Advancements and Future Prospects in Hypocrellins Production and Modification for Photodynamic Therapy

Abstract

Hypocrellins (HYPs), naturally occurring 3,10-xylene-4,9-anthracene derivatives sourced from Shiraia bambusicola and Hypocrella bambusae, exhibit significant photobiological activities. Despite their capability for generating a high yield of reactive oxygen species, including singlet oxygen and superoxide anion radical, their application in photodynamic therapy (PDT) is constrained. This limitation is due to their low dark phototoxicity, weak absorption within the therapeutic window of PDT (600–900 nm), and inherent hydrophobicity, which hinder their immediate use in amphipathic PDT applications. This review comprehensively discusses the research advancements in the bioactivities and biosynthesis of HYPs, alongside the reported chemical and physical modifications that enhance their water solubility and extend their therapeutic window. Additionally, it explores potential strategies for developing pharmaceuticals, photocatalytic agents, and photosensitive pesticides based on HYPs.

1. Introduction

Hypocrellins (HYPs) are natural 3,10-xylene-4,9-anthracene-perylenequinoneide (PQD) derivatives from the stroma of the Ascomycota fungi Hypocrella bambusae and Shiraia bambusicola [1] that are used for treating stomach pain, rheumatoid arthritis, limb numbness, anemia, and headache in Chinese traditional medicine. HYPs generally include four comparable compounds, viz. hypocrellin A (HA), hypocrellin B (HB), hypocrellin C (HC), and hypocrellin D (HD) [1] (Figure 1). The HA and HB content occupy up to 95% of the HYPs extracted from the stroma of H. bambusae [2]. HA and HB have excellent photosensitive activities against Gram-positive bacteria and fungi, with a good thermal stability in weakly polar neutral solutions.

Studies have proven that HYPs have emerged as a new generation of photosensitizers. Due to their unique molecular structure, the maintenance of the structural stability and photosensitive activity of HYPs is subject to intramolecular proton transfers. When irradiated by light, HYPs promote intramolecular proton transfer, as well as intramolecular motions of the aromatic skeleton and the side chains to the excited state. Through energy transfer by the HYPs, singlet oxygen (¹O₂), superoxide anion radical (O₂·⁻), hydroxyl radical (·OH), and hydrogen peroxide (H₂O₂) can be generated in an aqueous solution. The photodynamic generation of these reactive oxygen species (ROS) by HYPs relies on two reaction types. Upon light irradiation of the HYPs, the type I reaction involves the excited-state triplet HYPs transferring hydrogen or electrons directly to the substrate or solvent molecules from their surroundings, leading to free radicals and anion radicals. The generated radicals and anion radicals can react with ground-state oxygen (³O₂) to generate O₂·⁻. The type II mechanism proposed is a direct energy transfer from the excited HYPs to ³O₂ to generate ¹O₂ with a high oxidation reactivity. In the photosensitized reaction, both processes take place simultaneously and in competition. H₂O₂ and ·OH can be formed, while their divalent anions reduce O₂ after photosensitization by the HYPs [3,4] (Figure 2).

2. Bioactivities in Photodynamic Therapy (PDT)

HYPs are promising photosensitizers with a high phototoxicity and a short metabolic half-life in animals [4]. In addition, they show high antitumor, antiviral, and antibacterial activities.

2.1. Antibacterial and Antifungal Activities

HYPs significantly increase the on-site ROS level by photostimulation, enhancing antibacterial activity [5]. HA and HB have shown excellent light-induced growth-inhibitory activity against most Gram-positive bacteria, such as azole-resistant Candida albicans, methicillin-resistant Staphylococcus aureus, Mycobacterium intracellulare, Bacillus subtilis, and Listeria monocytogenes, but without apparent dark toxicity [6,7]. With low toxicity and high efficiency, it is expected that HA and HB can be helpful PDT agents for the inactivation of resistant microorganisms. In addition, antifungal activity against phytopathogenic fungi by HYPs has been reported [7]. HA is also considered a promising photodynamic candidate that could be applied in treating human fungal infections by C. albicans or developed as a green fungicide for agriculture and antiseptics for food (

Table 1. The antibacterial and antifungal activities of HYPs.

Groups	Species	Origins	Compounds	Methods of Application	Inhibition Efficiency	References
Bacteria, G+	B. subtilis	Plant pathogen	HA	5000 Lux for 120 min	NA	[4]
			HA	1 μM, 50 W halogen–tungsten lamp, as 30 mW/cm for 120 min	>99.9%	[8]
			HYPs	4 mg/mL, fluorescent light, 2000 Lux for 2 h	MIC 3.0 μg/mL; MBC 6.0 μg/mL	[9]
			HYP	NA	IC50 3–10 μg/mL	[7]
			solvent extracts by petroleum ether, ethyl acetate, and methanol	1 mg/mL, light resource NA	17.88 ± 0.06%	[10]
	Bacillus pumilis	Plant pathogen	25% acetone extract from stroma of H. bambusae	5000 Lux for 120 min	Significant inhibitory effect	[4]
	Corynebacterium pekinense	Plant pathogen
	Sarcina flava	Plant pathogen
	Staphylococcus albus	Plant pathogen
	S. aureus (methicillin-resistant)	Human pathogen	HA	NA	IC50 3–10 μg/mL	[7]
			HA	1 μM, under 50 W halogen–tungsten lamp, as 30 mW/cm2 for 120 min	99.98%	[8]
			HA	1.5 μg/mL, under halogen–tungsten lamp, 600 Lux for 120 min	100%	[5]
			HYPs	4 mg/mL, under 2000 Lux fluorescent light for 2 h,	Zone of inhibition 18.5 ± 0.5 μg/mL; MIC 0.75 μg/mL; MBC 1.5 μg/mL	[9]
	L. monocytogenes	Human pathogen	HYPs	4 mg/mL, fluorescent light, 2000 Lux, 2 h,	Zone of inhibition: 12.5 ± 0.5 μg/mL; MIC 6.0 μg/mL; MBC 12.0 μg/mL	[9]
Bacteria, G−	M. intracellulare	Human pathogen	HA	NA	IC50 3–10 μg/mL	[7]
	E. coli	Human pathogen	25% acetone extract from H. bambusae	5000 Lux for 120 min	0	[4]
			HYPs	4 mg/mL, under 2000 Lux fluorescent light for 2 h	0	[9]
			HA	1 μM HA, 0.05 M CaCl2, 50 W halogen–tungsten lamp, as 30 mW/cm2 for 60 min	98.80%	[8]
			HA	1 μM HA, 0.05 M CaCl2, 50 W halogen–tungsten lamp, as 30 mW/cm2 for 60 min	98.30%	[8]
	S. typhimurium	Human pathogen	HYPs	4 mg/mL, under 2000 Lux fluorescent light for 2 h	0	[9]
			HA	1 μM HA, 0.05 M CaCl2, 50 W halogen–tungsten lamp, as 30 mW/cm2 for 60 min	98.80%	[8]
			HA	1 μM HA, 0.05 M CaCl2, 50 W halogen–tungsten lamp, as 30 mW/cm2 for 60 min	98.30%	[8]
	P. aeruginosa	Human pathogen	25% acetone extract from H. bambusae stroma	5000 Lux,120 min	0	[4]
			HA	NA	IC50 3–10 μg/mL	[7]
	P. vulgaris	Human pathogen	25% acetone extract from H. bambusae stroma	5000 Lux for 120 min	0	[4]
Fungi	C. albicans	Human pathogen	HA	NA	IC50 0.65 ± 0.14 μg/mL, MIC 1.41 ± 0.22 μg/mL, MFC 1.41 ± 0.22 μg/mL	[7]
			Methanol extract of S. bambusicola stroma	1 mg/mL, light resource NA	20.69 ± 0.26%	[10]
			HA	1.0 μg/mL, 8 W incandescent lamp (400–780 nm), 1128 Lux for 30 min	48.76 ± 4.39%	[11]
	Gibberella zeae	Plant pathogen	Methanol extract of S. bambusicola stroma	1 mg/mL, light resource NA	54.64 ± 0.55%	[10]
	Fusarium oxysporum f. sp. vasinfectum	Plant pathogen	Methanol extract of S. bambusicola stroma	1 mg/mL, light resource NA	74.78 ± 0.49%	[10]
			HA	7200 Lux, light resource NA	IC50 7.2223 mg·L−1	[12]
	Rhizoctonia solani	Plant pathogen	Methanol extract from S. bambusicola stroma	1 mg/mL, light resource NA	61.42 ± 0.57%	[10]
			HA	7200 Lux, light resource NA	IC50 6.9161	[12]
	Fusariumgraminearum	Plant pathogen	HA	7200 Lux, light resource NA	IC50 4.2933 mg·L−1	[12]
	Fusariumgraminearum Schwabe	Plant pathogen	HA	7200 Lux, light resource NA	IC50 6.8587 mg·L−1	[12]
	Lecanostictaacicola	Plant pathogen	HA	7200 Lux, light resource NA	IC50 2.5773 mg·L−1	[12]
	Botrytis cinerea	Plant pathogen	HA	7200 Lux, light resource NA	IC50 3.9497 mg·L−1	[12]
	Valsa mali	Plant pathogen	HA	7200 Lux, light resource NA	IC50 4.2511 mg·L−1	[12]
	Sclerotinia sclerotiorum	Plant pathogen	HA	7200 Lux, light resource NA	IC50 6.4766 mg·L−1	[12]
	Exserohilum turcicum	Plant pathogen	HA	10 mg·L−1, 7200 Lux, light resource NA	46.81%	[12]
	Fusarium lateritium	Plant pathogen	HA	10 mg·L−1, 7200 Lux, light resource NA	44.96%	[12]
	Fusarium oxysporum f. sp. cucumeris	Plant pathogen	HA	10 mg·L−1, 7200 Lux, light resource NA	47.28%	[12]
	F. oxysporum	Plant pathogen	HA	10 mg·L−1, 7200 Lux, light resource NA	46.15%	[12]
	Botryosphaeria dothidea	Plant pathogen	HA	10 mg·L−1, 7200 Lux, light resource NA	35.65%	[12]
	Altemaria Solani	Plant pathogen	HA	10 mg·L−1, 7200 Lux, light resource NA	49.88%	[12]

