biotechnology of rhodococcus for the production of valuable … · 2020. 9. 12. · mini-review...

MINI-REVIEW

Biotechnology of Rhodococcus for the productionof valuable compounds

Martina Cappelletti1 & Alessandro Presentato2 & Elena Piacenza2,3 & Andrea Firrincieli1 & Raymond J. Turner4 &Davide Zannoni1

Received: 9 June 2020 /Revised: 14 August 2020 /Accepted: 26 August 2020# The Author(s) 2020

AbstractBacteria belonging to Rhodococcus genus represent ideal candidates for microbial biotechnology applications because of theirmetabolic versatility, ability to degrade a wide range of organic compounds, and resistance to various stress conditions, such asmetal toxicity, desiccation, and high concentration of organic solvents. Rhodococcus spp. strains have also peculiar biosyntheticactivities that contribute to their strong persistence in harsh and contaminated environments and provide them a competitiveadvantage over other microorganisms. This review is focused on the metabolic features of Rhodococcus genus and their potentialuse in biotechnology strategies for the production of compounds with environmental, industrial, and medical relevance such asbiosurfactants, bioflocculants, carotenoids, triacylglycerols, polyhydroxyalkanoate, siderophores, antimicrobials, and metal-based nanostructures. These biosynthetic capacities can also be exploited to obtain high value-added products from low-costsubstrates (industrial wastes and contaminants), offering the possibility to efficiently recover valuable resources and providingpossible waste disposal solutions. Rhodococcus spp. strains have also recently been pointed out as a source of novel bioactivemolecules highlighting the need to extend the knowledge on biosynthetic capacities of members of this genus and their potentialutilization in the framework of bioeconomy.

Key points• Rhodococcus possesses promising biosynthetic and bioconversion capacities.• Rhodococcus bioconversion capacities can provide waste disposal solutions.• Rhodococcus bioproducts have environmental, industrial, and medical relevance.

Keywords Rhodococcus . Antimicrobials . Bioflocculants . Biosynthesis . Bioconversion . Biosurfactants . Carotenoids .

Lipids .Metal-based nanostructures . Siderophores

Introduction

Rhodococcus genus belongs to the Actinobacteria phy-lum and comprises genetically and physiologically di-verse bacteria, which are distributed in various water,soil, and marine habitats, also including harsh ecologicalniches such as arctic, desert, and heavily contaminatedsites (Cappelletti et al. 2019a, 2019b). The wide distribu-tion of Rhodococcus spp. strains is due to their extraor-dinary metabolic versatility, which is comparable to thatdescribed only in a few other bacterial genera, and totheir unique environmental persistence and robustness(LeBlanc et al. 2008; Cappelletti et al. 2016).

* Martina [email protected]

1 Department of Pharmacy and Biotechnology (FaBiT), University ofBologna, Bologna, Italy

2 Department of Biological, Chemical and Pharmaceutical Sciencesand Technologies (STEBICEF), University of Palermo,Palermo, Italy

3 National Interuniversity Consortium of Materials Science andTechnology (INSTM), Florence, Italy

4 Department of Biological Sciences, Calgary University,Calgary, AB, Canada

https://doi.org/10.1007/s00253-020-10861-z

/ Published online: 12 September 2020

Applied Microbiology and Biotechnology (2020) 104:8567–8594

http://crossmark.crossref.org/dialog/?doi=10.1007/s00253-020-10861-z&domain=pdfhttp://orcid.org/0000-0002-6238-8296mailto:[email protected]

Bacterial strains belonging to Rhodococcus genus havebeen largely studied for their application in bioremediation,biotransformation, and biocatalysis due to their capacity tobiodegrade a wide range of organic compounds, includingtoxic and recalcitrant molecules such as chlorinated aliphaticand aromatic hydrocarbons, N- and S-heterocyclic com-pounds, and synthetic polymers (e.g., polyethylene)(Martínková et al. 2009; Cappelletti et al. 2015, 2017, 2018;Ciavarelli et al. 2012; Krivoruchko et al. 2019). During thelast decade, it has become obvious that various species of thisgenus have also interesting capabilities regarding the biosyn-thesis of lipids and other valuable compounds. These biosyn-thetic capacities are often associated to the ability ofRhodococcus to resist to different environmental stresses(LeBlanc et al. 2008; Orro et al. 2015; Cappelletti et al.2016) and allow these bacteria to cope with the presence ofhydrophobic substrates in the growth medium, limited nitro-gen sources, oxidative stressors, among others. The most ex-tensively reported compounds that are biosynthesized byRhodococcus spp. strains are glycolipid biosurfactants, carot-enoids, triacylglycerols, and polyhydroxyalkanoates (PHAs)(Krivoruchko et al. 2019). Recently, novel siderophores andnew antibiotics have been reported to be produced byRhodococcus spp. strains as possible competition strategies.Further, several Rhodococcus spp. strains have recently beendescribed to be able to produce metal-based nanostructures asthe result of metal and metalloid [metal(loid)] bioconversioninto their elemental state (Presentato et al. 2016, 2018a,2018b; Firrincieli et al. 2019).

Compared to the many papers summarizing the biotechno-logical potential of Rhodococcus cells in association with theirbiodegradative enzymes for bioremediation and biocatalysis(Martínková et al. 2009; Kim et al. 2018; Krivoruchko et al.2019; Jiao et al. 2020), this review gives an overview on therelevant biosynthetic capacities of bacteria belonging to thisgenus. These anabolic functions are at the basis ofRhodococcus-mediated bioproduction of commercially valu-able compounds and bioactive molecules, as well as biocon-versions of organic and inorganic contaminants/wastes intohigh value-added products.

Biosurfactants

Biosurfactants are a heterogeneous group of amphiphilicsurface-active compounds that are produced by various mi-croorganisms including bacteria, yeast, and fungi and findwide applications in several industries as demulsifiers; wet-ting, foaming, and spreading agents; functional food ingredi-ents; and soaps/detergents (Santos et al. 2016). Biologicalcompounds with surfactant activities can generally be classi-fied into low-molecular-weight (glycolipids, lipopeptides, andphospholipids) and high-molecular-weight (polysaccharides,

proteins, lipoproteins, or polymeric compounds) substances,the first being considered actual biosurfactant and the secondgenerally described as bioemulsifiers (Franzetti et al. 2010).They possess several advantages as compared to the chemicalsurfactants including biodegradability, lower critical micelleconcentrations (CMC), reduced toxicity, higher stability, andthe possibility to be produced by renewable raw material(Banat et al. 2010).

Several Rhodococcus spp. strains produce biosurfactants inresponse to the presence of water-insoluble substrates (e.g.,hydrocarbons) (Whyte et al. 1999; Cappelletti et al. 2019a),and in some cases also on water-soluble substances (e.g., eth-anol and glucose) (Table 1) (Pirog et al. 2004; Ciapina et al.2006). The formation of biosurfactants is believed to improvethe utilization (bioavailability) of these water-insoluble com-pounds as growth substrates by facilitating their entry intocells (Lang and Philp 1998; Yakimov et al. 1999; Philp et al.2002; Peng et al. 2007; Cappelletti et al. 2019b).

Rhodococci typically produce trehalose-based glycolipidbiosurfactants, which possess various hydrophobic moietiesand chemical compositions (Kuyukina and Ivshina 2010)(Fig. 1). Trehalolipids (TP) are a class of glycolipidbiosurfactants with interesting physicochemical and biologi-cal properties that have been studied for i) environmental ap-plications as emulsifiers in bioremediation (e.g. oil-spill treat-ment), ii) microbial-enhanced oil recovery, and iii) cosmeticand food industries (Christofi and Ivshina 2002; Pacheco et al.2010). Besides, these compounds can be used for medicalapplication for their immune-stimulating, antitumor, and anti-viral properties (Ortiz et al. 2008; Kuyukina et al. 2015).

TP are generally produced by rhodococci as either extra-cellular or cell wall-associated compounds during the growthon hydrocarbons (Lang and Philp 1998; Yakimov et al. 1999).Nitrogen limitation was also shown to favor the formation ofanionic trehalose tetraesters using these growth substrates(Ristau and Wagner 1983; Kim et al. 1990). In thoseRhodococcus spp. strains where the biosurfactants are excret-ed from the cell, the emulsification of the hydrocarbons withaqueous culture medium results in a very large surface area ofcontact between the cell and these compounds. Nevertheless,TP are more frequently retained by the Rhodococcus cell andlocalized on the outer cell surface (Rapp et al. 1979), increas-ing the hydrophobicity of the cell surface that facilitates theattachment and subsequent uptake of the hydrophobic com-pounds to be used as growth substrate (Kretschmer andWagner 1983; Bredholt et al. 1998; Lang and Philp 1998;Cappelletti et al. 2019a). These biosurfactants, which bind tothe cells, have more limited industrial applications because ofproblems with their recovery. The mechanism involved in theproduction and retention of biosurfactants by Rhodococcuscells appeared to be related to the type of substrate and thecell growth phase (Bredholt et al. 2002). Particularly, a marineRhodococcus strain grown in the presence of sunflower oil,

