Science Reviews - Biology, 2024, 3(4), 7-23 Prasanta Chakraborty

Gene cluster regulators from plants to microbes: Key

role in vesicular targeting, transport of intermediate

compounds and secondary metabolite biosynthesis

Prasanta Chakraborty, PhD

Indian Institute of Chemical Biology, Kolkata, West Bengal, India

Scopus: https://www.scopus.com/authid/detail.uri?authorId=57200536237

https://doi.org/10.57098/SciRevs.Biology.3.4.2

Received October 10, 2024. Revised October 25, 2024. Accepted November 05, 2024.

Abstract: Plants and microbes (e.g., bacteria, fungi) produces various types of secondary metabolites (SMs)

that may be used as nutraceuticals, defense molecules, drugs/antibiotics etc. Efficient biosynthesis of these

SMs depend upon the transport and accumulation of biosynthetic intermediates, enzymes and transporters in

a specific compartment of cells of the organism. Signals for biosynthesis of these important SMs often comes

from the related gene clusters and their regulators situated inside or outside (global) the cluster. In bacteria,

cluster situated regulators (CSRs), like SARPs (Streptomyces antibiotic regulatory proteins) and other

regulators regulates biosynthesis of many important drugs and antibiotics, in fungi, 40% CSRs and 60% global

regulators control the SM biosynthesis, whereas in plants mainly global regulators works. In this review, how

these regulators play their part in the transport and efficient biosynthesis of some important compounds in

plant and microbes will be discussed.

Keywords : Regulators; Gene cluster; Secondary metabolites; Biosynthesis; Plants; Microbes

Introduction

Secondary metabolites from plant and mi-

crobes are considered indispensable for survival,

growth, and metabolism, and to perform many

other numerous functions. To counter the resistance

issue, drug discovery is essential. Hence secondary

metabolites are important natural sources which are

focused to find new drugs. Many valuable mole-

cules like defense molecules, anti-nutritional com-

pounds, drugs/antibiotics have already been ob-

tained from these metabolites, e.g., cyanogen glyco-

sides, steroidal alkaloids from crop plants, anti-can-

cer drugs vinblastine/vincristine, noscapine from

medicinal plants (1-5), penicillin, tetracycline and

many more from microbes (6-9). Efficient produc-

tion of any valuable molecules/drugs in plants and

microbes depends upon the localization of their sec-

ondary metabolite biosynthetic intermediates, bio-

synthetic enzymes and transporters in the specific

vesicles of the organism e.g., biosynthetic enzymes

for penicillin biosynthesis, isopenicillin N-acyl-

transferase along with their substrate was localized

in peroxisome of fungus Penicillium chrysogenum

(10,11), biosynthetic intermediates vindolines,

catharanthine along with their transporters for anti-

cancer drug vinblastine/vincristine was localized

in internal leaf cells and leaf surface of medicinal

plant, Catharanthus roseus (12-14), and MATE(multi-

drug and toxin extrusion) transporter, SbMATE2

was localized along with cyanogen glucosides in

vacuolar membrane of food plant sorghum (15).

Targeting and subcellular localization of the trans-

porters and the biosynthetic enzymes are tightly

regulated by the expressions and regulations of

their genes. Though the recent literature indicates

that the genes of the biosynthetic enzymes of sec-

ondary metabolites and many transporters in plant

and microbes are organized as gene cluster in the

genome, e.g., metabolic gene clusters for terpenoids

and alkaloids in plants (16-20), gene clusters for

penicillin, tetracyclines in microbes (21-24); there

are very scanty reports about their regulations.

Many global and cluster situated regulators (CSRs)

may be operative in the regulation/ expression of

Prasanta Chakraborty Science Reviews - Biology, 2024, 3(4), 7-23

these genes responsible for secondary metabolite bi-

osynthesis.

The regulators are family of activator-re-

pressor type of transcription factors which plays

important role in the regulation of plant and mi-

crobe’s secondary metabolism. In plants, R3-MYB

(MYOBLASTOSIS), bHLH (helix-loop-helix) and

Apetala2/ Ethylene Response Factor (AP2/ERF)

families known to regulate secondary metabolism,

e.g., regulation of anthocyanin biosynthesis and

transport of MATE3 transporter in vacuolar mem-

brane in grapevine fruit by MYB-type transcription

factors (25), and in microbes, specifically in Strepto-

myces, SARP (Streptomyces antibiotic regulatory

proteins) regulators contain HLH motif through

which they bind DNA during expression of biosyn-

thetic genes. SARPs are a large family of CSRs and

its homologues are encoded by many biosynthetic

gene clusters (BGCs) including beta-lactam antibi-

otic BGCs and works in the expression of genes

within the same cluster (26-28). However, the global

regulators (GRs) work from a distant site and in ad-

dition to biosynthetic gene clusters; they can also in-

duce or repress gene activity of those not belonging

to secondary metabolism (29-31). A considerable

number of global regulators have been character-

ized in Streptomyces till 2022 (28,31). These regula-

tors may regulate directly or indirectly in the local-

ization and expression of many transporters and

other necessary enzymes in the specific vesicles. In

fungus, the global regulator VeA regulates the pen-

icillin biosynthesis in P.chrysogenum (32,33);