NA: Data not available; MBC: Minimum Bactericidal Concentration; MIC: Minimum Inhibitory Concentration; MFC: Minimum Fungicidal Concentration; IC₅₀: Half-Maximal Inhibitory Concentration.

).

The antibacterial mechanism of HA primarily focuses on disrupting the structural integrity of microbial membrane, explored through changes in permeability and morphological alterations (Figure 3). Key factors considered may include (1) fragmentation of the microbial cell wall; (2) depolarization of cell membrane potential, influencing apoptosis-related parameters such as mitochondrial DNA fragmentation and cytoplasmic calcium levels; and (3) disruption of membrane integrity, resulting in the leakage of cellular contents like reducing sugars and proteins, coupled with the inhibition of crucial respiratory enzymes like dehydrogenase.

While HYPs demonstrate inhibitory effects against Gram-positive bacteria and fungi, their efficacy against most Gram-negative bacteria, such as Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Proteus vulgaris, is moderate [8]. Distinct to the Gram-negative bacteria, the unique composition of Gram-negative bacteria, characterized by an outer membrane rich in lipoproteins, lipopolysaccharides, and a bilayer of phospholipids, may hinder HYP penetration (Figure 4A,B). This membrane allows free passage to molecules smaller than 100 Da but restricts larger molecules like HYPs (>500 Da), reducing their cytotoxic potential due to limited proximity to internal cellular structures and the transient nature of the generated ROS. The potential enhancement of HYPs’ photodynamic activity against P. aeruginosa, when combined with agents like CaCl₂ or MgCl₂, supports this observation and suggests outer membrane permeability significantly influences HYP effectiveness [8].

2.2. Antitumor and Antiviral Activities

The PDT applications of HA significantly reduce malignant cell invasiveness and promote apoptosis, particularly noted in keloid fibroblasts (KFs) during red-light treatment [13]. This effect is achieved through the suppression of TGF/Smad signaling and autophagy, leading to reduced cell proliferation and decreased collagen and extracellular matrix (ECM) production. Studies such as those by Hudson et al. and Fehr have further demonstrated HA’s capacity to inactivate pathogens like human immunodeficiency virus (HIV), emphasizing the requirement of oxygen for effective antiviral response [14,15].

The integration of HA with other agents, such as HB, HC, nystatin, curcumin, and oleanolic acid, could significantly promote the inhibitory activities of HYPs against pathogenic microbes and tumor cells [16]. Song et al. showed that HYP was able to significantly inhibit the mycelium growth, biofilm formation, and adhesion of C. albicans [17]. The combination of HA, HB, HC, and nystatin has a more substantial inhibitory effect on nystatin-resistant C. albicans strains than the addition of HA, HB, or HC alone [17]. Li et al. found that curcumin could significantly enhance the photokilling ability of HB and form an effective PDT system mediated by a two-photon mechanism [18]. Chio et al. reported that the combination of imatinib mesylate (IM) and HA could reverse the IM drug resistance phenotype in epithelial cell lines [19]. Compared to oleanolic acid (OA) or HA alone, the combination of HA and OA has a higher inhibition rate, so as to significantly inhibit the migratory ability of HCC cells [20]. The evidence highlights the benefits of combinatory therapies in overcoming drug resistance and improving treatment efficacy against resistant strains of pathogens and tumor cells.

HYP activity to induce apoptosis and necrosis in various human malignant cell lines, including HIC, MGC-803, HeLa, and BGC-823, underscores their potential in PDT [21] (

Table 2. The antitumor activities of HYPs.

Cells	Compounds	Methods for Application	Inhibition Efficiency	References
MDA-MB-231 cell breast cancer	HB	2.5 μM, by 0.72 W/cm2 ultrasound treatment	42.69%	[25]
A549 cells human lung adenocarcinoma	HA	Irradiation @ 470 nm for 24 h	IC50 < 0.05 μM	[26]
human melanoma cancer cells SK-MEL-28	HYPs	Under 100 J/cm2 broadband visible-light for 16 h	EC50 = 10.3–176.0 nM	[27]
hepatocellular carcinoma HepG2 cells	biodegradable HB nanoparticles coated with neutrophil membranes	20 μg·L−1, under 0.8 W cm2 laser stimulation	50%	[28]
human non-small-cell lung cancer cell lines H460, PC-9, and H1975	HA	48 h	IC50 = 0.22–0.62 μM	[29]

IC₅₀: Half-Maximal Inhibitory Concentration; EC₅₀: Half-Maximal Effective Concentration.

). The effectiveness of HA-PDT is particularly pronounced in the HIC cell line, where it significantly reduces cell viability and disrupts cellular functions such as DNA synthesis. In addition, HB-PDT produces ROS through photosensitization and disrupts red blood cell membranes [22]. In this process, the more lipids that contain unsaturated double bonds, the more susceptible they are to attack and photosensitization. It has been demonstrated that HYPs are photosensitive to the spatial structure of DPPC liposomes [23]. The production of ROS and subsequent disruption of cellular membranes rich in unsaturated lipids further illustrates the broad-spectrum cytotoxicity of HYPs against viruses, cancer cells, and membrane structures. At present, HYPs are widely used to treat skin diseases such as vulvar leukoplakia and vulvar intraepithelial nonneoplastic lesions. They have achieved ideal therapeutic effects and are recognized as an effective and safe drug [24].