8568 Appl Microbiol Biotechnol (2020) 104:8567–8594

Table1

Biosynthetic

activ

ities

ofthemostrepresentativeRhodococcus

spp.strainsandgrow

thsubstrateor

wasteresidueused

fortheproductio

nof

each

valuablecompoundcategory

Productcategory

Strain/s

Substrateand/or

grow

thconditionsa

Features

oftheproduct

and/or

biosyntheticprocess

Reference

Biosurfactants

R.erythropolis

DSM

43215

Cellsgrow

ingon

C14–C

15n-alkanes

orkerosene

Productio

nof

extracellular

trehalose-monom

ycolates

and

trehalose-dimycolates

Kim

etal.1990

Rhodococcus

H13-A

Cellsgrow

ingon

n-alkanes

orfatty

alcohols

Productio

nof

glycolipid

which

isableto

solubilizepolyarom

atichydrocarbons

Pageetal.1999

R.opacus1C

PCellsgrow

ingon

C10,C

12,C

14,C

16

n-alkanes

Productio

nof

trehalose

dinocardiomycolates

with

doublebonds

Niescheretal.2006

R.erythropolis

ATCC4277

Cellsgrow

ingon

glycerol

orn-hexadecane

Productio

nof

extracellularbiosurfactant

onglycerol,and

cell-bound

biosurfactanto

nn-hexadecane

Ciapina

etal.2006

R.erythropolis

strain

EK-1

Cellsgrow

ingon

hydrocarbons

(C16

n-alkane

andparaffin),ethanolo

rglucose

Productio

nof

cell-boundnon-ionic

trehalolipidson

hydrocarbons,produc-

tionof

bioemulsifier

onsolublesubstrates

Pirog

etal.2008

R.erythropolis

SD-74

Cellsgrow

ingon

n-hexadecane

Productionof

succynoiltrehalolip

ids

Tokum

otoetal.2009

R.erythropolis

3C-9

Cellsgrow

ingon

n-hexadecane

Productionof

extracellulartrehalose

lipidsandfree

fatty

acids

Pengetal.2007

R.erythropolis

16LM.UST

HB

Cellsgrow

ingon

sunflower

frying

oil

Productio

nof

extracellularglycolipids

Sadouk

etal.2008

Rhodococcus

sp.B

S32

Cellsgrow

ingon

rapeseed

oil

Productio

nof

extracellularbiosurfactants

Ruggerietal.2009

R.erythropolis

sp.P

6-4P

Cellsgrow

ingon

fish

wastecompost

Productio

nof

biosurfactantthatis

mainlycomposedof

fatty

acids

Kazem

ietal.2009

Bioflocculants

R.erythropolis

S-1

Cellsgrow

ingon

sorbito

l,mannitol,

ethanol,glucose,or

fructose

Productio

nof

thepeptidicbioflocculant

named

NOC-1,itisoneof

thebest

performingbioflocculantd

escribed

upto

date

Kuraneetal.1994

R.erythropolis

ATCC10543

Cellsgrow

ingon

pre-treatedsludge

andliv

estock

wastewater

Productio

nof

apolysaccharidic

bioflocculant

Pengetal.2014

R.erythropolis

Cellsgrow

ingon

potato

starch

wastewater

Productio

nof

apolysaccharidic

bioflocculant

Guo

etal.2018

Carotenoids

R.luteus,R

.coprhilu

s,R.lentifragm

entus,R.m

aris

Cellsgrow

ingon

Sauton

agar

medium

Productio

nof

β-carotene

Ochiyam

aetal.1989

R.equi,R.rubroperctin

ctus,

R.aichiensis,R.sputi,

R.chubuensis,R.obuensis,

R.bornchialis,R

.roseus,

R.rhodochrous,R

.rhodnii,

R.terrae

Cellsgrow

ingon

definedmedium

L-asparagineandglycerol

asmain

nitrogen

andcarbon

sources,

respectiv

ely

Productio

nof

γ-carotene-lik

ecompound

Ochiyam

aetal.1989

R.erythropolis

IBBPo1

Cellsgrow

ingon

n-alkane

Productionof

lycopene

athigher

level

Stancu

etal.2015

R.rhodochrous

RNMS1

ND

Productionof

γ-carotenederivativ

esTakaichietal.1990

R.erythropolis

AN12

Cellsgrow

ingon

rich

medium

(i.e.,nutrient

broth-basedmedium)

Productio

nof

γ-carotenederivativ

esTao

etal.2004

Rhodococcus

sp.C

IPCellsgrow

ingon

rich

medium

Osawaetal.2011

8569Appl Microbiol Biotechnol (2020) 104:8567–8594

Tab

le1

(contin

ued)

Productcategory

Strain/s

Substrateand/or

grow

thconditionsa

Features

oftheproduct

and/or

biosyntheticprocess

Reference

Productio

nofOH-chlorobactene

glucoside

hexadecanoateandrelatedrare

caroten-

oids

R.opacusPD

630

Glyceroland

ammonium

acetateas

maincarbon

andnitrogen

sources,

respectiv

ely

Productio

nof

uncharacterizedcarotenoids

Thanapimmetha

etal.2017

R.pyridinivoransNT2

Cellsgrow

ingon

2,4-dinitrotoluene

Productio

nof

acarotenoid

mixture

composedby

4-keto-γ-caroteneandγ

-carotene,lycopene,and

β-carotene

Kundu

etal.2015

Triacylglycerols(TAGs)

R.opacusPD

630

Cellsgrow

ingon

phenyldecane

Accum

ulationof

TAGscontaining

phenyldecanoicacid

residues

Alvarez

etal.2002

Cellsgrow

ingon

n-alkane

Accum

ulationof

fatty

acidswith

thesame

carbon

skeleton

ofthealkane

used

for

grow

th

Alvarez

2003

Cellsgrow

ingon

citrate,succinate,

propionate,valerate,saturated,and

mono-unsaturatedfatty

acidswith

C15andC17chainlength

Accum

ulationof

TAGswith

increased

fractio

nof

odd-numberedfatty

acids

Alvarez

etal.1997

Cellsgrow

ingon

C15–C

18n-alkanes,

phenylaceticacid,phenyldecane,

gluconicacid,acetic

acid,propionic

acid,fructose,andoliveoil

Firstw

orkreportingtheability

ofPD

630

strain

toproduceandaccumulateTAGs

usingdifferentcarbonsources

Alvarez

etal.1996

Cellsgrow

ingon

sugarbeetmolasses,

andsucrose

Productio

nof

largeam

ountsof

TAGswas

demonstratedusingahigh-cell-density

cultivatio

nin

a500-lp

ilot-plantscale

VossandSteinbüchel2001

R.opacusMITXM-61(i.e.,the

spontaneousmutanto

fR.opacus

PD630carrying

exogenousgenes

ofxylA

andxylB

from

Streptom

yces

padanus)

Cellsgrow

ingon

xylose/glucose,or

corn

stover

hydrolysates

Robustg

rowth

andTAGbiosynthesison

high

concentrations

ofxylose

and

simultaneousutilizatio

nof

xylose

and

glucosein

thecorn

stover

hydrolysate

Kurosaw

aetal.2013,2014

R.opacusPD

630

pEC-K

18mob2::bglABCTF

Cellsgrow

ingon

glucoseand

cello

biose

Acquisitio

nof

cello

bioseutilizatio

nfor

grow

thandTAGaccumulation

Hetzler

andSteinbüchel2

013

R.opacusMITGM-173

(i.e.,adap-

tivelyevolved

R.opacusPD

630)

Cellsgrow

ingon

glycerol/glucose/xylose

Higherglycerol

utilizatio

nas

comparedto

theparentalstrain

Kurosaw

aetal.2015b

R.erythropolis

strainsDSMZ

43060,17

andDM1–21;

R.fasciansstrainsF7

,S1.17b,

123andD188–5;

R.opacus

strainsPD630andMR22;

R.jostii

strainsRHA1and602;

R.equiA

TCC6939;R

.opacus

PD630pT

ip-Q

C2/glpK1D

1 F7

Cellsgrow

ingon

glycerol

Rhodococcus

strainsdiffered

interm

sof

numberof

days

required

forTAGs

productio

n;R.equip

roducedlower

amountsof

TAGsas

comparedto

the

otherspecies;R.opacusPD

630

improved

TAGproductionwhen

expressing

glpK

1D1from

R.fascians

Herrero

etal.2016

Rhodococcus

sp.602

Silv

aetal.2010


Tab

le1

(contin

ued)

Productcategory

Strain/s

Substrateand/or

grow

thconditionsa

Features

oftheproduct

and/or

biosyntheticprocess

Reference

Cellsgrow

ingon

gluconate,benzoate,

n-hexadecane,naphthalene,or

naphthyl-1-dodecanoate

TAGswith

fatty

acidsof

differentchain

length

wereaccumulated

dependingon

thegrow

thsubstrate,also

underresting

cellcondition

R.jostii

LGK(R.jostii

RHA1

bearingplasmid

pTAC-lg

k)Cellsgrow

ingon

glucose,levoglucosan

RHA1expressing

lgkgene

from

Lipomyces

starkeyiYZ-215

acquired

thecapacity

toutilize

levoglucosan

for

grow


Xiong

etal.2016a

R.jostii

RHA3(R.jostii

RHA1

bearingboth

araB

AD

andaraF

GH)

Cellsgrow

ingon

arabinose

RHA1expressing

araB

ADandaraF

GH

from

E.coliacquiredthecapacity

toutilize

L-arabinose

forgrow

thandTAG

accumulation

Xiong

etal.2016b

R.opacusstrainsPD630andMR22,

R.w

ratislaviensisV,R

.jostii

RHA1,R.erythropolis

DSMZ

43060,R.fasciansF7,R.equi

ATCC6939,R

.jostii

RHA1

pJAM2/ltp

1

Cellsgrow

ingon

olivemill

wastes

R.opacus,R.w

ratislaviensis,andR.jostii

weremoreefficientatp

roducing

cell

biom

assandlip

idsthan

R.fascians,

R.erythropolis,and

R.equi.The

overexpression

ofltp

1(agene

encoding

afatty

acid

importer)in

R.jostii

RHA1

prom

oted

anincrease

of3.4-fold

inlip

idsproductio

n

Herrero

etal.2018

Rhodococcus

sp.Y

HY01

Cellsgrow

ingon

oilp

alm

biom

assand

barley

strawlig

nin

Utilizationof

variousarom

aticcompounds

derivedfrom

lignocellu

losicbiom

asses

forgrow


Bhatia

etal.2017,2019

Polyhydroxyalkanoate(PHAs)

R.ruber

NCIM

B40126

Cellsgrow

ingon

acetate,lactate,

succinate,fructose,glucose,

andmolasses

Accum

ulationof

PHAwith

3HVand

3HBmonom

ers.The

relativ

eam

ountof

each

monom

erchangeddependingon

thesubstrate,butH

Vwas

generally

the

predom

inant

Haywoodetal.(1991)

R.ruber

NCIM

B40126

Cellsgrow

ingon

4-hydroxybutyrate

and1,4-butanediol

Accum

ulationof

PHAmainlycomposed

by3HVand3H

Bbutalso

incorporating4H

B

Haywoodetal.(1991)

