Microorganisms function as biocatalysts in the development of novel drug leads by producing compounds with functionalized moieties due to their ability to facilitate controlled oxidation reactions in C − H bonds of compounds [13, 21, 43, 47, 48]. One of the sought-after biocatalysts used include those possessing P450s which Alternaria species possess [26,27,28, 45]. These biocatalysts have the potential of oxidizing sp3 and sp2 C − H bonds with a high degree of chemo, regio, and stereoselectivity and other compounds with varying complexities in their structures [29, 30]. The enzymes are also responsible for the metabolic breakdown of drugs in humans, the biodegradation of pesticides, and environmental pollutants [28, 29]. The monooxygenation on sp3 and sp2 hybridized carbons is usually denoted by oxidation, epoxidation, methylation reactions amongst others, while the C = C double bonds oxidation is one of the widely known oxidation reactions by P450s [29, 45].

P450s are a large group of hemeprosthetic monooxygenase enzymes linked to a protein [29]. The enzymes are responsible for a plethora of oxidative reactions against highly diverse range of substrates. They activate molecular oxygen (O2), by simultaneously incorporating one oxygen atom into an organic substrate, while producing a water molecule with the remaining oxygen atom as shown in Eq. 1[51]. This mechanism uses the universal cellular cofactors NADH or NADPH as a reducing agent to deliver electrons via iron-sulfur protein or via flavoprotein [49, 51].

$$}\left( } \right)}\,\,\,\,}_ \to }\left( } \right)\, + \,}\,\,}_ }$$

(Reaction 1)

$$}_} + } \to }_$$

(Reaction 2)

$$}_ + } + }} \to } + O}}$$

(Reaction 3)

Equation 1: Stoichiometric reaction in P450s facilitated oxidation reaction [51].

Reports on the mechanisms involved in the oxidation of molecules using P450s to understand the reactivity and regioselectivity properties of these enzymes are widely available [45, 51]. Also, many studies have been conducted to understand the mechanisms involved in the catalytic cycle of P450s [45]. Briefly (Fig. 1), step 1 shows how the substrate is indirectly bound near the inactive heme iron. Step 2 shows the delivery of electrons by a di-flavin reductase followed by the binding of molecular oxygen to the inactive ferrous iron to form a Fe–O complex (step 3). Step 4 shows the formation of a Fe3+-O2¯ complex also known as compound 0. The formation of complex 0 is associated with the delivery of electrons from the reductase and is widely postulated to be involved in certain oxidation reactions. Step 5 and 6 involves the protonation of compound 0, and H2O elimination from the complex to obtain compound 1 previously described as FeO3+. The unstable compound 1 is mainly involved in most oxidation reactions involving P450s. Step 7 shows how compound 1 when near a substrate, is responsible for a hydrogen atom removal/ elimination from the substrate. Oxygen rebound occurs in step 8 via radical recombination to afford an oxygenated molecule and the formation of an enzyme–substrate complex taking place in step 9 [27, 45, 51, 52].

Catalytic Cycle of Cytochrome P450s. [27, 45, 51, 52]

The efficiency of these enzymes, compared with chemical methods, in catalysing the insertion of oxygen into unactivated C − H bonds under mild reaction conditions has sparked interest among researchers toward investigating and exploiting P450s for a variety of synthetic applications [26, 29, 30]. The capacity to which these enzymes can perform to biosynthesise molecules is highly dependent on the substrates used [53, 54]. Below is a proposed route to which P450s produce biotransformation products. The illustration below makes use of the known taxane compound paclitaxel from beccatin under the same conditions as described in Fig. 2.

Biotransformation of a taxane derivative via a P450s mediated reaction to afford paclitaxel. [53, 54]

Most oxygenation reactions during microbial transformation take place in phase I. Where a substrate-inducible oxygenase in the presence of molecular oxygen and the cofactor NADPH allows the insertion of a reactive oxygen atom at specific positions on the substrate to produce oxygenated molecules. Additionally, this reaction produces unstable intermediates that undergo spontaneous nonenzymatic rearrangement such as the elimination of acetyl groups, methyl migration to produce compounds with different functionalities as end products. An example includes the microbial biotransformation of 4′-demethylepipodophyllotoxin (4) in the presence of A. alternata S-f6 as a transformation catalyst to biosynthesise 4′-demethylpodophyllotoxone (DMEP) (5). The fungi facilitated the conversion of the hydroxyl group at position C-4 to a carbonyl group. Additionally, the activating agent C = O present in DMEP subsequently underwent trans-amination with ligustrazine after incubation with an Alternaria species resulting in the formation of 4-(2,3,5,6-tetramethylpyrazine-1)-4′-demethylepipodophyllotoxin (4-TMP-DMEP) (6) as shown in Fig. 3.[55]

Oxygenation reaction facilitated by A. alternata [55]

As stated by many authors that biotransformation products have the capacity to exhibit elevated bioactivity compared to the parent compound, so in comparing the bioactivity of biotransformation products with the parent compound, the biotransformed products were reported with a 50% effective concentration (EC50) that was more than 5000 folds efficacious than (4) (EC50 = 529 μM) and (5)- (EC50 = 0.11 μM). Simultaneously, the EC50 of (6) against the normal human proximal tubular epithelial cell line HK-2 (i.e., 0.40 μM) was 66 times higher than that of (4) (i.e., 0.006 μM). Furthermore, compared with the parent compound 4 (i.e., log P = 0.34), the water solubility of biotransformation product (6) (i.e., log P = 0.66) was significantly enhanced by 94% [55].