2.3. Challenges of HYPs in PDT Application

Despite the promising properties of HYPs in PDT, such as a defined structure and high photosensitivity, as well as low dark toxicity or side effects, their application is limited by inherent drawbacks like poor solubility, inadequate absorption within the optimal light treatment spectrum (600–700 nm), and low thermal stability. To address these issues, derivatives and complexes of HYPs have been synthesized to enhance their physicochemical properties and performance as photosensitizers [30]. The scarcity of natural HYP resources also poses a significant challenge, making chemical synthetic routes and fermentation vital for sustainable production.

3. Techniques for Production of HYPs

3.1. Extraction and Chemical Synthesis

HYPs can be extracted from the stroma of S. bambusicola or H. bambusae using organic solvents due to their hydrophobic nature. Effective solvents include methanol, absolute ethanol, butanol, n-butanol, acetone, chloroform, n-hexane, petroleum ether, ethyl acetate, dichloromethane, and trichloromethane. Acetone, n-butanol, butanol, chloroform, and dichloromethane are particularly efficacious, enhancing both the extraction rate and product purity. This improvement can also be achieved by optimizing the solid–liquid ratio, extraction temperature, ultrasonic time, and chromatographic separation methods [31,32]. For instance, Zhang et al. achieved an extraction rate of 1.72% by processing wet mycelia with acetone at a 1:4 solid-to-liquid ratio, followed by a low-temperature drying method [32]. Similarly, Shao et al. reported an extraction rate of 4.83 mg/g with a purity of 98.20% using acetone for a 30 min sonication of freeze-dried hyphal powder at a 1:30 solid-to-liquid ratio [31].

The chemical synthesis of HYPs, specifically HA, remains challenging due to the complex nature of its extended conjugated pentacyclic core, which is a characteristic of perylenequinone compounds [16]. Traditional synthesis methods like the coupled oxidation of 12-naphthoquinone mediated by ferric chloride have shown limited efficiency [33]. Morgan synthesized the diastereomers (+)-phleichrome and (+)-calphostin D, which are structurally similar to HA, by enantioselective catalytic biaryl coupling in a 17-step reaction, and their yields were only 5.3% and 5.2%, respectively [34].

Innovative approaches, such as the enantioselective catalytic biaryl coupling and biomimetic dynamic stereochemistry transfer (DST) aldol reaction, have been explored to improve yields. The total synthesis of HA was firstly realized through a 19-step reaction containing the enantioselective coupling of highly functionalized naphthols, a low-temperature aromatic decarboxylation, and a 1,8-diketone aldol reaction with an overall yield of only 1.6% [34,35].

Currently, most of the HYPs on the market are generally produced through the extraction method. However, the restricted regional distribution and seasonal availability of the fungal stroma have challenged the sustainability of this approach. As mentioned above, chemical synthesis techniques are complex and costly, with a low overall yield of HYPs [36], making them less viable for large-scale production. Consequently, fermentation emerges as the most feasible method for producing HYPs, offering a more sustainable and scalable alternative.

3.2. Biological Production of HYPs Through Fermentation

3.2.1. The Biosynthesis Pathway of HYPs

Advancements in genetic engineering and sequencing technologies have facilitated detailed investigations into the metabolic pathways of HYP biosynthesis. The gene clusters responsible for the biosynthesis of compounds like cercosporin and elsinochrome have been shown to share multiple homologous genes, implicated in the synthesis of the pentacyclic quinone core and co-substitutions [37]. Despite these advances, key steps such as the oxidative coupling needed to form the pentacyclic nucleus, the enol coupling required for the six-ring system in elsinochrome, and the formation of the seven-ring system necessary for final HYP products remain elusive [38].

Research has highlighted several crucial enzymes involved in HA biosynthesis in Shiraia sp. strain SLF14, including polyketide synthase (PKS), O-methyltransferase, hydroxylase, and various oxidoreductases, such as FAD-dependent monooxygenase, FAD/FMN-dependent oxidoreductase, NADPH-dependent oxidoreductase, and multicopper oxidase (MCO) [39].

Transcriptomic studies have further refined our understanding of the pathway. For instance, Zhao et al. identified seven proteins essential for HA biosynthesis through different gene expression pattens between the wild-type S4201-W and mutant S4201-D1 strains. They are MCO, fasciclin, PKS, O-methyltransferase/FAD-dependent monooxygenase, O-methyltransferase, hydroxylase, and FAD/FMN-dependent oxidoreductase [40]. Lei et al. pinpointed that PKS, monooxygenase, and O-methyltransferase could be involved in these pathways [41]. The application of the CRISPR/Cas9 gene editing system has proven invaluable in these studies, enabling the functional analysis of these genes by observing the effects of the knockouts of the PKS and monooxygenase genes on HA synthesis [42]. By referring to the biosynthesis pathways for the similar structural compounds of cercosporin and elsinochrome C [38], it was suggested that a biosynthetic pathway for HA begins with acetyl-CoA and malonyl-CoA, extending through a series of condensations and cyclizations mediated by the PKS enzymes, ultimately leading to the formation of the perylenequinone core. Subsequent cyclization leads to the formation of nor-toralactone. This intermediate undergoes further transformations, as O-methyltransferase/FAD-dependent monooxygenase and FAD/FMN-dependent oxidoreductase catalyze a series of oxidation, hydration, and methylation reactions. These reactions generate cyclized polyhydroxynaphthalene units. The perylenequinone carbon core is eventually formed by the polymerization of two identical or different polyhydroxynaphthalene units. Finally, HA is formed, followed by other unknown modification reactions (Figure 5). Moving forward, the application of more sophisticated gene editing techniques and the exogenous expression of genes involved in HYP biosynthesis will be essential to elucidate these pathways and identify intermediate compounds more precisely [43].

3.2.2. Fermentation Techniques for Production of HYPs

Table 3. Techniques for the biological production of HYPs.