R.ruber

NCIM

B40126

Cellsgrow

ingon

5-chlorovalerate

Accum

ulationof

PHAmainlycomposed

by3HVand3H

Bbutalso

incorporating5H

B

Haywoodetal.(1991)

R.ruber

NCIM

B40126

Cellsgrow

ingon

hexanoate

or2-hexenoate

Productio

nof

PHAcontaining

3HB,

3HV,and

3-hydroxyhexanoate

(3HHx)

monom

erunits

Haywoodetal.(1991)

R.ruber

NCIM

B40126

Cellsgrow

ingon

valericacid

and

2-pentenoicacid

Accum

ulationof

almostp

ure

poly(3-hydroxyvalerate)

Haywoodetal.(1991)

R.ruber

NCIM

B40126

Cellsgrow

ingon

glucoseandvalerate

Accum

ulationof

thecopolymer

poly(3HB-co-3HV)as

mainPHA

Alvarez

etal.1997b

R.rhodochrous

ATCC19070

Cellsgrow

ingon

acetate,lactate,

fructose,glucose,and

sucrose

Accum

ulationof

PHAwith

3HVand

3HBmonom

ers.The

relativ

eam

ountof

each

monom

erchangeddependingon

Haywoodetal.(1991)


Tab

le1

(contin

ued)

Productcategory

Strain/s

Substrateand/or

grow

thconditionsa

Features

oftheproduct

and/or

biosyntheticprocess

Reference

thesubstrate,butH

Vwas

generally

thepredom

inant

R.erythropolis

DSM

Z43060,

R.fascians

D188–5,R.opacus

MR22

Cellsgrow

ingon

glucose,

gluconate,andvalerate

Accum

ulationof

smallamountsof

homopolym

erPH

BAlvarez

etal.1997b

R.aetherivorans

IAR

Cellsgrow

ingon

toluene

Accum

ulationof

thecopolymer

poly(3HB-co-3HV)as

mainPHA

Horietal.2009

Polyunsaturatedfatty

acids(PUFA

)R.aetherivorans

BCP1

Cellsgrow

ingon

cyclopentane

carboxylicacid,cyclohexane

carboxylicacid,and

glucose

Productio

nof

linoleicacid

increasedon

CPC

Aas

comparedto

theotherC

sources

Presentatoetal.2018a

Antim

icrobials

R.jostii

K01-B0171

Cellsgrow

ingon

rich

medium

with

mannitol,glucose,yeastextract,

ammonium

succinateas

carbon

and

nitrogen

sources

Productio

nof

lariantin

AandB

Inokoshi

etal.(2012)

R.erythropolis

JCM

6824

Cellgrowingon

succinate,sucrose,and

casaminoacids

Productionof

aurachin

RE

Kitigawaetal.(2018)

Rhodococcus

sp.A

cta2259

Cellsgrow

ingin

rich

medium

Productio

nof

four

aurachins

Nachtigalletal.2010

Rhodococcus

sp.M

er-N

1033

Cellsgrow

ingin

rich

medium

Productio

nof

rhodopeptin

sChiba

etal.1999

R.fascians307C

OCo-cultu

ring

with

Streptom

yces

padanus

Productio

nof

rhodostreptomycin

AandB

Kurosaw

aetal.2008

R.erythropolis

JCM

2895

Soft-agarassaywith

rich

medium

medium

Productionof

abacteriocin-lik

emolecule

Kitigawaetal.2018

R.equiand

R.erythropolis

strains,

Rhodococcus

enclensisNIO

-1009

Genom

e-basedanalyses

Productionof

humim

ycin

AandB

Chu

etal.2016

Siderophores

R.erythropolis

IGTS8

Cellsgrow

ingin

definedsaltmedia

underiron

limitatio

nconditions

Productio

nof

heterobactin

AandB

Carrano

etal.2001

R.erythropolis

PR4

Cellsgrow

ingon

glucose,under

iron-depletedconditions

Productio

nof

heterobactin

A,

heterobactinsS1andS2

Bosello

etal.2013

R.jostii

RHA1

Cellsgrow

ingon

glucoseunder


Productio

nof

rhodochelin

Bosello

etal.2011

R.rhodochrous

OFS

Cellsgrow

ingon

hexadecane

iniron-deficient

minim

almedium

Productio

nof

rhodobactin

Dhunganaetal.2007

R.erythropolis

S43

Cellsgrow

ingon

glucose,under


Productio

nof

siderophorebiding

trivalent

arsenic,AsO

33-[alsoreferred

toas

As(III)]

Retam

al-M

orales

etal.2018b

Metal(loid)

nanomaterials

Rhodococcus

sp.strain

Restin

gcells

andHAuC

l 4as

precursor

Productio

nof

intracellularcrystalline

AuN

Ps(ca.12

nm)

Ahm

adetal.2003

Rhodococcus

sp.N

CIM

2891

Cellsgrow

ingusingsodium

acetateor

cell-free

extracts,A

gNO3

asprecursor

Productio

nof

intracellularcrystalline

AgN

Ps(ca.10

nm)

Otarietal.2012

R.pyridinivoransNT2

Cellsgrow

ingin

rich

medium

orcell-free

extracts;Z

nSO4·H

2Oas

precursor

Productio

nof

extracellularsphericaland

hexagonalcrystallin

eZnO

NPs

(ca.100nm

)

Kundu

etal.2014


produced trehalose-based biosurfactants that were bound tocells during the first growth phase, while a strong increase ofextracellular trehalolipid level was observed after the onset ofstationary growth phase, reaching a maximum level of ~75%of the total trehalolipids present in the liquid medium (Whiteet al. 2013). Further, the formation of anionic trehalose ester inR. opacus 1CP was associated with both biomass growth andn-alkane consumption, whereas the trehalose lipid productionby R. erythropolis DSM43215 seemed to be uncoupled fromgrowth and occurred in the stationary phase or under restingcells condition (Kim et al. 1990). An additional aspect regardsthe substrate used for the biosurfactant production, asR. erythropolis ATCC 4277 produced extracellular glyco-lipids upon growth on glycerol as sole carbon source, whilepartially cell-bound biosurfactants were obtained when n-hexadecane was added as only carbon and energy source(Ciapina et al. 2006). The presence of hydrocarbons (liquidparaffin and hexadecane) favored the generation of cell-boundtrehalose-based glycolipids in R. erythropolis EK-1 cells.Conversely, strain EK-1 produced extracellular bioemulsifierswhen the cells were grown on soluble substrates (ethanol andglucose) (Pirog et al. 2008; Franzetti et al. 2010). These con-siderations highlight the wide diversity in biosurfactant pro-duction mechanisms among different Rhodococcus strainsand the possibility to drive the production of specific surfac-tant molecules by selecting the growth substrate. In this re-gard, supplying R. erythropolis SD-74 cultures with differentn-alkanes as carbon source led to the biosynthesis of succinoyltrehalolipids featuring acyl groups of the same carbon chainlength as the growth substrate (Tokumoto et al. 2009), sug-gesting the possibility to direct the synthesis of specificbiosurfactants by providing alkanes with specific chemicalstructure (Lang and Philp 1998; Inaba et al. 2013;Cappelletti et al. 2019a). This property is incredibly usefulfor the commercial production, as most biosurfactant pro-ducers do not synthesize specifically defined derivatives. Onthe other hand, different Rhodococcus spp. strains produceddiverse biosurfactants using the same hydrocarbon as a carbonsource. For instance, Rhodococcus sp. SD-74 generated extra-cellular succinoyl trehalose lipids when cultivated on n-hexadecane (Tokumoto et al. 2009), while the same substratein R. erythropolis 3C-9 favored the synthesis and release oftwo types of biosurfactants, i.e., trehalose lipids and free fattyacids—the latter being rarely reported in Rhodococcus spp.,with the exception of R. ruber (Kuyukina et al. 2001; Penget al. 2007). Other active compounds with surfactant activityproduced by Rhodococcus were extracellular polysaccharides(EPS), which, in the case of R. rhodochrous S-2, enhanced itstolerance to the aromatic fraction (AF) of crude oil and facil-itated the growth of indigenous bacteria resulting in the pro-motion of AF degradation in seawater-based medium(Iwabuchi et al. 2002).

Tab

le1

(contin

ued)

Productcategory

Strain/s

Substrateand/or

grow

thconditionsa

Features

oftheproduct

and/or

biosyntheticprocess

Reference

R.aetherivorans

BCP1

Cellsgrow

ingin

rich

medium;

TeO

32−as

precursor

Productio

nof

intracellularTeN

Rs

(from

100to

500nm

)Presentatoetal.2016

Restin

gcells;T

eO32−as

precursor

Productionof

intracellular

crystalline

TeN

PsandTeN

Rs

(from

200to

700nm

)

Presentatoetal.2018b

Cellsgrow

ingin

rich

medium;

SeO32−as

precursor

Productio

nof

SeN

PsandSeN

Rs

(from

50to

600nm

)Presentatoetal.2018a

R.erythropolis

ATCC4277

Cellsgrow

ingin

rich

medium

orin

astirredtank

reactor;sulfatemineral

coaltailingsas

precursor

Productio

nof

crystalline

Fe2O3NPs

(ca.50

or100nm

)Maasetal.2019a,2019b

aIn

Cx,xindicatesthenumberof

carbonsin

thealkane

chain


Biosurfactants produced by some Rhodococcus specieswere reported to be more efficient in reducing the surfaceand interfacial tensions between aqueous and oil phases andto have lower critical micelle concentrations (CMCs) thanmany synthetic surfactants (Lang and Philp 1998). For in-stance, the biosurfactant produced by Rhodococcus sp. strainH13-A was up to 35-fold more effective than the syntheticTween 80 counterpart in solubilizing polyaromatic hydrocar-bons from a complex mixture into an aqueous solution (Pageet al. 1999). Furthermore, in several cases, the biosurfactantsfrom Rhodococcus were also found to have extremely lowtoxicity, sometimes lower as compared to that ofbiosurfactants isolated from other bacterial genera. Indeed,biosurfactants produced by and extracted from R. ruber AC235 were 100–1000 times less toxic than synthetic commer-cial surfactants (Inipol EAP22, Corexit 9597, Finasol OSR-5)and 13 times less toxic than rhamnolipids from Pseudomonasaeruginosa (Kuyukina et al. 2001).