whereas an LaeA-, BrlA-dependent cellular net-

work governs tissue-specific secondary metabolism

in the human pathogen Aspergilus fumigatus (34,35).

Understanding the regulatory mechanism

will eventually help to learn how these pathways

contribute to the efficient production of valuable

compounds in plant and microbes specially in cases

of unexplored ones.

Organelle specific secondary metabolite bi-

osynthesis in plants and microbes

Biosynthetic intermediates along with biosyn-

thetic enzymes and transporter for any SM assem-

ble together in specific organelles of cells of plants

and microbes for efficient biosynthesis of valuable

molecules including drugs, antibiotics, nutraceuti-

cals and others. Though, the transport mechanism

in cases of many SM biosynthesis is still not very

clear, the major organelles in plant and microbes

and their role and involvement in the efficient bio-

synthesis of some important molecules will be dis-

cussed here.

Vacuoles are the major organelles, known as

the final storage point of and sites for biosynthesis

of secondary metabolites including many important

alkaloids in plant cells (36-40). In medicinal plant,

Catharanthus roseus, anti-cancer vinblastine and vin-

cristine are important dimeric terpenoid indole al-

kaloids, biosynthesized from the central intermedi-

ate strictosidine. The central intermediate is biosyn-

thesized in the vacuoles of leaf epidermal cells of

C.roseus from tryptamine and secologanin. Their

transport mechanism into the vacuole is not known,

but the formation of strictosidine is catalyzed by

strictosidine synthase localized in the vacuolar lu-

men (37). The biosynthetic pathway of vinblastine

and vincristine is shown in Fig.1.

Science Reviews - Biology, 2024, 3(4), 7-23 Prasanta Chakraborty

Figure 1: Role of the vacuole in the biosynthesis of secondary metabolite, vinblastine in medicinal plant C.roseus.

(a) In C.roseus leaf epidermal cells, two intermediates of vinblastine pathway, tryptamine and secolaganin, are trans-

ported into the vacuole, and converted to strictosidine by an enzyme strictosidine synthase (STR), located in the

vacuolar lumen. Strictosidine is the central intermediate of vinblastine biosynthetic pathway.

(b) (b) Carqueijeiro et al. have also shown that in C.roseus mesophyll cells, vacuolar accumulation of vindoline,

catharanthine and anhydrovinblastine prior to final step of vinblastine biosynthesis (Carqueijeiro et al., 2013).

As shown in Fig.1, final step of biosynthesis of

vinblastine takes place from two biosynthetic inter-

mediates, the monomeric monoterpenoid indole al-

kaloids catharanthine and vindoline (38). Before the

vacuolar accumulation of the mesophyll cells of the

main leaf of C.roseus, the biosynthetic intermediates

travel through different cells during biosynthesis of

vinblastine and vincristine. A transporter known as

ABC (ATP-binding cassette) transporter, CrTPT2

which localizes in plasma-membrane helps in the

efflux of catharanthine to the leaf surface. This phe-

nomenon helps in plant protection against herbi-

vores, the accumulation of catharanthine at leaf sur-

face inhibits the production of vinblastine, vincris-

tine as vindoline stays in internal leaf cells. Down

regulation of the expression of the transporter,

CrTPT2, inhibits the targeting of catharanthine at

the leaf surface and increases the availability of

catharanthine for the synthesis of dimeric alkaloids

in internal leaf cells. This indicates how organelle

specificity and transport engineering dictates the

production of valuable compounds at the high-level

(41,42).

Vacuoles in plant cells are also the site where

many important flavonoids are transported/ accu-

mulated. Flavonoids are accumulated in vacu-

oles/cell wall probably to attract different pollina-

tors through influencing their colors, and to protect

cells from different stresses (43,44). The coloration

of flowers and fruits specially different fruit berries

and their transition from growth to ripening, all

these depends upon the flavonoids, they, therefore,

should be properly transported and stored in dis-

tinct organelles, e.g., vacuoles, cell wall (43-45).