	Strain	Carbon and Nitrogen Sources (g/L or g/Kg)	Temperature (°C)	Fermentation Time (h)	Additive/Treatment	Product	Production Yield	Reference
SMF	S. bambusicola LBR-SB6	30 glucose, 20 potato juice	26	168	NA	HYP	40–50 mg/L	[46]
	S. bambusicola UV-62	1.93 glucose, 45.7 (NH4)2SO4	25	120	NA	HA	196.94 ± 6.93 mg/L	[47]
	S. bambusicola P. Henn.	20 glucose, 2 NaNO3	27	72	NA	HYP	28.04 mg/g	[48]
	Shiraia sp. ZZZ816	40 maltose, 20 yeast extract, 4 urea	25	144	NA	HA	921.6 mg/L	[49]
	Shiraia sp. SUPER-H168	20 glucose 3 yeast extract	30	72	0.6% Triton X-100	HYP	780.6 mg/L	[50]
	S. bambusicola S8 (CGMCC3984)	Potato extract from 200 fresh potato, 20 glucose, 3 yeast extract, 10 peptone	28	72	2.5% Triton X-100	HA	96 mg/L	[41]
	Shiraia sp. S9 (CGMCC 16369)	Potato extract from 100 fresh potato, 20 starch, 100 potato, 4 NaNO3	28	192–240	2.5% Triton X-100	HA	206.2 mg/L	[51]
	S. bambusicola S8 (CGMCC3984)	Potato extract from 100 fresh potato, 20 starch, 100 potato, 4 NaNO3	28	192–240	ultrasound	HA	247.67 mg/L	[52]
	Shiraia sp. Slf14	Potato extract from 200 fresh potato, 10 sucrose, 5 yeast extract	28	336	6 g/L Ca2+	PQ	1894.66 ± 21.93 mg/L	[53]
	Isolation from the stroma of S. bambusicola	20 glucose, 3 NaNO3	27	96	0.03% Ca2+	HYP	8.12 mg/g	[54]
	S. bambusicola S8 (CGMCC3984)	Potato extract from 100 fresh potato, 20 starch, 4 NaNO3	28	192	1 g/L La3+	HA	225.05 mg/L	[55]
	S. bambusicola S8 (CGMCC3984)	Potato extract from 100 fresh potato, 20 starch, 4 NaNO3	28	192–240	light/dark shift (24:24 h)	HA	181.67 mg/L	[56]
	S. bambusicola S8 (CGMCC3984)	Potato extract from 100 fresh potato, 20 starch, 4 NaNO3	28	192	red LED light (627 nm) at 200 lux	HA	175.53 mg/L	[57]
	Shiraia sp. SUPER-H168 (CCTCC M 207104)	Potato extract from 200 fresh potato, 20 glucose, 3 yeast extract, 10 peptone	30	72	10 mM H2O2	HYP	increased by 27%	[58]
	S. bambusicola S4201	Potato extract from 200 fresh potato, 20 glucose	28	120	0.01 mM sodium nitroprusside (nitric oxide donor)	PQ	increased by 156%	[59]
	S. bambusicola GZUIFR-11K1	3 beef extract, 10 peptone	26	168	80 μg/mL Aspergillus niger elicitor	HYP	approximately 90 mg/L	[60]
	S. bambusicola GZUIFR-11K1	32.45 glucose, 2.99 yeast extract	26	168	81.40 μg/mL Trametes sp. elicitor	HYP	102.60 mg/L	[61]
	S. bambusicola BZ-16 × 1	15 glucose, 1.5 NaNO3	26	288	5 nmol/L PB90 elicitor	HYP	278.71 mg/L	[62]
	Shiraia sp. S9 (CGMCC 16369)	Potato extract from 100 fresh potato, 20 starch, 4 NaNO3	28	192	400 cells/mL P. fulva SB1	HA	225.34 mg/L	[63]
	S. bambusicola S8 (CGMCC3984)	Potato extract from 200 fresh potato, 20 glucose,5 yeast extract	28	240	UV, 15 W	HA	28. 1 mg/L	[64]
	Shiraia sp. SUPER-H168	50 carbon sources (glucose, sucrose, maltose, amylose, amylopectin or corn flour) and 10 yeast extract	30	96	through overexpression of alpha-amylase gene	HYP	3521 mg/L	[65]
	Shiraia sp. SUPER-H168 modified by CRISPR/Cas9 system	Potato extract from 200 fresh potato, 20 glucose, 3 yeast extract, 10 peptone	30	120	NA	HYP	8632 mg/L	[66]
SSF	Shiraia sp. ZZZ816	Potato extract from 200 fresh potato, 20 dextrose	25	60	γ-irradiation, 100 Gy	HA	2018.3 mg/L	[67]
	S. bambusicola Henn. LBR-SB6	1% glucose, 20% rice bran 68.9% corn starch, 10% bran	26	168	NA	HYP	40 mg/kg	[68]
	Shiraia sp. SUPER-H168	20 g rice, moistened with 25 mL nutrient salt solution	30	432	NA	HA	2.02 mg/g dry solid substrate	[69]
	Shiraia sp. SUPER-H168	1.65 g glucose and 0.43 g NaNO3 with 100 g corn	30	432	NA	HA	4.7 mg/g	[70]
	Shiraia sp. SUPER-H168	1.65 g glucose and 0.43 g NaNO3 with 40 g corn	30	360	overexpression of alpha-amylase gene	HYP	71.85 mg/g	[65]

NA: Data not available.

shows the strains, fermentation methods, and HYP yield during different fermentation techniques. The fermentation processes of HYPs include solid-state fermentation (SSF) and submerged fermentation (SMF). Although SSF has the advantages of high productivity, product stability, and a low production cost [44], an insufficient mass and heat transfer and prolonged fermentation time are still challenging the SSF technique in HYP production [45].

Solid-state fermentation (SSF). SSF contributes to about 20% of the research on fermentation-based HYP production. The key raw materials in SSF include rice bran, rice, glucose, dextrose, NaNO₃, potato, and cracked corn. Typically, the temperature for SSF ranges from 25 °C to 30 °C, with most studies opting for 30 °C. The general pH value maintained is around 6.0. Despite its advantages, SSF usually requires extended fermentation periods, ranging from 168 to 432 h (Table 3). A notable exception is the mutant strain treated with Co⁶⁰-γ irradiation, which reduces the fermentation time to about 60 h. Recent studies have explored various strategies to enhance SSF productivity and reduce the process time, such as adding nutrient solutions, using a low dosage of radiation, and overexpressing the alpha-amylase gene. Initial outputs of HYP yields are typically around 40 mg/kg, but with optimized processes, yields have increased significantly from a range of 2.02 mg/g to 71.85 mg/g. With further process improvements, SSF has the potential for broader application in industrial-scale production due to its high productivity and low cost.

Submerged fermentation (SMF). SMF is the predominant method for HYP production, accounting for 80% of the production detailed in the existing reports. Its popularity stems from several advantages: shorter production times, higher yields, greater productivity, and reduced labor costs. The fermentation time in SMF usually spans from 72 h to 336 h, with efforts generally made to keep it under 200 h to minimize costs. The pH for HYP production in SMF typically ranges from 5.5 to 7.5, with 6.0 being the most common. Glucose and potato extract are the preferred carbon sources, although alternative sources like maltose, starch, and sucrose have shown beneficial effects on HYP yields. Yeast extract and NaNO₃ are commonly used as organic and inorganic nitrogen sources, respectively. As summarized in Table 3, HYP yields vary widely across different studies, with typical productivity ranging from 28.1 mg/L to 921.6 mg/L. Modifications such as enhancing cell membrane permeability have boosted yields to 780.6 mg/L by incorporating 0.6% Triton X-100 into the fermentation broth. Furthermore, genetic modifications targeting the PKS, zftf, and MCO genes in Shiraia sp. SUPER-H168 (CCTCCM 207104) have significantly increased the HYP productivity to 8632 mg/L. Alongside yield improvements, future research should also focus on production stability, a crucial aspect for scaling up to pilot plant practices.

3.3. Methods for Improving the Biosynthesis of HYPs

3.3.1. Medium and Process Optimization

Optimizing the fermentation medium and process parameters is crucial to maximize the HYP yield and enhance fermentation efficiency. In SSF, critical factors such as time, temperature, initial moisture content, initial pH, and inoculum amount significantly influence efficiency. Current research identifies optimal conditions for SSF as a 15-day fermentation cycle at 30 °C, with 50% initial water content, a pH of 7.5, and using 30 g of a corn–glucose–NaNO₃ substrate mixture [49]. The optimization of these factors, especially water content, is essential, as low levels can impede mass transfer, while high levels may restrict oxygen availability, thus inhibiting microbial growth. Post-optimization, HYP yields have improved significantly from a baseline of 40 mg/kg to between 2.02 mg/g and 4.7 mg/g.

In SMF, variables such as culture medium composition, culture duration, initial pH, volume, rotation speed, and inoculum size all play roles in optimizing HA yield. It is necessary to explore these variables across multiple gradients to identify the optimal growth and biosynthesis conditions for the mycelium, aiming to minimize raw material and time wastage while maximizing productivity. Key conditions for effective SMF include using a 5% maltose solution as the carbon source, yeast extract or urea as the nitrogen source, a fermentation duration optimized to peak HA production at 144 h, and an initial pH range of 5.5–7.0, with an agitation speed of 180 rpm [69]. Optimal conditions yield HA concentrations up to 921.6 mg/L, highlighting the importance of the carbon source and pH as primary optimization factors.