To reduce the cost of producing biosurfactant withrhodococci, specific approaches, such as the exploitation oflow-cost substrates, were used. Raw material accounts foralmost 30% of the overall cost of a microbial surfactant pro-duction and the investigation on potential usage of low-costraw materials (e.g., industrial and/or municipal wastes) is nec-essary to develop economically sustainable processes (Georgeand Jayachandran 2013). In this context, R. erythropolis 16

LM.USTHB showed the capacity to convert residual sunflow-er frying oil, a cheap renewable substrate, into extracellularglycolipids, which effectively lowered the surface tension ofthe crude broth (Sadouk et al. 2008). Ruggeri et al. (2009)isolated Rhodococcus sp. BS32, a strain able to produce ex-tracellular biosurfactants growing on rapeseed oil. Fish wastecompost was an effective source of nutrient-rich organic mat-ter for the growth of R. erythropolis sp. P6-4P and the produc-tion of biosurfactants (Kazemi et al. 2009).

Despite promising features of the biosurfactants produced byrhodococci as compared to the well-understood biochemical andmolecular bases of rhamnolipids synthesized by Pseudomonasstrains (George and Jayachandran 2013), less information isavailable for the genetic and biochemistry of TL generation inRhodococcus. It seems that the hydrophilic (the trehalosemoiety)and hydrophobic (the mycolic acid moiety) portions of thetrehalolipid molecules are synthesized independently and aresubsequently esterified (Kretschmer and Wagner 1983;Kuyukina and Ivshina 2010). During biosynthesis, trehalose isfirst esterified with mycolic acids synthesized in the cytoplasm toform the trehalose monomycolate, which is believed to be theprecursor of the di- or trimycolates produced at the plasmamem-brane (Lang and Philp 1998). Moreover, an enoyl-acyl carrierprotein reductase, InhA, was indicated to be part of the fatty acidsynthase-II system (FASII) involved in the process of the elon-gation of medium-sized fatty acids leading to long-chained

Fig. 1 Chemical structures of the main biosurfactants produced by Rhodococcus spp. strains (modified from Franzetti et al. 2010; Kuyukina and Ivshina2010)


hydrophobic moieties of trehalose mycolates in Rhodococcusspp. (Asselineau et al. 2002). The trehalose-6-phosphate syn-thase, OtsA, was reported to be the key enzyme involved in thesynthesis of the sugar residue of the trehalose lipids biosynthesis(Tischler et al. 2013). The presence of an alternative trehalosebiosynthesis pathway mediated by maltooligosyltrehalose syn-thase (TreY) and maltooligosyltrehalose trehalohydrolase(TreZ) enzymes was predicted in the biosurfactant producerR. erythropolis B7g (Retamal-Morales et al. 2018a). This path-way for trehalose biosynthesis uses oligo/polymalto-dextrins/gly-cogen as substrate (Tropis et al. 2005).

Bioflocculants

Flocculants are used as additives that induce aggregation oragglomeration of colloidal and other particles to form largeparticles (flocs) which settle allowing the clarification of thesystem in sedimentation and clarification processes (Lee et al.2014). In this context, flocculants have been extensively ap-plied for removing turbidity, suspended and dissolved solids,colors and dyes, and chemical oxygen demand (COD) in tap-and wastewater treatment processes. Compared with tradition-al inorganic and organic synthetic flocculants such as poly-acrylamides, bioflocculants are more readily biodegradable,as well as less toxic to humans and environments(Salehizadeh et al. 2018).

Bioflocculants produced by Rhodococcus spp. strains re-ceived a large amount of attention due to their strong floccu-lating activities (Peng et al. 2014; Jiang et al. 2019). Theywere reported to mainly consist of polypeptide and lipid as-semblies, especially mycolate-containing glycolipids(Finnerty 1992), which should allow the bacteria to more eas-ily access the growth substrate. Specifically, bioflocculantsseemed to be produced by those Rhodococcus spp. strains thatdo not synthesize biosurfactants during the utilization of long-chain alkanes (Bouchez-Naïtali et al. 2001). One of the firststudies on this topic indicated that the production of an extra-cellular flocculant by R. erythropolis S-1 cells was stronglydependent on the type of substrate added. Although glucose,fructose, sorbitol, and mannitol were all efficiently used forbioflocculant production by S-1 strain, ethanol resulted to bethe best performing carbon source for this purpose (Kuraneet al. 1994), whereas organic acids and phenol did not induceany bioflocculant generation. The bioflocculant produced byS-1 resulted to be usable on a wide range of suspended solids,such as acid and alkaline soils, as well as India ink (Kuraneet al. 1994). The same Rhodococcus strain could also use n-pentadecane as a carbon source, yet in this culture medium,the flocculant was found to be located on the cell surface,influencing the cell appearance during S-1 growth. Indeed,cells forming fibrous flocs that floated at the surface wereobserved upon S-l cultivation in the medium containing n-

pentadecane, whereas when glucose was used as the carbonsource, the cells were dispersed (Takeda et al. 1991).Characterization analyses performed on the bioflocculant pro-duced by S-1 revealed that its main component was a protein(Kurane et al. 1994; Takeda et al. 1991). This bioflocculantwas named NOC-1 and it has been used in both environmentalpollution control and industrial wastewater treatment.R. erythropolis S-1 is currently recognized as one of the bestflocculating bacteria and was used as a reference strain instudies on microbial flocculant production (Jiang et al. 2019).

Several studies investigated the possibility to reduce thecost of bioflocculant development, by using economical cul-ture media for bacterial growth (Table 1). For instance, usingpre-treated sludge and livestock wastewater as growth sub-strate , R. erythropolis ATCC 10543 produced apolysaccharidic bioflocculant featuring a strong flocculatingpotential on the same wastewaters within a wide pH range (2–12) (Peng et al. 2014). A recent study reported on the use ofpotato starch wastewater by another R. erythropolis strain toproduce, parallelly to its growth, a bioflocculant, whichshowed a polysaccharide nature. The effectiveness of thebioflocculant was harnessed by treating the potato starchwastewater at neutral pH and analyzing the residual organics(COD and BOD), ammonium, phosphorus, turbidity, andchroma. This bioflocculant could be successfully applied tothe potato starch wastewater treatment in a sequencing batchreactor (SBR), indicating that the compound was active in areal application scenario (Guo et al. 2018).

Carotenoids

Carotenoids are pigments ranging in color from yellow toorange and deep red that are soluble in lipids. Chemically,they are usually tetraterpenoids composed of a 40-carbonatom polyene skeleton, which is either acyclic or terminatedby one or two cyclic end groups. In nature, carotenoids pro-tect cell membranes against damage by light, oxidation, andfree radicals (Thanapimmetha et al. 2017); further, severalcarotenoids have been proved to play an important role inthe prevention of human diseases and maintaining goodhealth, due to antioxidant and anticancer properties (Raoand Rao 2007). Moreover, in humans and animals, α-caro-tene, β-carotene, and β-cryptoxanthin are precursors of vita-min A; thus, carotenoids are widely used as food additives,as well as pharmaceuticals, poultry, and cosmetic products(Saini and Keum 2017). Nowadays, there is an increasingdemand for “natural” carotenoids to replace the chemicallysynthesized ones due to the impelling scientific interest to-wards green technologies and the public awareness about thepossible toxicity of synthetic food additives. The microbialproduction of carotenoids is considered as a promising alter-native, as carotenoid-rich microbial biomass can be grown in


bioreactors using inexpensive substrates and under controlla-ble culture conditions to maximize production (Agarwal andRao 2000; Thanapimmetha et al. 2017).

ManyRhodococcus spp. strains can produce different typesof carotenoid pigments (Table 1) (Takaichi et al. 1990; Taoet al. 2004), which are located either intracellularly (e.g., inlipid droplets or in the vicinity of the plasma membrane) oraround the hydrophobic Rhodococcus cell wall. As they arenon-photosynthetic bacteria, the role of carotenoids was gen-erally associated with cell protection from various oxidativedamages, as demonstrated by testing cell response to H2O2treatment and to methylene blue-sensitized photo-oxidation(Osawa et al. 2011; Bequer Urbano et al. 2014). Further, theincrease of carotenoid accumulation in these bacteria or themodification of carotenoid profile was associated with thetype of growth and the presence of organic solvents. Indeed,carotenoids were suggested to have an antioxidant role inRhodococcus cells grown as biofilm, whose developmentcould induce an oxidative stress in individual cells withinthe community due to an increase of reactive oxygen species(ROS) levels (Zheng et al. 2013; Kundu et al. 2015).Rhodococcus erythropolis IBBPo1 cells exposed for 1 or24 h to 1% alkanes instead showed a modification of thecarotenoid profile with an increased level of lycopene (theintermediate in the biosynthesis of γ-carotene andβ-carotene)as compared to the control, likely as a response to these or-ganic solvents (Stancu 2015). Lastly, carotenoid formation ina Rhodococcus strain isolated from Azolla symbiotic cavitieswas reported to be induced by cells’ exposition to the light,which led to a sevenfold higher accumulation of a yellowish-orange pigment than dark-incubated cells (Cohen et al. 2004).