However, till now, the knowledge about the

transport and accumulation of the flavonoids is

very limited (46). Anthocyanins, one major class of

flavonoids, found in grapevine tissues and other

Prasanta Chakraborty Science Reviews - Biology, 2024, 3(4), 7-23

plants, whose transport and accumulation in vacu-

oles have been well studied (47,48). Various trans-

porters, e.g., ABC transporters, MATE transporters

have been involved in the transport and accumula-

tion process of anthocyanins. Glycosidic form of an-

thocyanins and their further modified forms e.g. hy-

droxylated, methylated and esterified forms in-

creases their stability, colour variation of the pig-

ments, as well as they facilitate the binding of the

transporters. Anthocyanin glucosides when in mal-

onylated forms binds strongly with MATE trans-

porters, and thus transportation of the pigment into

vacuoles of leaf cells in Medicago truncatula (49) is

fecilitated, and consequently this helps in pigmen-

tation of leaves and in flower coloration. ABC trans-

porters are also involved in anthocyanin transport.

This transporter prefers the glutathione conjugated

anthocyanin and transport them into vacuoles

through the hydrolysis of ATP. This is based by the

finding that a mutant defective in GST (glutathione

S- transferase) unable to accumulate anthocyanin

into vacuoles of maize (50). These studies indicate

that specific localization of MATE and ABC- trans-

porters on the vacuolar membranes facilitates the

transport of this flavonoid in the very organelle.

How this transport is regulated by various regula-

tors/transcription factors that will be discussed in

the next section.

Peroxisome is a spherical intracellular orga-

nelle found in all eukaryotes from microbes to

plants and animals and are the sites for accumula-

tion and biosynthesis of many important molecules.

These organelles contain at least one H

-produc-

ing oxidase and H

-decomposing catalase and

thus protect cells via generation of toxic metabolites.

The organelles are also the sites for acetyl CoA gen-

eration through β-oxidation of fatty acids. The val-

uable and important β-lactam antibiotic penicillin

in it’s final step is synthesized in this compartment

of fungus, P.chrysogenum (51). The enzymes isopen-

icillin N-acyltransferase (IAT) and phenylacetyl-

CoA ligase (PCL) responsible for conversion and li-

gation of isopenicillin N (IPN) to penicillin G are lo-

calized in this compartment (Fig.2).

Figure 2: Beta-lactam antibiotic penicillin synthesis in peroxisome of the fungus P. Chrysogenum

In it's final step of biosynthesis, the enzymes isopenicillin N-acyltransferase (IAT) and phenylacetyl-CoA ligase (PCL)

are involved in conversion and ligation of isopenicillin N (IPN) to penicillin G are localized in peroxisomes. IPN (the

beta-lactam nucleus) is formed into the cytosol and then transported to peroxisome.

Science Reviews - Biology, 2024, 3(4), 7-23 Prasanta Chakraborty

IPN (the β-lactam nucleus) is formed into the cyto-

sol and then transported to peroxisome. The en-

zyme IAT helps in the exchange of α-amino adipyl

side chain of IPN with CoA-activated phenylacetic

acid and the enzyme PCL acts in the activation of

side chain precursor during formation of penicillin

G (52). These enzymes also help in the formation of

other forms of penicillin through the exchange of

other carboxylic acid substrates. These results indi-

cate peroxisomes play important role for efficient

production of penicillin. High penicillin producing

strains expresses a greater number of peroxisomes

(53). Another important antibiotic, tetracycline, the

most used molecule in livestock and human world-

wide, is produced by the Streptomyces genus of Ac-

tinobacteria. Despite having importance of these

bacteria in the production of the antibiotic, the or-

ganelles of the bacterial cells for accumulation and

biosynthesis of this antibiotic is not yet known. In

fact, the cellular features of Streptomyces and their

industrial strains are very poorly understood. How-

ever, in some industrial strain of Streptomyces vina-

ceus L-6 which produces viomycin, a large number

of organelles with electron-dense dark contents

were found. Viomycin, a nonribosomal peptide an-

tibiotic having affinity for heavy metals strongly

visible as dark material under electron microscope

(54), and these types of organelles also been ob-

served in other Streptomyces strains, e.g., S.erythreus,

S.melanochromogenes (54,55). Finding these types of

organelles in bacterial cells will really help in iden-

tifying the sites of antibiotic accumulation and bio-

synthesis. The biosynthesis of tetracyclines in Strep-

tomyces aureofaciens was studied using its mutant

culture and substrate feeding experiments. Feeding

of pretetramid and 6-methylpretetramid in mutant

culture could restore the biosynthesis of tetracycline,

however, feeding of C4-dimethylamino-pretetra-

mid did not restore tetracycline biosynthesis indi-

cating pretetramid and 6-methylpretetramids are

key intermediates in the biosynthesis (56). Finally,

as we know, for it’s action as antibiotic in target cells,

tetracycline has to move to the cell cytoplasm,

where it binds to 30S ribosomal subunit and blocks

the protein synthesis of the target cell. For this, tet-

racycline penetrates the cell walls of organism e.g.,

gram-negative bacteria with the formation of posi-

tively charged magnesium-tetracycline complexes

and use the OmpF and OmpC porin channels to

cross the outer membrane (57,58). Then, they enter

the periplasmic space and tetracycline dissociates

and accumulates as uncharged tetracycline. This in

short covers the transport mechanism of the antibi-

otic tetracycline around the cell.