The anti-fungal and anti-bacterial monoterpene α-terpinol 7, a volatile component of numerous pharmaceutical preparations, is another compound of interest in biotransformation studies. The metabolic pathway of (7) in mammals has been investigated with reports showing P450s as reaction catalysts. Additionally, biotransformation capabilities of A. alternata with 7 produced two oxidative products namely 4R-oleuropeic acid (9) and (1S,2R,4R)-p-menthane-1,2,8-triol (10) as shown in Fig. 4. [56] The biotransformation resulting to (9) is reported as a one step process while the reaction for (10) is via an intermediate 7-Hydroxy-α-terpineol (8) [56].

The biotransformation of α-terpenol by A. alternata to produce two oxidative products namely 4R-oleuropeic acid (9) and (1S,2R,4R)-p-menthane-1,2,8-triol (10). [56]

There is no available data on the bioactivity of (9) yet, but its derivatives are reported to exhibit anti-bacterial, anti-inflammatory, and analgesic properties. Additionally, the biotransformation of 7 is shown to be stereoselective with observed stereochemistry on (9) and an unstable diol intermediate that is later acetylised to form (10) [57].

Solidagenone (11) isolated for the first time from the rhizomes of Solidago chilensis Meyen (Asteraceae) is reported to have potent proliferative properties against different cancer cell lines [57]. Additionally, the gastroprotective properties and low oral toxicity of (11) led to further studies with focus on the structure activity relationship of the compound along with its derivatives [40, 58]. The biotransformation of (11) in the presence of A. alternata ATCC 44501 led to the production 3-oxosolidagenone (12) [40, 58]. The biotransformation was reported to be regioselective as the oxygenation took place at position C-3 of the molecule as shown in Fig. 5. The reaction is notably initiated by ring A hydroxylation with no report on the formed intermediate [40].

Biotransformation of solidagenone using A. alternata to yield 3-oxosolidagenone (12) [40]

The bioactivity of 12 is attributed to the furan moiety [58]. Additionally, derivatives possessing a hydroxyl group at position C-3 also possess some form of bioactivity. Some more examples of compounds that are reported to have experienced oxygenation mediated reaction in the presence of an Alternaria species are listed in Table 1.

Enzymatic hydroxylation reactions involve the bioconversion of a C-H bond to form a C–OH bond, with the assistance of the hydrolase enzymes [30, 53, 63]. The isolation of hydroxylase enzymes is reported to be a challenging process due to their unstable nature in pure form [27,28,29, 64]. However, the use of microorganisms possessing P450s is known to facilitate the hydroxylation of natural products [28]. A study on the stereospecific hydroxylation of platensimycin and its biosynthesis reportedly shows how P450s facilitated reactions results to a C-7 α-OH orientation via a dehydrogenase installation as shown in Fig. 6 [63].

Stereospecific hydroxylation mechanism facilitated by P450s [63]

Ursolic acid (21) a pentacyclic triterpenoid with medicinal properties including anti-inflammatory, analgesic and anti-cancer activities. The compound is also known for its water solubility problems, hence numerous selective biotransformation studies on (21) have been done to improve its solubility properties [41]. A biotransformation of ursolic acid by A. alternata resulted in 8 biotransformation products namely corosolic acid (22), urs-12-en-2α,3β,28-triol (33), 3β,23-dihydroxyurs-12-en-28-oic acid (24), 2α,3β,23-trihydroxyurs-12-en-28-oic acid (25), 2α,3β,23,24-tetrahydroxyurs-12-en-28-oic acid (26), 3β,28-dihydroxy-12-ursene (27), urs-12-en-3β-ol (28), and urs-12-en-2α,3β-diol (29) with some possessing a common hydroxyl group at position C-2 as shown in Fig. 7 [41]. Upon examining the compounds for their antiproliferative properties, the authors observed an increase in the activity when comparing the anti-proliferative properties of (21) with compounds having multiple hydroxyl groups. The observed increase in efficacy was later attributed OH groups at positions C-2,23 and 24.

Proposed biotransformation of ursolic acid by A. alternata

Cyclocanthogenol (30) is a cycloartane type triterpenoid, belonging to a class of compounds that possess anti-inflammatory, analgesic, sedative, and hypotensive properties [65]. The biotransformation of (30) by an Alternaria species afforded 8 compounds. However, only 3β,6α,12α,16β,24(S),25-hexahydroxycycloartane (31), 3β,6α,16β,22,24(S),25-hexahydroxycycloartane (32) and 3β,6α,16β,17α,24(S),25-hexahydroxycycloartane (33) underwent hydroxylation [66]. The hydroxylation on the compounds was observed at different carbon positions C-12,17, and 22 as shown in Fig. 8. Notably, the stereochemistry of the compounds was not altered, implying that the enzyme did not facilitate the C-H bond rotation. Additionally, there is no record on the biological activity of the biotransformed compounds reported in this study [66].

Biotransformation of cyclocanthogenol by A. eureka [66]

Enhanced neuroprotective properties of a cycloartane-type sapogenin (34) were reportedly observed after incubating (34) with A. eureka 1E1BL1. The reaction afforded 17 biotransformation products (35–51) possessing hydroxyl groups in different carbon positions as shown in Fig. 9. Additionally, the hydroxylation on (34) mainly occurred at positions C-11 and/or C-12 for most compounds. A noticeable stereoselectivity of the reaction was observed, based on the α and β oriented hydroxyl groups reported on the molecules [24, 67]. Of the 17 biotransformation products only compound (50) possessed a hydroxyl group at both positions, also the orientation of the hydroxyl bonds was reported as α and β. The compounds were further reported to regulate the reduction of H2O2-mediated oxidative stress and inhibition of H2O2-induced mitochondrial damage [24]. Of particular interest about the biotransformation products of this reaction was the observed different reactions that were facilitated also included epoxidation, methyl migration, ring expansion, ring cleavage and so on [24, 67].