3.3.2. Surfactant Additives and Ultrasonic Stimulation

The late stages of fermentation often see a reduced intracellular production due to high product concentrations and complex downstream processing challenges. Surfactant addition can mitigate product inhibition and prevent degradation [71]. Research indicates that Triton X-100 significantly enhances HA production compared to other surfactants like Tween-40, Triton X-114, and SDS. Specifically, adding 0.6% Triton X-100 can increase HYP yields fourfold to 780.6 mg/L [50]. By comparing the performance of several nonionic surfactants, including Pluronic F68, Pluronic F-127, Tween-40, Tween-80, SDS, Brij 52, Span 80, and Triton X-100, Lei et al. found that 2% (w/v) Triton X-100, added after 36 h of fermentation, significantly boosted HA production to 1.88 mg/g DW and facilitated its release into the medium to 70.9 mg/L, cumulatively yielding 96 mg/L [41]. Li et al. introduced 25 g/L of Triton X-100 into the fermentation broth after 36 h, resulting in a 15.6-fold increase in HA exudate and a 5.1-fold increase in HA content within the mycelium. This intervention elevated the final HA yield to 206.2 mg/L. The significant increase in HA yield during extractive fermentation is largely attributed to enhanced permeability and cellular signaling mechanisms, specifically involving nitric oxide (NO) and ROS [59].

Furthermore, moderate ultrasonic treatment is proven to enhance mass transfer and cell wall permeability, thus improving both growth rates and yields [72]. For instance, the ultrasonic treatment of Phellinus igniarius at 0.28 W/cm² at 40 kHz increased the HA yield to 247.67 mg/L on the eighth day of fermentation [52].

3.3.3. Metal Ion Additives

It has been suggested that appropriate concentrations of Ca²⁺, Cu²⁺, and La³⁺ in the medium, which are crucial for enzymatic activity in microorganisms, can dramatically enhance HYP formation. For instance, Liu et al. demonstrated that adding 6 g/L Ca²⁺ increased the total PQ content to 21.93 mg/L, nearly six times the control level [53]. Studies by Xiang et al. highlighted that ions like Ca²⁺ and Cu²⁺ are particularly effective. A 0.05% Ca²⁺ in the medium could significantly increase the formation of HYPs to 8.12 mg/g mycelium. Cu²⁺ boosted mycelium growth as the mycelium dry weight reached 15.28 g/L when 0.03% Cu²⁺ existed in the broth. Lu et al. utilized lanthanum chloride (LaCl₃) as an initiator in the fermentation of S. bambusicola to enhance HA production. Culturing S. bambusicola in a medium containing 1.0 g/L of La³⁺ for six days facilitated a 2.5-fold increase in HA content within the mycelium, with the HA yield reaching 225.05 mg/L. Additionally, further investigations revealed that La3+ enhances HA production by promoting the intracellular accumulation of ROS [55].

3.3.4. Light Stimulation and Oxidative Stress

Light plays a crucial role as an environmental signal that regulates developmental and physiological processes across animals, plants, fungi, and bacteria [73], with S. bambusicola being no exception. Light has been shown to induce HA production, stimulating its accumulation and release in the mycelium through optimized light conditions. Sun et al. discovered that when S. bambusicola was cultured under a 24:24 h light/dark cycle for eight days, the maximum yield of HA reached 181.67 mg/L, which was 73.42% higher than that observed under constant dark conditions. This light/dark regime promoted the formation of smaller, denser mycelial pellets without inhibiting growth, which is advantageous for metabolite production in mycelial cultures. Additionally, the lighting condition triggered the production of ROS in the mycelia by upregulating genes associated with O₂·⁻ production, such as NADPH oxidase and cytochrome C peroxidase. These ROS, in turn, were involved in the upregulation of HA biosynthesis genes, including PKS, O-methyltransferase/FAD-dependent monooxygenase, and FAD/FMN-dependent oxidoreductase, thereby enhancing HA production [56]. Further studies by Ma et al. examined the effects of different light wavelengths and found that red LED light at 627 nm and 200 lux significantly promoted HA synthesis in the mycelium, with the maximum yield reaching 175.53 mg/L after an eight-day fermentation—approximately 3.82 times higher than the yield under dark conditions. An RNA-seq analysis revealed that many differentially expressed genes (DEGs) were linked to changes in membrane transport and composition, with an increase in the proportion of unsaturated fatty acids in the membrane. The enhanced secretion of HA induced by red light was attributed to alterations in the transport function and permeability of the mycelium membrane [57].

Fungal processes are predominantly aerobic, necessitating an ample oxygen supply and effective O₂ transfer rates within bioreactors [74]. Oxygen, however, acts as a double-edged sword for fungal producers. On the one side, the presence of oxygen facilitates the production of intracellular ROS, which, when imbalanced with intracellular antioxidant defenses, can hinder fungal growth [75,76]. Conversely, transcription factors linked to oxidative stress play a crucial role in regulating secondary metabolism; increased ROS levels can significantly enhance the formation of secondary metabolites. Thus, a carefully controlled oxidative stimulation may be used to boost the HA yield. For example, Deng demonstrated that incubating for 72 h with 10 mM and 20 mM H₂O₂ increased HYP yields by 27% and 25%, respectively [58]. As we know, the incorporation of Triton X-100 into the culture has been shown to enhance HA biosynthesis through redox changes, increasing ROS content within the mycelia and upregulating antioxidant enzyme gene expression. This increase in ROS may enhance HA production by influencing key biosynthesis genes such as PKS, monooxygenase, and O-methyltransferase [41].

3.3.5. Temperature Regulation

In addition to light, temperature is a critical environmental factor influencing the production of secondary metabolites in filamentous fungi [77,78]. In a recent study, we explored the impact of varying the fermentation temperatures during HA production in S. bambusicola. Our findings showed that at 32 °C, the HA content in the mycelia was five times and three times higher than at 26 °C and 28 °C, respectively. At 32 °C, the mycelia formed tightly wound and dense mycelial pellets, which affected nutrient and oxygen transfer to the inner layers, leading to autolysis due to nutrient and oxygen scarcity in the central mycelia [79,80]. The extent of autolysis at 32 °C was greater compared to that observed at 26 °C and 28 °C. Further insights were gained through RNA-seq analysis, which identified a co-expression network of highly expressed DEGs at 32 °C [81]. Notably, the Zn (II)Cys6 type zinc finger protein/fungal-specific transcription factor (MH01c06g0046321), and LIM domain protein/GTPase activating protein (MH01c11g0073001) acted as hub genes within the same submodule, potentially influencing HA biosynthesis and cellular machinery [43,82]. Additionally, qRT-PCR validation confirmed that key HA biosynthesis genes for FAD-dependent oxidoreductase (MH01c01g0006641), O-methyltransferase (MH01c03g0017691) FAD-binding monooxygenase (MH01c04g0029531), cytochrome P₄₅₀ (MH01c04g0030701), and PKS (MH01c22g0111101) were significantly upregulated at 32 °C [83,84]. These findings suggest that higher temperatures may significantly enhance HA synthesis by altering the mycelial morphology and upregulating critical biosynthetic genes.

3.3.6. Microbial Cocultivation

Secondary metabolites generally require specific chemical and physical signals for optimal production, which are challenging to replicate under standard laboratory conditions. One effective strategy to increase the yield of these metabolites is to mimic natural environmental signals through the addition of inducers and coculturing techniques [85]. For instance, after treating them with 50 g/mL of A. niger for four days, the yield of HYPs increased to approximately 90 mg/L, a 6.2-fold increase compared to the control [61]. You et al. explored the potential of endophytic bacteria and fungi isolated from bamboo stems infected by S. bambusicola. They identified the bacterial trigger TX4 and fungal trigger GZUIFR-TT1 (Trametes sp.), which significantly enhanced HYP production in Bambusa lutea, achieving a final yield of 102.60 mg/L—about 7.90 times higher than the control [5]. Additionally, You et al. achieved a HYP yield of 278.71 mg/L by introducing PB90, a protein activator from Phytophthora boehmeriae. PB90 not only enhanced metabolite production but also increased cell wall permeability and enzyme activity, while reducing the biomass of S. bambusicola [62]. Ma et al. reported on the diversity of bacteria associated with the stroma of S. bambusicola and their role in HA biosynthesis. Their study characterized the bacterial community within the stroma, dominated by Bacillus sp. and Pseudomonas sp. Further research revealed that certain Pseudomonas isolates could significantly boost HA production. Treatment with Pseudomonas sp. SB1 upregulated the expression of PKS involved in HA biosynthesis, as well as ATP-binding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters critical for HA secretion, resulting in a high yield of HA (225.34 mg/L), approximately 3.25 times higher than the control [63]. Moreover, Yan et al. discovered that the endophytic fungus Arthrinium sp. AF-5, when cocultured with S. bambusicola (GDMCC 60438) and other endophytic fungi isolated from bamboo, enhanced HA production significantly. Cocultivation with Arthrinium sp. AF-5 for 84 h resulted in an HA yield of 66.75 mg/g of carbon source, four times the yield when S. bambusicola was cultured alone [86].