Aflatoxisomes are another important orga-

nelles responsible for aflatoxin biosynthesis and its

transport to the cell exterior. Aflatoxins are synthe-

sized by several fungal species in the genus Asper-

gillus. The biosynthetic pathway for this mycotoxin

has been studied very thoroughly and this pathway

has now become model system to study secondary

metabolism in eukaryotes (59,60). Using purified af-

latoxisome vesicles and analysing proteomic pro-

files of the vesicles, it has been shown that various

aflatoxin pathway enzymes are present in the orga-

nelles (61). In addition, in the past few years, the

genes involved and the entire aflatoxin gene cluster

of the aflatoxin biosynthetic pathway have been

characterized (62,63).

In the following section, the regulatory role of

various regulators in the regulation of transport

process of the intermediates/biosynthetic enzymes

and subsequently biosynthesis of the SMs in differ-

ent organelles of the cells will be discussed.

Role of regulators (cluster situated and

global) in SM-specific gene cluster regulation,

vesicle targeting of biosynthetic enzymes, inter-

mediates and transporters

The drugs/antibiotics and other important

SMs discussed above are all derived from specific

gene clusters. How different regulators regulate

these clustered genes which directly or indirectly af-

fects in the localization of the biosynthetic enzymes,

intermediates, transporters of the secondary metab-

olites to the specific vesicles will be discussed here.

In plant specialized metabolism, biosynthetic

gene clusters for several secondary metabolites in-

cluding nutraceuticals, glycoalkaloids, drugs, e.g.,

noscapine, vinblastine have been reported (18,19,

64-66). From the genomic research of medicinal

plant, C. roseus, the important anticancer drug vin-

blastine was discovered. of this plant Surprisingly,

gene cluster in the genome encoding vinblastine

pathway biosynthetic enzymes and the MATE

transporter genes were found to be coexpressed (67).

MATE was also found to be coexpressed as

SbMATE2 in gene clusters encoding cyanogenic

Prasanta Chakraborty Science Reviews - Biology, 2024, 3(4), 7-23

glucoside biosynthetic enzymes in the Sorghum bi-

color genome (68). MATEs, a class of transporters in-

volved in transport of natural product biosynthetic

intermediates whose coexpression is quite common

in bacterial and fungal biosynthetic gene clusters

(69-71). The coexpression of these transporter genes

in plant biosynthetic gene clusters might also have

significance in transport of specific biosynthetic en-

zymes and intermediates in specific organelles for

specific secondary metabolite biosynthesis. In

C.roseus genomes, partial clustering of genes of bi-

osynthetic enzymes TDC/STR of vinblastine bio-

synthetic pathway occurs. TDC is a Tryptophan de-

carboxylase that generates tryptamine from trypto-

phan, and STR is strictosidine synthase responsible

in the generation of strictosidine, the central inter-

mediate in vinblastine biosynthetic pathway (37).

There are now plenty of evidences that vin-

blastine pathway gene clusters/genes are strictly

regulated by various regulators/transcription fac-

tors. ORCA2 and ORCA3 are two transcription fac-

tors of large family AP2/ERF transcription factors

characterized as critical regulators in vinblastine bi-

osynthesis (72-74 ). In C.roseus, ORCA2 to ORCA6

forms a cluster (75,76) that regulates the biosynthe-

sis of important terpenoid indole alkaloids (TIA).

Though, ORCAs are key regulators in TIA biosyn-

thetic pathways, there are some TFs, e.g., BIS2,

ZCT2 and WRKY2 act as negative regulators in the

pathway. The ORCA regulators are highly ex-

pressed in flowers, roots and stems and induced by

different inducers. These regulators bind specifi-

cally to the elicitor-responsive element in STR pro-

moter region and interferes in changes of the ex-

pressions of STR and modulates in the expression of

other biosynthetic genes of vinblastine pathway

(Fig. 3).

Figure 3: Critical regulators of vinblastine biosynthetic pathway.