The first study on the ability of Rhodococcus strains toproduce carotenoids was performed by Ichiyama et al.(1989), who cultivated 16 Rhodococcus strains, each belong-ing to a different species, using glycerol as the main carbonsource, at pH 7 and at 37 or 28 °C (Ichiyama et al. 1988,1989). Based on thin-layer chromatography, the strains pro-duced 11 different carotenoid-type pigments, whose naturewas only partly identified. Nevertheless, Rhodococcus specieswere divided into three groups depending on the type of pig-ment synthesized, i.e., β-carotene, a γ-carotene-like sub-stance, or neither of these carotenes; some species also pro-duced derivatives of β- and γ-carotene, such asmyxoxanthophyll-like, zeaxanthin-like, and β-citraurin-likecarotenoids. In this work, the biosynthesis of β-carotene wassuggested to originate from a conversion of γ-carotene. Thepresence of the enzyme catalyzing the transformation of γ-carotene into β-carotene distinguished the species under anal-ysis (Ichiyama et al. 1989). Among these, R. maris andR. ruber were subsequently reported as β-carotene (bicycliccarotenoid) producers without being further characterized(Ichiyama et al. 1989). On the other hand, R. rhodochrousRNMS1 and R. erythropolisAN12 were described to produce

only monocyclic carotenoids such as γ-carotene derivatives(Takaichi et al. 1990; Tao et al. 2004). This aspect was relatedin the AN12 strain with the peculiar activity of the lycopeneβ-monocyclase encoded by the crtLm gene, which producedalmost exclusively γ-carotene from lycopene. In relation tothis, Rhodococcus is one of the few bacterial genera, whichare able to produce only monocyclic carotenoids such as γ-carotene derivatives. More frequently, monocyclic caroten-oids are part of the mixture formed during the synthesis ofbicyclic carotenoids (e.g., β-carotene). The asymmetricallyacting lycopene β-cyclase from AN12 strain is therefore ofgreat biotechnological significance to obtain asymmetric ca-rotenoids of commercial interest, which are generally verydifficult to produce chemically (Tao et al. 2004). An addition-al work by Tao and Cheng (2004) further described the re-maining of the carotenoid synthesis genes (crt genes) inAN12. Among these genes, crtO encodes a β-caroteneketolase, whose heterologous expression in an Escherichiacoli strain accumulating β-carotene resulted in the productionof canthaxanthin (β,β-carotene-4,4′-dione) (Tao and Cheng2004), which is an orange-to-red keto-carotenoid exhibitingstrong antioxidant activities and the ability to reduce and pro-tect against UV light-induced tumor/types of cancers (Rao andRao 2007). Additionally, members of genus Rhodococcus areamong the few selected microbes producing carotenoids withconjugated keto functions (Ichiyama et al. 1989). In particular,keto-carotenoids are characterized by high polarity which fa-cilitates their absorption and distribution once ingested andpositively influences their antioxidant activity in membranes(Borroni et al. 2017). Recently, during a screening for antiox-idative carotenoids of bacterial origin, a novel aromatic carot-enoid (OH-chlorobactene glucoside hexadecanoate) and rarecarotenoids (OH-chlorobactene glucoside, OH-carotene glu-coside, and OH-4-keto-carotene glucoside hexadecanoate)were found to be produced by the orange-pigmentedRhodococcus sp. CIP isolated from a soil sample (Fig. 2).The produced carotenoids showed potent antioxidative activ-ities, as indicated by analyzing the 1O2 quenching abilities ofthe compounds. The biosynthetic pathway for these promisingcarotenoids showed the γ-carotene as the main metabolic in-termediate, which is further modified by the activities of se-quential enzymes, such as a desaturase (CrtU), a hydroxylase(CrtC), and a glucosyltransferase (CruC), an acyltransferase(CruD), or a β-carotene ketolase (CtrO) (Osawa et al. 2011).

In the view of setting the bases for a possible industrialprocess, the carotenoid content of the biomass, the cultureproductivities, and biomass yields were evaluated in batchand fed-batch cultures of R. opacus PD630 using glyceroland ammonium acetate as cheap carbon and nitrogen sources,respectively. These experiments indicated that carotenoid pro-duction was dependent on the biomass growth, while no effectwas observed upon modifications of the growth medium pH(Thanapimmetha et al. 2017). Under similar growth


conditions, adding glucose as a co-substrate in a glycerol-based culture medium and controlling the nitrogen supplyimproved the production of biomass, lipid, and carotenoids.Particularly, a repeated fed-batch operation with controlledfeeding of carbon- and nitrogen-substrates increased the pro-ductivity of carotenoids in PD630 up to 3.5-fold, and a 5-foldincrease in the final lipid concentration (Suwaleerat et al.2017). In addition to the use of glycerol as cheap substrate,organic contaminants are promising carbon sources for thesustainable production of carotenoids using Rhodococcusspp. strains. In this respect, a few studies have reported thecapacity of Rhodococcus spp. strains to accumulate caroten-oids growing on hydrocarbon and nitroaromatic compounds(Table 1) (Stancu 2015; Kundu et al. 2015).

Intracellular accumulation of storagecompounds

Rhodococcus spp. strains are able to synthesize and accumu-late storage lipids in the form of polyhydroxyalkanoates(PHAs) (Anderson and Dawes 1990), triacylglycerols(TAGs) (Alvarez et al. 1996), or wax esters (WEs)(Lanfranconi and Alvarez 2017; Alvarez and Steinbüchel2019) as an adaptation response to specific growth conditionsand nutrients.

In addition to the accumulation of lipid-based molecule,Rhodococcus spp. can also produce inclusions enriched in

polyphosphate (polyP) and/or carbohydrates (i.e., glycogenand trehalose) (Hernández et al. 2008). In this respect, the mi-crobial polyP production has been described as a promisingapproach to remove or recover phosphate from wastewaters(Wang et al. 2018). PolyP granules are formed as a responseto unfavorable environmental conditions, such as osmotic andoxidative stresses, low environmental phosphate load, desicca-tion, and heat shock. Indeed, oligotrophic growth conditionsdetermined the accumulation in R. erythropolis N9T-4 ofpolyP granules, which were named oligobodies due to theiranalogies with acidocalcisomes of eukaryotic cells (Yoshidaet al. 2017; Presentato et al. 2018a). Similar polyP granuleswere intracellularly generated by R. aetherivorans BCP1 cellsupon utilization of cyclohexane carboxylic acid (CHCA, anaphthenic acid molecule) as the sole carbon and energy source(Fig. 3). Recently, the intracellular accumulation of polyP wasproposed as an energy sink to replace ATP, which may have aregulatory effect towards specific enzymatic activities(Hernández et al. 2008). On the other hand, the accumulationof glycogen and trehalose in Rhodococcus was considered acarbon and energy storage strategy occurring in response toeither N-limiting conditions of growth or hyperosmotic stress(Hernández et al. 2008).

Triacylglycerols

The ability to accumulate triacylglycerols (TAGs, glycerolesters with long-chain length fatty acids) and wax esters

Fig. 2 Chemical structures of the main carotenoids produced by Rhodococcus sp. 1CP


(WE, esters of primary long-chain fatty acids and primarylong-chain fatty alcohols) has been reported for only somebacterial genera including Streptomyces, Rhodococcus,Mycobacterium, and Nocardia (Alvarez et al. 2019).Bacterial TAGs were proposed to function as reserve com-pounds for carbon and energy, although other roles have beenalso discussed in relation to the regulation of cellular mem-brane fluidity and with the sink for reducing equivalents(Alvarez and Steinbuchel 2002). These lipids have relevantapplications in the production of food additives, cosmetics,lubricants, oleochemicals, candles, and biofuels (Alvarezand Steinbüchel 2002). Alternative sources for TAG produc-tion are derived from the agriculture, although the advantagesof using microorganisms over agricultural sources are numer-ous and mainly relating to the high variability of the fatty acidcomposition produced, as well as the better accessibility ofmicroorganisms to genetic and metabolic engineering.

Among the TAG accumulating genera, Rhodococcus isone of the most promising since bacterial strains of this genuscan accumulate significant amounts of lipids (above 20% ofthe cell dry weight), being for this reason referred to as “ole-aginous” (Table 1). In particular, R. opacus and R. jostiistrains are considered oleaginous models as they producelarge amounts of TAGs (up to 87% of the cellular dry weightinR. opacus PD630) (Fig. 4) (Alvarez et al. 1996; Alvarez andSteinbüchel 2002; Alvarez 2016), which are visible inside thecells as insoluble inclusions surrounded by a thin membrane(Alvarez et al. 1996; Alvarez 2003). They also showed theability to accumulate unusual acyl moieties in TAGs, suchas phenyldecanoic acid (Alvarez et al. 2002).

As compared to R. opacus and R. jostii, R. fascians,R. erythropolis, and R. equi generally produced loweramounts of TAGs during cultivation on sugars, organic acids,or hydrocarbons (Table 1) (Alvarez et al. 1997; Alvarez 2003;

Herrero et al. 2018). The different biotransformation perfor-mance is due to genetic and physiological differences amongRhodococcus spp. strains. The high lipid production perfor-mance of R. opacus, R. jostii, and R. wratilaviensiswas attrib-uted to their large genomes and high number of genes codingfor transporters and enzymes involved in the lipid metabolism(Cappelletti et al. 2019a). On the other hand, R. fascians andR. erythropolis were the most efficient species in TAG accu-mulation from glycerol. Despite the smaller genome, theseRhodococcus spp. possessed the genes glpFK1D1, whichare involved in glycerol degradation and are absent inR. opacus and R. jostii (Herrero et al. 2016).

In addition to the species/strain itself, other factors influ-ence the amount, composition, and structure of lipids inRhodococcus such as the carbon source used, the time ofcultivation, and the amount of carbon and nitrogen presentin the culture medium (Alvarez et al. 1997; Wältermannet al. 2005). Different Rhodococcus spp. strains have shownthe ability to produce and accumulate TAG using differentsubstrates including defined carbons sources like sugars andorganic acids (Alvarez et al. 1996, 1997, 2000; Silva et al.2010), single organic contaminants (e.g., aliphatic and aro-matic hydrocarbons) (Alvarez et al. 1997; Silva et al. 2010)but also complex carbon sources present in agro-industrialwastes, such as sugar beet molasses, orange, olive mill wastes,whey, oil palm biomass and corn stover (Voss andSteinbuchel 2001; Kurosawa et al. 2014; Bhatia et al. 2017;Herrero et al. 2018).