3.3.7. Mutagenesis and Genetic Engineering

Classical strain development typically involves mutation, selection, or gene combination as foundational elements of commercial fermentation processes [87]. For S. bambusicola, targeted screening can be executed following random mutagenesis to exploit specific metabolic traits. For instance, Dong et al. applied ultraviolet light and a high temperature to deactivate the protoplasts of mutant strains UV4 and NU12, followed by polyethylene glycol (PEG)-mediated protoplast fusion. This process led to the selection of mutant strain FIII-21, which exhibited stable genetic traits and produced an HA yield of 80.4 mg/L, a 58.9–167.1% increase over the original strain [88]. Similarly, a 100 Gy dose of Co⁶⁰ γ-irradiation was applied to mutate spores of S. bambusicola, with a resulting 77.2% spore lethality and a 35% positive mutation frequency, culminating in a strain that produced 2018.3 mg/L of HA, which represents a 414.9% increase over the original strain and significantly surpasses other industrial HA-producing strains [70]. Du et al. treated the spores of the wild-type strain S. bambusicola GZ19 with ethyl methanesulfonate (EMS), obtaining mutant strain GZ19M1, with a notably enhanced HA production; after further optimization, this strain’s HYP yield was 8.7 times that of the wild type, achieving 498.89 mg/L [89].

While conventional mutagenesis can be inefficient and difficult to control, advances in molecular biology and the ongoing exploration of the genomic information of S. bambusicola have facilitated the application of genetic engineering. This includes the overexpression of regulatory factors and key enzymes within the HYP biosynthesis pathway. For example, Li et al. employed Agrobacterium-mediated transformation (AMT) to overexpress the cluster-specific transcription factor SbTF1, enhancing the HYP yield by 210.65% compared to the original strain [90]. Further, the genetic regulation of key enzymes in the HYP synthesis pathway proves effective; Deng et al. used polyethylene glycol and CaCl₂ to introduce a lentiviral PgfpHyg vector in S. bambusicola SUPER-H168, overexpressing MCO and achieving a yield approximately five times that of the control [91]. Additionally, overexpressing carbon metabolism-related genes has shown to increase HYP production by expanding the polyketide precursor pool. Gao et al. created strains overexpressing four glucosidase genes, significantly boosting liquid fermentation yields of HA; notably, the Amy365-1 and Amy130 co-expressing strain produced an HA yield of 71.85 mg/gds, 2.83 times that of the wild type [65]. Li et al. optimized the polyethylene glycol transformation system through plasmid transfection, successfully overexpressing the double-domain protein and hydroxylase gene, which increased hypocrellin production threefold and twofold, respectively [92].

The advent of CRISPR/Cas gene editing technology has revolutionized genetic engineering in filamentous fungi, enhancing the synthesis of secondary metabolites. Significant strides have been made in transforming engineering strains for enhanced production capabilities. Deng et al. developed an efficient CRISPR/Cas9 system by optimizing sgRNA transcription elements and disrupting non-homologous end-joining genes. They simultaneously edited genes related to precursor supply, the central hypocrellin pathway, and the antioxidant system, achieving a remarkable 8632 mg/L of hypocrellin, 12 times the yield of wild-type strains [66].

3.4. Challenges and Solutions in HYPs Production Techniques

Although significant strides have been made in enhancing the fermentation of PQDs or HYPs by Shiraia sp., several critical areas remain underexplored. One notable challenge is the inability to accurately assess and compare the potential for large-scale production due to the absence of standardized criteria for measuring HYP productivity and the lack of detailed information on strain stability. Most strains were not conserved in third-party preservation centers at the time of publication, which complicates the replication and verification of results. Establishing uniform experimental criteria is essential for validating, comparing, and assessing the reliability of the productivity of selected strains. Despite the increased interest in using mutagenesis and genetic engineering to accelerate HYP production, the underlying biosynthesis pathways and mechanisms that significantly improve HYP productivity through cocultivation remain largely unknown. These need further investigation.

The application of genetic engineering in transforming S. bambusicola faces challenges, including low transformation efficiency and low frequency of homologous recombination. Current transformation methods, primarily PEG-mediated and AMT, rely on protoplasts prepared from single-cell spores. Protoplast preparation in some filamentous fungi is problematic, depending heavily on the growth state of the mycelium and the type, dose, and batch of reagents. AMT requires specific conditions related to the type and amount of Agrobacterium and coculture conditions. Future efforts should focus on improving the conversion efficiency of editing tools and editing efficiency in S. bambusicola mycelia that do not produce single-cell spores. For multinucleated cells in mycelia that produce multinucleated spores and protoplasts, the efficiency of simultaneous editing is low, making it extremely difficult to obtain homozygous offspring. Zou et al. used RNPs-mediated CRISPR/Cas9 gene editing and PEG-mediated transformation methods, controlling the mitotic cycle with inositol and phenylalanine, and significantly improved transformation efficiency by adding surfactant Triton X-100 and extending the culture time. This optimization significantly enhanced the efficiency of obtaining homozygous transformants [93]. Given the varied growth statuses and physiological characteristics of different S. bambusicola mycelia, it is crucial to consider a range of factors when selecting genetic transformation and modification methods to enhance the yield of positive clones. Using RNPs-mediated CRISPR gene editing in polykaryotic mycelia could be a viable approach.

New editing systems like the Base Editor (BE), Prime Editor (PE), CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), and CRISPR/Cas-based marker-free gene editing systems are evolving and being applied in fungi. The use of plasmids carrying the AMA1 sequence, which does not integrate into the genome and can self-replicate, allowing multiple rounds of recycling under non-selective culture conditions, addresses the challenge of the lack of screening markers in filamentous fungi such as S. bambusicola.

As the biosynthetic gene cluster (BGC) of HYPs has been proposed, suitable model fungi like Aspergillus, known for its clear genetic background and well-defined metabolic pathways, can be used to reconstruct the BGC of HYPs [7,8]. Some auxotrophic strains of Aspergillus require only positive clone screening on selection media, simplifying the system and facilitating the rearrangement of intracellular carbon flux towards the precursor supply of HYPs.

While the BGC of HYPs offers a foundation, the complete biosynthetic pathway and the involved genes and enzymes are not fully elucidated. Modifying non-model filamentous fungi like S. bambusicola genetically remains challenging. Ongoing research should aim to further decode the biosynthetic pathway and regulatory mechanisms of HYPs to establish a research platform for model fungi of S. bambusicola, develop high-titer production strains, and combine metabolic engineering with fermentation process optimization to enhance industrial HYP production.

4. Techniques to Improve the PDT Performance of HYPs

4.1. Chemical Modification Methods

To enhance the water solubility, light absorption, and ROS yield, chemical modifications of HYPs typically target the hydroxyl, carbonyl, aromatic ring, and cyclomethoxy groups through amination and sulfonation reactions (Figure 6).

However, these modifications often result in a significant reduction in photosensitizing properties, such as decreased ROS yields, compared to the unmodified HYPs. Among these, amine-substituted HYP derivatives are extensively documented. These derivatives, including compounds 2, 3, 7, 8, 11, 12, 27–29, 31–33, generally exhibit greatly enhanced water solubility (

Table 4. Key characteristics of HYP derivatives *.