The regulators ORCA of AP2/ERF transcription factor family form cluster and regulate biosynthesis of terpenoid in-

dole alkaloids in medicinal plant C. roseus. In the vinblastine biosynthetic pathway genes, TDC/STR are partially

clustered in the genome where MATE1 transporter genes are also co-expressed. Co-expression may facilitate specific

localization of biosynthetic enzymes and intermediates to specific organelles. Now, ORCA regulators bind in the pro-

moter region and interfere in the expression of STR and modulate in the expression of other biosynthetic genes of the

vinblastine pathway. Upregulation of these regulators also helps in the transport and accumulation of many interme-

diates of the vinblastine pathway, e.g., catharanthine, tabersonine, to the specific organelle of biosynthesis.

The upregulation of ORCA2 in the cell also

plays an important role in the transport and the ac-

cumulation of various biosynthetic intermediates

e.g., catharanthine and tabersonine of vinblastine

pathway in the specific organelles of the cell via the

regulation of vinblastine/vincristine metabolism

(77,78). ORCA2, ORCA3 and other signaling cas-

cades/mechanism induced biosynthesis of vinblas-

tine/vincristine and vacuolar localization of inter-

mediates in C.roseus cells requires lots of energy.

These might require the induction of MATEs which

Science Reviews - Biology, 2024, 3(4), 7-23 Prasanta Chakraborty

could be used as secondary transporters (79). How-

ever, further studies will be required to support this

hypothesis. In addition, many other transporters

which play important role in plant and microbe sec-

ondary metabolism and whose expressions are reg-

ulated by various regulators are shown in Table 1.

Table 1. Transporters in plant and microbes involved in the transport of biosynthetic enzymes, intermediates to a

specific organelle in the biosynthesis of secondary metabolites.

Transporter

Family

Function

Source

Concerned

secondary

Metabolite

References

MATE

vacuolar targeting of alkaloids

N.tabacum

Alkaloid

(Shitan et al.2015)

MATE

transport catechin and

anthocyanin

A.thaliana

Phenolic

polyketides

(Marinova et al.2007)

MATE

vacuolar accumulation of nicotine

N.tabacum

Alkaloid

(Shoji et al. 2008)

ABC

transport of mono-

terpenes, indolealkaloids

C.roseus,

H.irregulare

Vinca alkaloids,

terpenoids

(Shitan,N. 2016,

Baral et al.2016)

ABC

Drug efflux

S.rimosus

Phenolic/polyk

etides

(Petkovic et al.2006)

PUP

Purine, nicotine uptake

N.tabacum

Nicotine/alkalo

ids

(Kato et al. 2014)

MFS

Cephalosporin transport

A.chrysogenum

NRPs

(Ullan et al.2010)

MFS

transporter in antibiotic

biosynthesis

P.chrysogenum

NRPs

(Juan 2020)

Table 1a. Co-expressed transporters with biosynthetic gene clusters of secondary metabolites

Transporters

Biosynthetic gene clusters

Plant/microbes

Secondary

Metabolite

MATE

TDC/STR, partial clustering vinblastine

biosynthetic pathway

C.Roseus

Vinblastine

MATE/SbMATE2

Cyanogenic glucoside

Gene cluster

Sorghum bicolor

Cyanogenic

glycosides

MFS/cefM, cefP

Cephalosporin gene cluster

A.chrysogenum

Cephalosporin

Another important secondary metabolite, an-

thocyanin, is responsible for skin color of various

fruits/crops, whose regulatory system of biosyn-

thesis is controlled by MYB-type transcription fac-

tors (80-82). In purple color rice plants, OsC1, a gene

encoding R2R3-MYB transcriptional factor specifi-

cally binds to a pigmentation gene OsPa to activate

OsDFR encoding dihydroflavonol 4-reductase, and

other anthocyanin biosynthesis genes (83). All these

proteins are localized in the nucleus or cytoplasm

and plays important role in the regulatory mecha-

nism of anthocyanin biosynthesis in rice (84). In

pears, the expression of anthocyanin biosynthetic

genes and R2R3 MYB transcription factor, PcMYB10

found to be strongly correlated with anthocyanin

accumulation during developmental stages of fruits.

The expression patterns during clustering analysis

showed that most of the anthocyanin biosynthetic

genes and PcMYB10 genes are related to the same

cluster (85). In grapevine (V.vinifera) fruit, the dark

skin color of the fruit is due to the accumulation of

anthocyanins. Accumulation of anthocyanins and

their transport in cell organelles via MATE trans-

porters, is regulated by MYB-type transcription fac-

tors (86). In V.vinifera, MATE transporters are di-

rectly or indirectly transcriptionally regulated by

MYB-transcription factors. The MYB-transcription

factor, VvMYBA1, transcriptionally regulate the en-

zyme, V.vinifera vacuolar H

PPase 1;2, VvVHP1;2.

