucidation program COCON [15] confirmed isomers **1a** and **1b** as allowed structures, but delivered >7600 additional alternatives! Most of them were highly strained (cyclobutenes, non-linear allenes, or bridged aromatic systems) and therefore excluded.

**Table 2.** 13C (150 MHz) and 1H NMR spectroscopic data of compounds **1a**–**3a** in DMSO-*d*6 (*δ* in ppm, *J* in [Hz]).

(a) 300 MHz; (b) 600 MHz. See Supplementary Materials for NMR spectra. (c) Small signals; the assignment was confirmed by their HMBC correlations.

**Figure 2.** 2D NMR correlations of mansouramycins E–G (**1a**–**3a**). Blue arrows = 2*J*, 3*J* HMBC correlations; green arrows = 4*J* HMBC correlations, red bonds = COSY correlations.

Amongst 24 plausible indoloquinoline- and indoloisoquinoline-quinones, only 6 (**1a**, **1b**, **S1c**, **S1e**, **S1f**, **S1h**) \* were found by COCON and therefore only these are in agreement with the COSY and HMBC correlations. For mansouramycin E, the isomer **1a** showed the best agreement of experimental NMR data with shifts calculated by SPARTAN'20 [16] using ab initio methods on a high level of theory. This structure was therefore assumed for mansouramycin E (see Supplementary Materials). Further applications of this technique have been described previously [17]. \* Formula numbers with a leading bold letter "**S**" are refering to structures in Supplementary Materials.

For the dark red mansouramycin F (**2a**) the molecular formula C12H9N3O2 (ESI-HRMS) was determined, which entails 10 DBE. The 1H and 13C NMR shifts (Table 2), as well as the HMBC couplings, confirmed again an *N*-methyl-isoquinolinequinone substructure as in all other mansouramycins (Figure 1). According to the chemical shifts and 2D correlations, the unassigned residual atoms C2H3N were belonging to an annulated pyrrole ring, which was confirmed by the 1H triplet at *δ*H 7.93 and the dd signal at 6.70 with the expected small coupling constants (~5 Hz). The pyrrole ring can be fused with the isoquinolinequinone core in three different ways, yielding structures **2a**, **2b**, **S2m**, and the respective isomers with the N-methyl group at C-6 instead at C-7 (see Supplementary Materials).

With COCON using atom types, 19 isomers were found. Four of them were quinones (**2a**, **2b**, **S2c**, **S2d**). The other structures were azepin-2-ones or highly strained bridged systems. Isomers of type **S2m** were excluded by COCON as well, and also *o*-quinones were not predicted for mansouramycin F.

H-3 in **2b** should show a 3*J* correlation with C-4a, which is missing in the experimental spectrum and therefore better fitting on **2a**. In **S2c**/**S2d**, the quinonoid proton H-7 (*δ* ~5.6) should show a 3*J* correlation with C-8a at *δ* ~147. However, this was also not observed, so that only structure **2a** was left. For further confirmation, we compared the experimental with calculated shifts of all possible pyrrolo-quinoline- and pyrroloisoquinoline-5,8- quinones. The results (Table S2) pointed again clearly to structure **2a** for mansouramycin F. This conclusion was further confirmed by comparison with similarly fused pyrrolo-pyridine skeletons [18,19].

Compound **3a** was obtained as a red solid as well, which displayed isoquinolinequinone-like UV/vis and other physicochemical properties. The molecular formula of **3a** was established as C15H11N3O4 by ESI-HRMS and 1H and 13C NMR analysis, entailing 12 DBE. The 1H and 13C NMR spectra confirmed a further isoquinoline-quinone, which showed, however, remarkable differences compared with **1a** and **2a**. Instead of one *N*-methyl residue (7-NHCH3) and one quinonoid proton (6-H), as in the other mansouramycins, the 1H NMR spectrum showed each two of these signals. In addition, the 13C NMR spectrum displayed four carbonyl groups (*δ*C 178.2, 179.8, 177.6, and 180.9) instead of two carbonyls as in **1a** and **2a** (Table 2). Interpretation of the HMBC spectrum of **3a** (Figure 2) revealed an isoquinoline-quinone and an *N*-methylaminobenzoquinone substructure, which can be connected in two different ways only, resulting in **3a** or **3b**, respectively (Figures 1 and 2). The alternative **3b** was excluded, however, based on the significant 3*J* HMBC correlations of H-6 (*δ*H 5.75) and NH-9 (*δ*H 7.84) with CO-8 (*δ*C 179.8) and not with CO-5 (*δ*C 178.2); the position of the second *N*-methyl group at C-3 was determined in a similar way. All the remaining HMBC and COSY correlations (Figure 2) were in full agreements with structure **3a**, a novel azaphenanthrene diquinone, which we named mansouramycin G (see also Supplementary Materials).