Compounds	Aqueous Solubility	Characteristic Wavelength	Quantum Yield		Reference
			1O2 Generation (Φsinglet oxygenType II)	O2·− Generation (ΦSuperoxide Type I)
2	++	++	-		[94]
3	++	+	-	+++	[94]
4	++	NA	--	+++	[95]
5	NA	-	--	+	[95]
6	++	-	-	++	[96]
7	++	--	--	+	[96]
8	++	-	+	+	[97]
9	NA	+	NA	NA	[98]
10	+	--	+		[99]
11	++	+	--	-	[99]
12	++	+	--	--	[99]
13	+	+	--	--	[100]
14	+	++	--	-	[100]
15HA	Insoluble	-	+	+	[101]
16HB	Insoluble	-	+	+	[101]
17	Insoluble	++	+	--	[102]
18	Insoluble	+++	+	+++	[102]
19	++	++	+	--	[102]
20	++	+++	-	+++	[102]
21	++	-	+	NA	[101]
22	++	--	--	NA	[101]
23	++	-	--	++	[101]
24	++	-	---	+++	[101]
25	+	-	---	+++	[101]
26	++	-	---	+++	[101]
27	++	++	--	NA	[103]
28	++	++	--	NA	[103]
29	++	++	---	NA	[103]
30	+	+		NA	[104]
31	++	+	---	NA	[105]
32	++	+	---	NA	[105]
33	++	+	--	NA	[105]
34	NA	NA	+	NA	[106]
35	++	+	--	NA	[106]
36	+	+	--	NA	[107]
37	+	+	--	NA	[107]
38	+	-	---	NA	[107]
39	+	-	---	NA	[107]
40	+	-	---	NA	[107]
41	+	-	---	NA	[107]
42	+	-	---	NA	[107]
43	+	-	---	NA	[107]
44	+	+	---	NA	[107]
45	+	+	--	NA	[107]
46	++	+	-	NA	[108]
47	++	++	+	NA	[108]
48	++	+	NA	NA	[108]
49	++	++	-	NA	[108]
50	++	++	-	NA	[108]
51	++	-	NA	NA	[109]

* Aqueous solubility: soluble (+); very soluble (++). Characteristic wavelength: <450 nm (--); 450–600 nm (-); 600–625 nm (+); 625–680 nm (++); >680 nm (+++). ¹O₂ generation quantum yield (Φ_{singlet oxygen}): high (+); medium (-); low (--); very low (---). O₂·⁻ generation (Φ_Superoxide): high (+); medium (-); low (--); very low (---). NA, not available.

). Despite this improvement, there is a corresponding decrease in ¹O₂ yield, particularly when the amine substitution occurs on the pentacyclic perylenequinone core structure. Notably, compound 8, which features the amino group on the seven-membered ring outside the perylenequinone core, does not show a significant reduction in photosensitizing properties. Conversely, derivatives where the amino group substitutes a methoxy group on the seven-membered ring (such as compounds 31–33) also show diminished photochemical properties. Additionally, many amine-substituted HYP derivatives exhibit a red-shift in their absorption wavelengths, aligning more closely with the optimal wavelength window for PDT. Similarly, sulfonate-substituted derivatives improve in water solubility by forming organic salt structures (compounds 22 and 35), yet their ¹O₂ yield decreases, even when the sulfonation is outside the perylenequinone core. Thiol-substituted derivatives also enhance water solubility but at the expense of ¹O₂ yield. However, these derivatives show a significant increase in O₂·⁻ yield. Moreover, modifications involving sugars and amino acids not only enhance water solubility but also impart properties like an affinity for DNA or cell membranes, evident in compounds 36–45. These modifications showcase the potential of chemical alterations to balance the solubility and functional efficacy of HYPs in clinical applications.

4.1.1. Amination

Amination allows HYPs to react with various amines, forming derivatives with enhanced water solubility. Typically, amination does not occur at the steric hydroxyl group or the anthracycline structure, thus preserving the photodynamic activities of the derivatives. As a result, the photodynamic properties and absorption under the PDT window are generally improved. However, the high reactivity of HYPs with amines often leads to the complex reaction products and low yields (between 5% and 15%) of the amine-derived HYPs, complicating their separation and purification. Some amine-derived HYPs require mild reaction conditions, necessitating extreme dryness and prior drying of the solvent, which limits their practical application. Deng et al. targeted the aldehyde group on the seven-membered ring for substitution, producing amino-sulfonated HB with a carbon–nitrogen double bond. This derivative, compared to the 2-substituted amino-sulfonated HB, exhibited better amphiphilicity and a higher distribution coefficient, suggesting potential for human body absorption without the need for drug carriers [110]. Lee synthesized 2-ethanolamino-2-demethoxy-HB (EAHB1) and 2-thanolamino-2-demethoxy-17-ethanolamino-HB (EAHB2) through a mild reaction [100]. UV–Vis spectra of the EAHBs show enhanced absorption in the red spectral range (600–900 nm), with significant potential to generate ¹O₂ and O₂·⁻. Their improved hydrophilicity and potential specific affinity for malignant tumors make EAHBs promising candidates for PDT applications, warranting further photobiological investigations [100].

4.1.2. Sulfonation

Sulfonated HYPs, classic derivatives in the family, maintain a high water solubility without compromising absorption efficacy within the PDT spectrum [111]. The sulfonation reaction produces few byproducts, simplifying the separation and purification processes. According to ESR spectroscopy, 14-dehydroxy-15-deacetyl-HA-13-sulfonate (13-SO₃Na-DDHA) engages in both type I and type II photochemical reactions [111]. Additionally, mercaptan-substituted HB derivatives (MHBDs) are prepared through nucleophilic addition and oxido-reduction reactions on the perylenequinone core, significantly enhancing red absorption and efficiently generating ¹O₂ through the Type II reaction [112].

4.1.3. Derivation with Biomacromolecules

Carbohydrate-modified HYPs have been synthesized using improved classical Königs–Knorr reactions, with mono-mercaptoethanol or di-mercaptoethanol substituting on the perylenequinone core (compounds 36–45). These modifications confer a specific affinity for malignant tumor cells and enhance membrane interactions, thereby increasing the efficacy of HYPs in targeting and destroying tumor cells [107]. Additionally, the inherent biological affinity of amino acids has been leveraged to produce amino acid-sustained HYP derivatives, significantly enhancing their photodamage capability against tumor cells [113]. For instance, a tyrosine-modified HB (TYHB), composed of a 1:1 molar ratio of 5- and 8-substituted HB, was synthesized via the photoaddition of mercaptoacetic acid to HB, followed by amidation with tyrosine methylester hydrochloride. This derivative has demonstrated a unique affinity and enhanced photodamage capability over calf thymus DNA (CT-DNA) [114].

4.1.4. Chelation with Metal Ions

The lone pair of electrons on the carbonyl oxygen of HYPs enables them to chelate with various metal ion ligands, forming metal chelates of HYPs, as shown in Figure 6 and detailed in Table 4. These macromolecular HYP chelates are capable of forming one-, two-, and three-dimensional structures, which increase their cellular and DNA affinity, thus enhancing the photodynamic response [111]. HA and HB can form chelates with a wide range of metal ions, including Mg²⁺, Ca²⁺, Pr³⁺, Ti³⁺, V³⁺, Cr²⁺, Fe³⁺, Fe²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ga³⁺, and La³⁺ (Figure 7). These coordination compounds exhibit a high thermal and photochemical stability in neutral media and a strong DNA affinity [108,115]. However, these chelates disintegrate in extremely acidic (pH < 1) or alkaline (pH > 12) environments. Notably, the solubility of Mg²⁺-HA and Mg²⁺-HB is particularly high. Recent developments include the design of five new HB-dinuclear Cu²⁺ chelates ([Cu₂(HB)(X)₂](PF₆)₂, where X = bpy, phen, tmp, dpq, dppz), illustrated in Figure 8 which have shown high efficiency in photocleaving supercoiled pBR322 DNA [116]. Additionally, a new Co(III)HB complex, [Co₂(HB)(tmp)₄]⁴⁺, has been synthesized and tested (see Figure 9). This complex demonstrates good affinity for dsDNA and superior photocleavage ability compared to the dinuclear Cu²⁺ chelates [117].