The overexpression of the enzyme led to the in-

creased expression of VvMATE3. In V.vinifera, an-

thoMATE1 and anthoMATE3 were found to be to-

noplast-localized MATE transporters that transport

acylated anthocyanins in the vacuole (80,87). These

results collectively show that regulators-biosyn-

Prasanta Chakraborty Science Reviews - Biology, 2024, 3(4), 7-23

thetic genes/clusters-transporters network func-

tions altogether for transport, accumulation and bi-

osynthesis of secondary metabolites.

The regulation mechanism of important anti-

biotic tetracycline from Streptomyces recently got

importance due to having not only it’s enormous

potential against various bacteria but also for it’s

anticancerous activity (88). Therefore, tetracycline

molecules and their derivativesall are significantly

important from pharmaceutical point of view. Oxy-

tetracycline and chlorotetracycline are two such no-

table molecules in tetracycline family and both are

derived from their respective gene clusters. Now re-

garding regulation of these gene clusters, a gene

known as otcR (oxytetracycline regulator) encoding

a SARP protein, was discovered in Streptomyces ri-

mosus (89) which is located immediately adjacent to

otrB gene of oxy cluster (90). Later, the SARP-bind-

ing sequences of the promoter regions of oxyclus-

ters were characterized and it has been shown that

otcR directly activated the transcription of oxy pro-

moters (91). Interestingly, during working with

chlorotetracycline (CTC), Wang et al. (92) found

that the SARP regulator, encoded by ctcB from CTC

gene cluster was able to activate the transcription of

oxy cluster in heterologous host. It was proposed

that since chloro and oxy-tetracyclines are structur-

ally similar antibiotics, they might share the similar

regulatory mechanisms.

The regulation of biosynthesis of another im-

portant β-lactam antibiotic, penicillin in filamen-

tous fungi is controlled by global regulators (93,94).

The penicillin pathway biosynthetic enzymes en-

coded by genes, penDE, pcbC and pcbAB are clus-

tered in the genome of Penicillium chrysogenum. No

regulators of penicillin biosynthesis are found in

this gene clusters; however, global regulators, e.g.,

LaeA , PacC etc. have been identified (Fig.4).

Figure 4: Global regulators in the regulation of penicillin, cephalosporin gene clusters in P.chrysogenum, Acremonium and

Streptomycetes.

Global regulators LaeA and PacC control the expression of penicillin genes pcbAB, pcbC, and penDE, whereas LaeA

and VeA control the cephalosporin genes pcbAB, pcbC, cefD1, and cefD2 expression. Genes pcbAB and pcbC encoding

the first two enzymes valine synthetase and isopenicillin N synthase of the cephalosporin pathway, are very similar to

those involved in penicillin biosynthesis. Two genes, cefT and cefM, of transporter MFS family, are involved in the

transport of intermediates and the secretion of cephalosporins are also co-expressed in the cluster. The regulator LaeA

regulates the synthesis through a SAM binding site, unique for methyltransferases. Thus it regulates the gene clusters

through chromatin modification and heterochromatin repression.

Global regulators LaeA and PacC control the

expression of penicillin genes pcbAB, pcbC, and

penDE, whereas LaeA and VeA control the cepha-

losporin genes pcbAB, pcbC, cefD1, and cefD2 ex-

pression. Genes pcbAB and pcbC encoding the first

Science Reviews - Biology, 2024, 3(4), 7-23 Prasanta Chakraborty

two enzymes valine synthetase and isopenicillin N

synthase of the cephalosporin pathway, are very

similar to those involved in penicillin biosynthesis.

Two genes, cefT and cefM, of transporter MFS fam-

ily, are involved in the transport of intermediates

and the secretion of cephalosporins are also co-ex-

pressed in the cluster. The regulator LaeA regulates

the synthesis through a SAM binding site, unique

for methyltransferases. Thus it regulates the gene

clusters through chromatin modification and heter-

ochromatin repression.

PacC activates penicillin biosynthesis at alka-

line pH (93), while LaeA was found in light-de-

pendent fungal morphology and development to

secondary metabolism as in Aspergillus nidulans (94).

About 50% of BGCs in fungi was shown to be af-

fected by LaeA and reduced secondary metabolite

formation including penicillin was observed with

the loss of LaeA (95). Deletion of these regulators

blocks the expression of penicillin and other gene

clusters and conversely overexpression of this reg-

ulator induces penicillin and other gene transcrip-

tion and corresponding product formation (95, 96).