The growth substrate supplied to the culture medium influ-enced the amount and chemical nature of the accumulatedTAGs. For instance, in R. opacus PD630 culture, the supplyof growth substrates which are intermediates of the TCA cycle(i.e., citrate and succinate) or which are delivered to the TCAcycle, i.e., acetate, and odd-numbered organic acids (i.e., pro-pionate or valerate), induced an increase of the fraction of odd-numbered fatty acids (both saturated and mono-unsaturated,

Fig. 3 Transmission electron microscopy (TEM) image of RhodococcusaetherivoransBCP1 cells grown on naphthenic acids. The electron-denseintracellular body represents a possible polyP granule (from Presentatoet al. 2018a)

Fig. 4 Transmission electron microscopy (TEM) image of Rhodococcusopacus PD630 cells containing TAG granules (from Alvarez et al. 2013)


with chain length of C15 and C17) in TAG compared to thegrowth on fructose or gluconate (Alvarez et al. 1997).Conversely, when the cells were cultivated on n-alkanes, theaccumulated fatty acids were related to the carbon skeleton ofthe respective alkane used for growth. For instance, whenR. opacus PD630 cells were cultivated on hexadecane as thesole carbon source, palmitic acid (C16:0) was the predominantfatty acid occurring in cells, whereas during PD630 growth onpentadecane, cells accumulated only odd-numbered fattyacids directly related to the carbon chain of the specific alkaneand to its β-oxidation derivatives (Alvarez 2003). These re-sults suggested that β-oxidation pathway does not only repre-sent a catabolic route in oleaginous Rhodococcus spp. strains,but it is also a source of fatty acids. Further, the alkanes thatare incorporated into cellular lipids are not completely degrad-ed to acetyl-CoA (Alvarez 2003; Cappelletti et al. 2019b).These studies highlighted the possibility to drive lipid storageto specific branched-chain and odd-numbered fatty acids inR. opacus by only changing the growth substrate, making thisspecies a valuable TAG producer for next-generation biofuels(Tsitko et al. 1999).

The limitation of the nitrogen source in the presence of anexcess of carbon in the culture medium was reported to be themain trigger for TAG accumulation, causing an imbalancebetween C and N in cells, which redirects the carbon metab-olism to lipogenesis (Alvarez et al. 2000). Early studies de-scribed the improved capacity of R. opacus PD630 to accu-mulate TAGs when the cells were growing under nitrogenlimiting conditions (ca. 20 times less nitrogen than control)and in the presence of high amounts of gluconate (Alvarezet al. 2000). These results indicated that, in contrast to whatwas observed in many bacteria, PD630 cells do not block lipidmetabolism under growth-limiting conditions and generateacyl-residues from the available carbon source leading toTAG production. Yet, TAGs were mobilized and used as car-bon and energy source when R. opacus PD630 cells wereincubated under carbon starvation conditions in the presenceof a nitrogen source (Alvarez et al. 2000; Alvarez andSteinbüchel 2002; Alvarez 2019). Finally, TAG biosynthesiswas dependent on the time of bacterial cultivation and, there-fore, the growth phase. Indeed, R. opacus PD630 demonstrat-ed shifts in the composition of TAGs during the progressionfrom exponential to stationary phase under unbalancedgrowth conditions (Alvarez et al. 2000).

Due to the capability of Rhodococcus spp. strains to con-vert l ignin-derived aromatic compounds (e.g., 4-hydroxybenzoate, benzoate, phenol, vanillate, guaiacol, andtrans-p-coumaric acid, p-coumaric acids, cresol, and 2,6-dimethoxyphenol) into TAGs, members of this genus arepromising microbial hosts for lignocellulosic biomass conver-sion into biofuels (Kosa and Ragauskas 2012; Bhatia et al.2019). Diverse genetic, physiological, and biochemical stud-ies along with more recent “omic”works provided indications

on the metabolic pathways and regulatory mechanisms in-volved in the biosynthesis and accumulation of TAGs fromdifferent carbon sources, including lignocellulose biomasses(Chen et al. 2014; Dávila Costa et al. 2015). On the basis ofthese studies, genetic and metabolic engineering strategieswere proposed to develop sustainable processes of biofuelproduction using Rhodococcus spp. strains, mostlyR. opacus PD630 and R. jostii RHA1 (Castro et al. 2016;Anthony et al. 2019). In this context, R. opacus PD630 andR. jostii RHA1 cells expressing specific heterologous genes,e.g., xylA and xylB from Streptomyces lividans TK23, andbglABC operon from Thermobifida fusca, were shown to ac-quire the capacity to degrade cellulose, arabinose, and xylosefrom lignocellulosic biomass and to simultaneously producelipids (Table 1) (Hetzler et al. 2013; Xiong et al. 2012, 2016a,2016b; Hernández et al. 2015; Kurosawa et al. 2013).Through Adaptive Laboratory Evolution (hereafter: ALE) pro-cedures, a series of R. opacus PD630 strains were generated,which were able to better utilize aromatics and produce lipids,to consumemultiple carbon sources simultaneously (i.e., glyc-erol, glucose, and xylose in R. opacusMITGM-173, Table 1),and to better tolerate inhibitors (e.g., phenolic compounds),which are usually present in lignocellulosic hydrolysates(Kurosawa et al. 2015a, 2015b; Yoneda et al. 2016; Hensonet al. 2018). Genome-based manipulation strategies based onCRISPR-Cas9 and recombineering, were successfully appliedin Rhodococcus spp. strains, opening new frontiers for geneticand metabolic engineering of members of this genus aimed atoptimizing biofuel production from lignocellulose bioconver-sion (DeLorenzo et al. 2018; Liang et al. 2020). In order toimplement metabolic engineering strategies for waste biocon-version into valuable compounds, a genome-scale model wasalso developed in R. jostii RHA1 to describe and predict theaccumulation rate of three types of carbon storage compounds(i.e., glycogen, polyhydroxyalkanoate, and triacyloglycerols)using different carbon sources (glucose or acetate) and undergrowth conditions typically occurring in activated sludge bio-reactor systems for wastewater recovery (Tajparast and Frigon2015, 2018).

Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of varioushydroxycarboxylic acids, which are produced by a variety ofbacterial species generally under nutrient-restricting condi-tions in the presence of carbon in excess (Anderson andDawes 1990). They are accumulated by bacteria as intracellu-lar hydrophobic inclusions as carbon and energy storage or aselectron sinks of redundant reducing power (Muhammadiet al. 2014). Accumulated PHAs are typically degraded byintracellular depolymerases and metabolized as carbon andenergy sources as soon as the supply of the limiting nutrientis restored (Steinbüchel et al. 1992). Based on the type of


monomer(s) constituent, PHAs can be homopolymers (e.g.,polyhydroxybutyrate [PHB]), copolymers (e.g., poly(3-hydroxybutyrate-co-3-hydroxyvalerate [poly(3HB-co-3 HV)or PHBV]), or terpolymers (e.g., poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) [P-3HB-co-3 HV-co-3HHx]) (Haywood et al. 1991). These polymersshow good biodegradability, insolubility in water, non-toxic-ity, thermoplasticity, and/or elastomericity, which supporttheir suitability for applications in the packaging industry,medicine, pharmacy, agriculture, food, and chemical indus-tries (Anderson and Dawes 1990). Indeed, PHA is consideredthe green polymers of the future, since they are expected togradually substitute conventional plastics (e.g., polypropylene(PP) and low-density polyethylene (LDPE)) with similarphysicochemical, thermal, and mechanical properties(Kourmentza et al. 2017). Since the chemical synthesis ofPHA is not feasible, microorganisms represent the only sourcefor these polymers.

Members ofRhodococcus genus are able to synthesize PHAsin variable amounts and composition depending on the bacterialstrain and the carbon source (Haywood et al. 1991; Pieper andSteinbüchel 1992; Alvarez et al. 1997a,b; Alvarez andSteinbüchel 2002; Alvarez 2003) (Table 1, Fig. 5). One of themost extensively studied strains is R. ruber NCIMB 40126

which was found to be able to accumulate a peculiar PHAcontaining a copolymer of 3-hydroxyvalerate (3 HV) and 3-hydroxybutyrate (3HB) monomer units (i.e., poly(3HB-co-3 HV) or PHBV) upon cultivation under N-limiting conditionsin the presence of different substrates such as acetate, lactate,succinate, fructose, glucose, and molasses (Table 1). In particu-lar, this strain, as well as other members of genus Rhodococcus(e.g., R. aetherivorans IAR), showed the peculiar capacity tosynthesize the commercially important PHBV from single car-bon sources which were characterized by chemical structureunrelated with that of the copolymer (Hori et al. 2009). Therelative amount of each monomer changed depending on thecarbon source, although HV was always predominant over HB(HV content range 60–91%) (Haywood et al. 1991). SeveralRhodococcus strains, including NCIMB 40126 andR. aetherivorans IAR, NCIMB 40126 was also capable of in-corporating 4HB and 5 HV monomer units into PHAs whenprovided with substrates which are precursors of these mono-mers (i.e., 4-hydroxybutyrate and 5-chlorovalerate, respectively)(Haywood et al. 1991). Further, this R. ruber strain accumulatedalmost pure poly(3-hydroxyvalerate) when supplied with valericacid and 2-pentenoic acid, while when hexanoate or 2-hexenoate were used as substrates, a PHA containing 3HB,3 HV, and 3-hydroxyhexanoate (3HHx) monomer units wasproduced (Haywood et al. 1991) (Fig. 5). In a different studyperformed by Alvarez et al. (1997b), R. ruber NCIMB 40126showed higher accumulation of PHA than TAGs on valerateand glucose, whereas it seemed to accumulate only TAGs onhexadecane (Table 1) (Alvarez 1997b). R. fascians D 188–5,R. erythropolisDSMZ 43060, and R. opacusMR22 accumulat-ed homopolymers of polyhydroxybutyrate (PHB) in addition tominor amounts of diacylglycerols and wax esters from variouscarbon sources, although the main carbon storage was TAG(Alvarez 1997b). R. opacus PD630 has been described to accu-mulate significant amounts of TAGs but not PHA from variouscarbon sources (Alvarez et al. 1996).