4.2. Physical Modification Techniques

4.2.1. Liposomes

Liposomes, known for their excellent amphiphilicity, have been extensively utilized in the physical modification of HYPs, as illustrated in Figure 10 [118]. Beyond improving their amphiphilicity, encapsulating HYPs in liposomes has enhanced their stability. Additionally, amino-substituted HB derivative (DPAHB) has been incorporated into self-assembling biodegradable nanovesicles using poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PEG-PLAG), resulting in water-dispersible DPAHB NVs, depicted in Figure 11 [98]. These nanovesicles have demonstrated superior PDT efficiency compared to chlorin e6. Both in vitro and in vivo studies indicate that DPAHB-NVs possess high photothermal stability, improved tumor accumulation, and a favorable biodegradation rate, showcasing their potential as effective carriers in PDT applications.

4.2.2. Nanoparticles

HYP-derivative nanoparticles, specifically APHB-NPs, were successfully self-assembled using polyethylene glycol polylactic-coglycolic acid (PEG-PLGA) and APHB, as depicted in Figure 12 [119]. These nanoparticles not only improve water solubility and PDT efficiency but also exhibit excellent biocompatibility, suitable biodegradation rates, and enhanced tumor targeting and accumulation. Additionally, a novel biodegradable nanoparticle system utilizing polyethylene glycol-modified gelatin (PEG-GEL) and polylactic acid (PLA) has been developed to further enhance the photodynamic effectiveness of cyclohexane-1,2-diamino-HB (CHA2HB) [120]. In vitro studies have demonstrated that CHA2HB-loaded PEG-GEL-PLA nanoparticles are effectively internalized by various cancer cell types, including human breast adenocarcinoma (MCF-7), human gastric sarcoma (AGS), and mouse-specific Dalton’s lymphoma (DLA), showcasing their potential as targeted delivery vehicles for PDT applications.

4.2.3. Cosolvent Additives

HYPs exhibit solubility in micellar media, which include mixtures of water with surfactants or organic reagents. For instance, micelles formed by acetonitrile and water have been used to prepare aqueous HYP micelles at a concentration of 2.0 × 10⁻⁴ mol/L, facilitating studies into the electrochemical properties of HYPs in aqueous solutions [121]. Enhancements in the water solubility of HYPs were achieved up to 2.0 × 10⁻⁵ mol/L with sodium dodecyl sulfate (5.0 × 10⁻⁵ mol/L), 3.0 × 10⁻⁴ mol/L with tetradecylpyridine bromide (5.0 × 10⁻⁵ mol/L), and 5.0 × 10⁻⁵ mol/L with Triton X-100 (1.0 × 10⁻⁵ mol/L) [122]. Among these, Triton X-100 demonstrated the most effective solubilization.

4.3. Challenges and Solutions in Enhancing HYPs PDT Performance

Physical techniques for forming HYP complexes generally result in higher recovery rates compared to chemical modification methods. For example, Xu et al. reported a 56% yield in synthesizing diamino-substituted HYPs using a one-step reaction [123]. Commonly, chemical modifications involving one-step or multistep reactions generate numerous byproducts, leading to lower yields and the potential wastage of HYPs. In contrast, complexes like HYPs/CuCo₂O₄ and SiO₂-HB were prepared by evaporating solvents under reduced pressure, a process that does not involve chemical reactions, thus avoiding the low yields associated with such modifications [124,125]. These complexes also tend to have more stable structures than chemically modified HYP derivatives. However, it is important to note that all chemical modification methods require alterations to the chemical structure of HYPs, which can disrupt their photochemical properties [123]. This highlights a significant challenge: maintaining the integrity of HYPs’ photochemical properties, while enhancing their solubility and stability through modifications.

5. Conclusions and Prospects

HYPs are distinguished by a highly conjugated aromatic pentacyclic dione with axial chirality. Notably, they exhibit noteworthy antibacterial, antiviral, and cytotoxic properties. Their compelling structural fragments and bioactivities have spurred our interest in further investigation.

5.1. Structure Modification for Improved Performance in PDT

Due to the high reactivity of HYPs, a wide range of chemical reactions are suitable for modifying these compounds, with mild amination being the most common and effective method. This adaptability allows HYPs to acquire novel properties such as an affinity for DNA on tumor cells, which has garnered significant research interest. However, the byproducts from these chemical reactions pose substantial limitations to the development of chemical modifications of HYPs. As such, there is a pressing need for high-yield chemical modification techniques to enhance the application of HYP derivatives. In contrast, physical modification techniques offer a higher atomic utilization, simpler separation and purification processes, and safer end products. Expanding the range of physical modification techniques, such as developing more HYP complexes, could further improve the water solubility and photochemical properties of HYPs.

5.2. Technique Development for Improved Productivity During Fermentation of HYPs

At present, a variety of physical and chemical methods have been employed to enhance the yield of fermentation products used in HA production, achieving some significant results. For instance, the highest recorded yield of HYPs in a laboratory setting was 8632 mg/L [66]. However, inconsistencies in the determination and calculation methods for productivity across different studies make it challenging to systematically compare technical achievements. A standardized set of calculation criteria is essential for future studies to enable meaningful comparisons. Given the multiple strategies available for increasing target product yields, a combination of methods should be pursued for further productivity enhancements, while also exploring the fundamental processes involved. Although the secondary metabolic pathway of S. bambusicola for producing HA remains unclear, advances in whole-genome sequencing and functional gene analysis could deepen our understanding of HA biosynthesis at the molecular level, potentially improving yields.

5.3. Biotechnology for Emerging New HYP Derivatives

Our research has demonstrated that the application of specialized fermentation techniques has facilitated the production of novel HYP compounds with structures resembling perylenequinone, highlighting an advanced antitumor activity. This represents a promising avenue for expanding the chemical diversity of HYPs and contributing to elucidating their biosynthetic pathway (unpublished data). Furthermore, the biosynthesized non-natural compound cercosporin A exhibits high photostability and minimal dark toxicity, while retaining potent photodynamic antimicrobial and anticancer activities [126]. Through the use of synthetic biology strategies, the biosynthetic pathway could be reprogrammed, enabling the biosynthesis of a range of perylenequinone derivatives with diverse configurations and substituents.

5.4. Industrialization of HYP Biosynthesis for Advanced Applications

Research into the pilot industrial fermentation of HYP is crucial for the future scaling up of fermentation processes. Novel regulatory techniques (e.g., particle loading, microbial coculture, light induction, nitrogen source control, and inducers), along with computational fluid dynamics-based reactor optimization and process enhancement (oxygenation, mass transfer), have been introduced to regulate HYP production through the deep fermentation of S. bambusicola at different scales. Simultaneously, the differentially expressed genes of S. bambusicola could be analyzed using a multi-omics strategy, and the functions of key enzymes and regulatory factors verified through metabolic flow analysis, CRISPR-Cas9 gene editing, and molecular cloning and expression techniques, thereby revealing the biosynthesis of HYPs and the associated regulatory mechanisms at the molecular scale. Based on this foundation, molecularly oriented modification should be conducted to further enhance the efficiency of HYP biosynthesis in S. bambusicola. This study could benefit from unveiling the regulatory mechanism of S. bambusicola response fermentation at multiple scales and developing control strategies for the optimized process.

Understanding the economic implications of pilot-level fermentation is crucial for transitioning the technology to larger scales and broadening the application of HYPs in areas such as environmental protection, agriculture, and the food industry. In these fields, HYPs could be utilized for the degradation of persistent organic pollutants, the biological control of pests and diseases in crops, and as food preservatives and coloring agents [127].