Penicillin biosynthesis also requires specific locali-

zation of biosynthetic enzymes and intermediates

to specific organelles and several transporters play

important roles in the process. In biosynthetic pro-

cess, phenylacetyl-CoA ligase and isopenicillin N

acyl transferase, the last two enzymes of penicillin

pathway along with the intermediates isopenicillin

N and phenylacetic acid were located in peroxi-

somes(97). Two MFS (major facilitator superfamily)

transporters PenM and PaaT were shown to be in-

volved in the import of intermediates into peroxi-

somes (98). Similar vesicle localization of the inter-

mediates was also seen in Acremonium chrysogenum

during cephalosporins (CPC), another class of β-lac-

tam antibiotic biosynthesis. Isopenicillin N is being

converted through a long pathway in A.chryso-

genum into penicillin N. In the conversion process,

Isopenicillin N is epimerized to penicillin N by the

action isopenicillin N-CoA ligase and the isopenicil-

lin N-CoA epimerase encoded by genes cefD1 and

cefD2 (99,100). These enzymes along with MFS

transporters were shown to be localized in peroxi-

somes. In the early cephalosporin gene cluster,

pcbC, pcbAB, cefD1, cefD2 genes along with two

genes encoding MFS transporters, cefM and cefP

were found to be present (Fig.4). Generally, these

MFS superfamily transporters are regulated by

Yap1 transcription factors, however, recently, Pe-

rez-Perez et al. (101) reports that Yap1 protein,

PcYap1 binds to the regulatory sequence TTAG-

TAA in the pcbAB gene promoter of P.chrysogenum.

This is also a first report showing a Yap1 protein in

addition to its other role regulating transcription of

a secondary metabolism gene. No doubt, these find-

ing indicates how the regulators of gene clusters

and transporters coordinately acts in the expression

of genes and localization of biosynthetic enzymes,

intermediates and transporters to the specific orga-

nelles. Consequently, these processes help in the ef-

ficient biosynthesis of antibiotics.

Do cluster situate regulators (CSR) and

global regulators (GR) act differently in the sec-

ondary metabolite biosynthetic pathway of plants

and microbes?

In the previous section it has been discussed

how various important secondary metabolites,

drugs/antibiotics derived from different gene clus-

ters are regulated by variety of CSRs and GRs. It

was also discussed how these regulators play im-

portant role in various transport process responsi-

ble for efficient biosynthesis of these important mol-

ecules. Now it will be discussed whether at all and

if yes, how these regulators work differently in

plant and microbes.

In bacteria, the cluster situated regulator is

more common; SARP (Streptomyces antibiotic regu-

latory protein) is one of the largest families of CSRs

present in BGCs of Streptomyces. This bacteria is

used to produce most of the today’s antibiotics in

use. The BGCs coding for many antibiotics from

Streptomyces contain SARP family regulators. As

discussed in the previous section that in oxytetracy-

cline cluster, a gene known as otcR (oxytetracycline

regulator) encoding a SARP protein was found im-

mediately adjacent to otrB gene of antibiotic cluster

in Streptomyces rimosus (89). Oxytetracycline regula-

tors thereby help in the activation of the transcrip-

tion of oxy promoters through the interaction of

SARP binding sequences and the promoter regions

of the clusters. The BGCs coding for another antibi-

otic actinorhodin in Streptomyces coelicolor (102) con-

tain SARP family regulators that activate the tran-

scription of biosynthetic genes within the cluster. In

addition, there are many other SARP family regula-

tors as CSRs found for many other antibiotics in

Streptomyces species (103-105), and most im-

portantly, beside SARPs there are other class of

CSRs found not only in Streptomyces but also in

Prasanta Chakraborty Science Reviews - Biology, 2024, 3(4), 7-23

Gram-negative proteobacteria (106, 107). Nonethe-

less, in some cases, the cluster situated regulators

may be controlled by global regulatory systems.

In fungi, approximately 60% of secondary me-

tabolite gene cluster do not contain genes for regu-

latory proteins, but they are controlled by global

regulators. That means for 40% gene clusters there

are cluster situated regulators. As described in the

previous section, global regulators, PacC, LaeA reg-

ulates penicillin biosynthesis in Penicillium chryso-

genum (93,94). PacC activates penicillin biosynthesis

at alkaline pH, whereas LaeA controls secondary

metabolism in A.nidulans and other aspergilli in

light dependent manner. Lae A is a universal regu-

lator protein in several aspergilli and control the

synthesis of penicillin and other antibiotics e.g.,

lovastatin, sterigmatocystin (108,109). This regula-

tor as PcLaeA also regulates the secondary metabo-

lism of P.chrysogenm (110). LaeA protein has methyl

transferase activity and is thought to function at the

level of chromatin modification (111). It was also

proposed that LaeA regulates several gene clusters

through repression of heterochromatin as sug-

gested for sterigmatocystin gene cluster in Aspergil-

lus (109,112), and in regulation of several genes in

gilitoxin gene cluster of Trichoderma reesei (113). And

for cluster specific regulators in fungi, they often

make a direct connection with secondary metabolite

formation network. A typical feature of cluster spe-

cific regulators is that they are mostly involved in

the transcriptional activation as in AflR of the afla-

toxin cluster in A.flavus (114) and in ApdR of aspyr-

idone biosynthesis cluster in A.nidulans (115). For

detailed actions of regulatory proteins in fungal sec-

ondary metabolism, an article by Knox et al. (116)

may be consulted.