The possibility to combine biodegradation of toxic and con-taminant molecules with PHA production was investigated toachieve a cost reduction of the biodegradable plastic productionprocess, along with the effective utilization of wastes or toxiccompounds. In this context, R. aetherivorans IAR was able toproduce PHBV from toluene, which is the volatile organiccompound (VOC) most abundantly emitted in the environmentof some countries (e.g., Japan) (Hori et al. 2009).R. aetherivorans BCP1 accumulated electron-transparent intra-cellular inclusions composed by neutral lipids during thegrowth on naphthenic acid models (Presentato et al. 2018a).Although the chemical nature of these inclusion bodies is stillundefined, recent experiments showed that, based on the differ-ent NAs used for growth, BCP1 cells accumulated either PHBor poly(3HB-co-3 HV) (Cappelletti, unpublished results).

Notably, IAR strain could also accumulate TAG along withPHA on toluene and acetate (Hori et al. 2009), being both

Fig. 5 Chemical structures of the main polyhydroxyalkanoates (PHAs)produced by Rhodococcus spp. strains


PHBV and TAGs simultaneously synthesized and accumulat-ed before the nitrogen source was exhausted. Subsequently,the accumulation of both the storage compounds continued,whereas only the TAG was synthesized even after the carbonsource depletion, although at a low rate. This lipid productionprofile was different from that ofR. ruberNCIMB 40126 cellsgrowing on glucose, during which only the accumulation ofPHBV started during the exponential growth phase (Alvarezet al. 2000). When the complete consumption of the nitrogensource occurred, PHBV content reached a maximum inNCIMB 40126 cells and the biosynthesis and accumulationof TAGs started. These results demonstrated that the accumu-lation of TAGs continued after carbon source exhaustion inboth the strains, whereas, after substrate depletion, PHBVwasmobilized and started to be degraded. With the aim of opti-mizing the bioprocess, this time course study gave indicationson the culture and harvesting conditions that resulted in bothhigh cellular content of these storage compounds and PHAsand TAGs of desired composition (Alvarez et al. 2000). Theutilization of specific metabolic inhibitors led to the produc-tion of either PHA or TAG, defining possible selective bio-synthesis strategies (Alvarez et al. 2000). These experimentsalso demonstrated that the biosynthetic route of PHA and fattyacids in R. ruber NCIMB 40126 compete for the commonprecursors, acetyl-CoA and propionyl-CoA, during cellgrowth under storage conditions (Alvarez et al. 2000).

PHA synthases represent the key enzymes of PHA biosyn-thesis, which catalyze the stereoselective conversion of (R)-3-hydroxyacyl-CoA substrates to PHA with the concomitantrelease of CoA (Hernández et al. 2008). The phaC gene fromR. ruber is the only gene encoding for a PHA synthase iden-tified and cloned from a member of Rhodococcus (Pieper andSteinbüchel 1992). This PhaC enzyme is a short-chain lengthclass I PHA synthase, which comprises only one type of sub-unit that utilize CoA thioesters of 3-hydroxy fatty acids with 3to 5 carbon atoms (Rehm 2003). Downstream of the R. ruberphaC, a gene coding for a putative PHA depolymerase (PhaZ)was identified, which is predicted to be the key enzyme forPHA mobilization. In the model strain R. jostii RHA1, threedifferent chromosomal loci including the phaC and phaZwererecognized. Despite being similar to that described inR. ruber,this pha gene organization is different from that of otherGram-negative bacterial strains accumulating short-chainlength PHA, which also typically include other genes in-volved in the PHA biosynthetic pathway such as phaA andphaB (Rehm and Steinbüchel 1999).

Polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are fatty acids containingmore than two double bonds. Important PUFAs includeomega-3 and omega-6 (ω-3 FA and ω-6 FA) fatty acids that

are named based on the position of the first double bond fromthe methyl-end in the fatty acid chain. They are critical nutri-ents for human health, as they modulate brain developmentand cognition, as well as many diseases, such as cardiovascu-lar disease, cancers, and diabetes (Lee et al. 2016).Furthermore, specific PUFAs are precursors of molecules reg-ulating inflammatory and immune responses (Calder 2013).Humans must acquire PUFAs via foods or nutritional supple-ments because they do not contain delta-12 desaturases andconvert molecules to PUFAs very slowly. Because of the nu-traceutical and pharmacological importance, biotechnologymethods have been used to clone and introduce numerousdesaturases into diverse organisms to yield PUFAs with crit-ical significance in diet. Many groups have investigated theuse of marine algae as a commercial source (Patel et al. 2019);however, bioengineered bacterial strains may result in superi-or producers, due to the large biomass accumulation and highgrowth rate. In this context, R. opacus was proposed as a hoststrain of polyunsaturated fatty acid synthase genes (pfa genesfrom marine bacterial strain Shewanella baltica) expressionfor PUFA production (Blakie 2015), because of the abilityof this species to accumulate high amount of fatty acids.

PUFA biosynthetic process has been mainly described inmarine bacteria (Nichols 2003), but recently, Rhodococcuserythropolis and R. aetherivorans strains showed the ability toaccumulate PUFAs, likely as a response mechanism associatedwith specific stress conditions, such as the exposition to a highsalt concentration, extremely low temperatures, toxic metals,and the growth on toxic organic compounds (e.g., naphthenicacids) (de Carvalho 2012; de Carvalho et al. 2014; Presentatoet al. 2018a). In particular, R. aetherivorans BCP1 cells accu-mulated significant amounts (around 7% of the total cell fattyacid content) of linoleic acid (omega-6, 9cis,12cis-C18:2) dur-ing the growth on the NA, cyclopentane carboxylic acid(CPCA). The molecular mechanisms supporting PUFA accu-mulation in Rhodococcus spp. strains have not been elucidatedyet. Recent RT-qPCR experiments on RNA extracted fromR. aetherivorans BCP1 cells grown on naphthenic acids didnot clearly show the upregulation of any specific gene amongthose predicted to encode desaturases and polyketide synthases(Cappelletti, unpublished results).

Antimicrobials

Antibiotics revolutionized the treatment and prevention of nu-merous types of infections and deadly diseases. As a side effect,many pathogenic bacteria have developed antibiotic resistance,which causes problems in the treatment and threatens modernhealthcare. Increasing efforts are in progress to find new anti-biotics or antimicrobial strategies to fight these strains.However, it is frequent to rediscover compounds that are al-ready known, and this aspect presently slows down substantial


developments in novel drug discovery. More than half ofthe presently known antibiotics have been isolated fromStreptomyces spp. (Nepal and Wang 2019). Despite be-longing to the same phylum (Actinobacteria), the knowl-edge on the production of antimicrobial compounds byRhodococcus spp. strains is only a very recent matter ofstudy (Kitigawa et al. 2018; Elsayed et al. 2017). Over thelast 15 years, a few studies have reported this genus to bea good source of novel antibiotics (Table 1). To date, thelariantin peptide antibiotics, the polyketide aurachin RE,the rhodopeptins, and the humimycins are the natural andimportant products with antimicrobial activity that havebeen isolated from Rhodococcus strains (Fig. 6).

One of the first studies on antimicrobial production byRhodococcus reported the strain R. jostii K01-B0171 to beable to produce lariantin A and B, which are antimycobacterialcyclic peptides consisting of 18 and 20 L-amino acid residues,respectively. They are defined as “lasso” peptides because oftheir structure having a macrolactam ring between the α-amino group of amino-terminal Gly1 and the γ-carboxylgroup of Glu8 and a carboxy-terminal tail of the peptidelooped back and threaded through the macrolactam ring

(Fig. 1). These two peptides selectively inhibited the growthof Mycobacterium smegmatis and Mycobacteriumtuberculosis, probably via targeting mycobacterial cell wallsynthesis (Iwatsuki et al. 2006, 2007). The main biosyntheticgene cluster of the lariatins in R. jostii K01-B0171 was de-scribed to consist of five genes, larABCDE, in which larAencodes the precursor peptide which is post-translationallymodified by LarB and LarD to produce lariatin. The larE geneencodes a possible ABC transporter involved in the maturelariatin export (Inokoshi et al. 2012).

By screening 80 Rhodococcus strains from several bacte-rial culture collections, Kitigawa et al. (2018) showed that18 strains of five different Rhodococcus species had antimi-crobial activities. Among these, a high number of strainsbelonging to R. erythropolis species were found to produceantibiotics. These R. erythropolis strains were divided intothree groups depending on the antibiotic production(Kitigawa et al. 2018). In particular, one of the antibiotic-producing strains, R. erythropolis JCM 6824, was describedto produce a new quinoline antibiotic aurachin RE whichexhibited a strong activity against a broad range of Gram-positive bacteria. From a chemical point of view, aurachin

Fig. 6 Chemical structures of the main antibiotics found to be produced up to date by Rhodococcus spp. strains


RE has a structure very similar to aurachin C, which derivesfrom the Gram-negative bacterium Stigmatella aurantiacaSga15 (Fig. 7). However, compared to aurachin C, aurachinRE exhibited a wide and strong antimicrobial spectrumagainst both high- and low-GC Gram-positive bacteria(Kitagawa and Tamura 2008). Additional four aurachin mol-ecules were isolated from Rhodococcus sp. Acta 2259(Nachtigall et al. 2010). These compounds showed inhibito-ry activities against the growth of numerous Gram-positivebacteria, such as Staphylococcus epidermidis DSM 20044,Bacillus subtilis DSM 347, and Propionibacterium acnesDSM 1897, but not against Gram-negative bacteria(Nachtigall et al. 2010). Conversely, the cyclic tetrapeptide

rhodopeptins that were first isolated from Rhodococcus sp.Mer-N1033 exhibited antifungal activity against Candidaalbicans and Cryptococcus neoformis, but they showed noantibacterial activity (Chiba et al. 1999).

The two antibiotics rhodostreptomycin A and B were in-stead isolated from culture broths of R. fascians 307CO cellsafter being subject to competitive co-culture experiments withthe antibiotic producer Streptomyces padanus. These antibi-otics were found to be biosynthesized following horizontalgene transfer of a large DNA segment derived from theStreptomyces strain. From a chemical point of view,rhodostreptomycins were identified as two isomers of a newclass of aminoglycosides, which greatly differed in the

Fig. 7 Structures of the mainsiderophores produced byRhodococcus spp. strains(modified from Bosello et al.2012)


structure from the Streptomyces-produced actinomycins(Kurosawa et al. 2008). Rhodostreptomycins exhibited goodantibiotic activities against an extensive range of Gram-negative and Gram-positive bacteria, such as Streptomycespadanus, Escherichia coli, Staphylococcus aureus, Bacillussubtilis, and Helicobacter pylori. Rhodostreptomycin B wasfound to be more potent than rhodostreptomycin A, suggest-ing that their stereochemistry difference influenced the biolog-ical activity (Kurosawa et al. 2008) (Fig. 7).