In plants, several cluster-specific transcription

factors (TF) are found, though the genes of these

transcription factors are not located within the met-

abolic gene clusters they control. These TFs re-

motely control the genes of metabolic gene clusters.

In some cases these TFs forms clusters, however

these clusters are not thoroughly identified and

characterized (117). In the previous section, TF clus-

ters of ORCA was discussed in the regulation of ter-

penoid indole alkaloid biosynthetic pathway (75,76).

These ORCA regulators were also shown in the ac-

cumulation and transport of many biosynthetic in-

termediates of the pathway. In the control of cucur-

bitacin gene cluster in cucumber, melon, and water-

melon (118), a novel basic helix-loop-helix, bHLH

TF cluster consisting of two genes were found.

These regulators strongly binds to the promoter re-

gion of the biosynthetic genes of cucurbitacin and

helps in the biosynthesis of bitter substances cucur-

bitacins C (CuC), B (CuB), and E (CuE) of the fruits.

Three clustered bHLH genes (BIS1, BIS2, and BIS3)

were also found in the regulation of iridoid biosyn-

thesis in terpenoid indole alkaloid biosynthetic

pathway in Catharanthus roseus (119). Furthermore,

other transcription factors have been found in the

regulation of many clusters, e.g., GAME9 regulates

the expression of steroidal glycoalkaloid gene clus-

ters in potato and tomato (120) and basic leucine

zipper domain,bZIP TF OsTGAP1 in momilactone

gene cluster in rice and oat (121).

The above findings clearly indicate that clus-

ter situated regulators over global regulators plays

an important role in the production of huge number

of valuable compounds as in bacteria. It may hap-

pen that for CSRs, due to the closer proximity of the

regulators to the genes of the metabolic gene cluster,

bacteria are able to regulate production of specific

valuable compounds/ secondary metabolites at a

much faster pace compared to plants and fungi. To

date, almost all antibiotics globally used in the clin-

ics are derived from bacteria and the contribution of

plants and fungi in that respect still remains a few.

Concluding remarks:

Plants and microbes are tremendous source of

natural products that may give rise to many valua-

ble compounds including new target drug mole-

cules. In the era of drug-resistance, continuous ef-

forts are necessary for achieving new drugs and an-

tibiotics. In fact, the gene clusters of plant and mi-

crobes may help develop new molecules, help ex-

plore the pathways wherein drugs can be made fit.

Drugs can be developed particularly keeping in

mind the specific pathway and/or specific reaction

regulated by gene clusters.

The efficient biosynthesis of any valuable

molecule/secondary metabolite(SM) in the specific

vesicles of cells of plant or microbe depend upon

the transport of all the ingredients like biosynthetic

intermediates, enzymes, transporters into the spe-

cific vesicles. Though it is known that the genes of

the biosynthetic enzymes, transporters in many

cases are clustered within BGC of the genomes of

the organisms, there are scanty of reports regarding

Science Reviews - Biology, 2024, 3(4), 7-23 Prasanta Chakraborty

the regulation/activation of the gene clusters. The

gene clusters and their regulators would definitely

play important role in the transport and efficient bi-

osynthesis of any valuable molecule/SM in specific

vesicles of the cells of the organism. Various regu-

lators like, cluster situated regulators and global

regulators would be the main players in the discov-

ery process. Cluster situated regulators are more

common in bacteria, like SARP-activated gene clus-

ters in Streptomyces, several thousands of antibiot-

ics and secondary metabolites have been identified

from these gene clusters. Still, researchers around

the globe are struggling to develop methods for the

activation of many silent or poorly expressed gene

clusters in various plants and microbes.

Elucidation of the regulatory mechanisms

and finding new regulators in the secondary metab-

olism specific gene clusters will provide deeper in-

sight into SM biosynthesis and ensure identification

of novel SMs.

Declaration of competing interest

The author does not have any conflict of interest.

Acknowledgments

The author gratefully acknowledges the over-

whelming support from American Center Library

and National Library, Kolkata.

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