Recently, the first bacteriocin-like molecule and its struc-tural gene (rap) were obtained from Rhodococcuserythropolis JCM 2895. A 5.4-kb circular plasmid harborsrapA and rapB genes encoding for RapA and RapB proteins.RapA is a small, heat-stable, water-soluble protein showingantimicrobial activity against other R. erythropolis strains,while RapB is an immunity protein against RapA that maybe located on the cell membrane (Kitigawa et al. 2018).Bacteriocins are usually peptides synthesized by ribosomesand have a narrow spectrum of activity; they are produced toinhibit the growth of competitor organisms in their environ-ment to outcompete for nutrients (Gillor et al. 2008).

Differently to the other antibiotic compounds, the structureof humimycins was initially predicted through a bioinformaticapproach (named syn-BNP, standing for synthetic-bioinformatic natural product), which scanned hundreds ofbacterial genomes residing in the human body for clusters ofgenes that were likely to produce non-ribosomal peptides thatform the basis of many antibiotics (Fig. 7) (Chu et al. 2016).Gene clusters encoding non-ribosomal peptide synthases pro-ducing humimycin A and B were detected from genomes ofRhodococcus equi and R. erythropolis strains. The genome ofRhodococcus enclensis NIO-1009 also showed a biosyntheticgene cluster coding for humimycin B (Ceniceros et al. 2016).The humimycin antibiotics are lipid II flippase inhibitorswhich hamper bacterial cell wall biosynthesis. They werefound to be particularly effective against pathogenic andmultidrug-resistant Staphylococcus and Streptococcus strains(Chu et al. 2016).

Siderophores

Siderophores are low-molecular-weight compounds, whichare released by some bacteria to chelate extracellular ironand transport it inside the cell. The production and release ofsiderophores allows bacteria to accumulate an intracellularpool of iron and to satisfy the nutritional iron requirement alsounder metal-l imiting conditions (Kraemer 2004).Siderophores were also reported to bind other essential metalssuch as zinc, manganese, molybdenum, and vanadium fortheir acquisition, as well as to interact with heavy metals toprevent their cellular entry (Johnstone and Nolan 2015).Further siderophore functions include the protection against

oxidative stress and the enhancement of antibiotic targetingand delivery (Johnstone and Nolan 2015). Based on the chem-ical structure, siderophores are classified as phenolate,hydroxamate, catecholate, (hydroxy-)carboxylate, and mixedtypes (Miethke and Marahiel 2007), and their biosynthesisoccurs via different mechanisms (Miethke and Marahiel2007). Areas of siderophore application include agriculture,medicine, pharmacology, bioremediation, biodegradation,and food industry. For instance, siderophores can be used toenhance plant growth due to their uptake by rhizobia, while inbioremediation strategies, siderophores can mobilize heavymetals and radionuclides (De Serrano 2017).

Rhodococcus spp. strains were described to be able to pro-duce chemically diverse siderophores, such as rhodochelin,rhodobactin, heterobactin A, rhequichelin, and rhequibactin(Table 1 and Fig. 7). Carrano et al. (2001) were among thefirst groups to isolate a new class of siderophores, namedheterobactins, from R. erythropolis IGTS8 culture. Amongthe three heterobactins that were produced by strain IGTS8,the two more abundant were named heterobactin A and B.From a structural point of view, heterobactins werecatecholate-hydroxamate mixed-type siderophores, whichcontained both hydroxamate and catecholate donor groups.Heterobactins were also isolated from R. erythropolis PR4byBosello et al. (2013), who, through a bioinformatic analysisof the bacterial genome, identified the gene cluster responsiblefor heterobactin A’s biosynthesis. The biosynthesis of thissiderophore was described to involve the activity of the mod-ular enzymes nonribosomal peptide synthetases (NRPSs), inwhich each module adds a specific monomer to the peptidebackbone. Similarly, a genomic study conducted on the otherfour Rhodococcus strains showed the presence of a high num-ber of gene clusters encoding for NRPSs compared to otherActinobacteria (Doroghazi and Metcalf 2013).

Analogously to heterobactin, rhodochelin andrhodobactin belong to the hydroxamate–catecholate mixedtype family and were isolated from Rhodococcus jostiiRHA1 and Rhodococcus rhodochrous OFS, respectively(Dhungana et al. 2007; Bosello et al. 2011). While the geneclusters which determine rhodobactin synthesis were notidentified, the gene clusters involved in the synthesis ofrhodochelin were described in RHA1. This biosyntheticprocess involved the functional cross-talk between threedistantly located NRPS gene clusters (Bosello et al.2011), which were identified in most of Rhodococcusspp. strains (Bosello et al. 2011; Bosello et al. 2012).

Additional works assessed the capacity of siderophores pro-duced by R. erythropolis S43 to bind trivalent arsenic, or arse-nite [As(III)]. Interestingly, the arsenic-binding activity of thesiderophore-like compounds from S43 was higher than theiron-chelating one. Although the capacity of the siderophoreto bind and sequester toxic metals could not be clearly associ-ated with arsenic resistance/tolerance mechanisms, the study


provided the basis for a first analysis of different actinobacterialstrains, including R. erythropolis S43, as a source of arsenic-binding compounds.When overproduced or in association withplants, this type of molecules could be used to decontaminatesoil or water, as well as for other potential biotechnologicalapplications (Retamal-Morales et al. 2018b).

Metal(loid)-based nanostructures

Nanotechnology is defined as the world of “very small mate-rial” and it is based on the manipulation of matter at eithermolecular or atomic level (Horikoshi and Serpone 2013). Theprefix nano is related to materials that feature at least onedimension in the nanorange (1–100 nm), where physical–chemical properties (i.e., high surface-to-volume ratio, largesurface energy, and high spatial confinement) become en-hanced as compared to their bulk counterparts (Cao 2004).Metal or metalloid (metal(loid)) nanostructures (NSs) havewidespread applications in biomedicine/biotechnologies, en-ergy production, environmental engineering, material science,and optoelectronics (Cao 2004; Horikoshi and Serpone 2013).Different physical–chemical methods efficiently producehigh-quality nanomaterials (NMs) of various compositionsand morphologies (including nanoparticles (NPs),nanocrystals (NCs), nanorods (NRs), nanowires (NWs), andnanotubes (NTs)) (Rao et al. 2004); however, these ap-proaches mostly rely on dangerous operational conditions(e.g., high temperature and pressure), as well as the use oftoxic and harsh chemicals (Zhang et al. 2006; CDC 2014).Moreover, there are still important challenges to be faced inthis industry, among which the most urgent are represented bythe generation of NSs featuring homogeneous size (i.e.,monodispersity) and shape, as well as thermodynamic stabil-ity (Piacenza et al. 2018). The natural ability of microorgan-isms to cope with metal(loid) compounds, simultaneously

generating NMs, has taken the form of an innovative andeco-friendly bioprocess.

For the most part, bacterial cells cope and interact withlarge amounts of metal(loid) compounds by exploitingbiosorption, bioaccumulation, and biotransformation pro-cesses, which are used for either detoxification or energypurposes (Li et al. 2011; Pantidos and Horsfall 2014).Bacteria belonging to the Rhodococcus genus tolerate thepresence of various metal(loid)s, in many cases, due to theirbiotic conversion of metal(loid)s into a less toxic form. Inthis context, Rhodococcus spp. have been shown tobioconvert several metal(loid) precursors including gold(Au), silver (Ag), zinc oxide (ZnO), tellurium (Te), seleni-um (Se), and arsenic (As) synthesizing NMs, which are ob-served as either intra- or extracellular products, as a functionof the strain and growth conditions (Fig. 8) (Ahmad et al.2003; Otari et al. 2012; Subbaiya et al. 2014; Kundu et al.2014; Presentato et al. 2016, 2018b, 2018c). The first studyreporting rhodococci for metal(loid) NS productionhighlighted the ability of a Rhodococcus sp. strain to syn-thesize relatively monodisperse spherical AuNPs having ca.12 nm as the average diameter and a good crystalline struc-ture, as compared to chemogenic AuNMs (Ahmad et al.2003). Similarly, Rhodococcus sp. NCIM2891 proved tobe an efficient biocatalyst for the generation of intracellular,small (ca. 10 nm), spherical, and crystalline AgNPs (Otariet al. 2012), which were also described both as an antimi-crobial, to be used in commercialized textiles and wounddressings, or an antitumor agent (Subbaiya et al. 2014). Ironoxide (Fe2O3) NPs were biosynthesized by R. erythropolisATCC 427 cultures during biomining processes (Maas et al.2019a, 2019b). In this context, parameters of the designedimplants, as the stirring rate and oxygen flow rate, appearedto determine the size of the produced Fe2O3 NPs, allowingto obtain fairly monodisperse populations (50 or 100 nm),as well as α-Fe2O3 and β-Fe2O3 crystalline structures(Maas et al. 2019a, 2019b).

Fig. 8 Tellurium-based nanorods (TeNRs) intracellularly produced byRhodococcus aetherivoransBCP1 and visible inside the cell (a) or in the cell-freeextract (after cell sonication) (b)


The role of rhodococci in the microbial nanotechnologyfield was deeper analyzed by Kundu and co-workers(2014), who focused on biosynthesis of extracellularZnO NPs by R. pyridinivorans NT2. The produced ZnONPs showed a quasi-spherical and hexagonal morpholo-gy, a large polydispersity (average size 100–120 nm), anda low tendency to aggregate. Nevertheless, these biogenicnanoproducts were as pure and crystalline as those com-mercially available, showing good absorbance and fluo-rescence properties (Kundu et al. 2014). BiogenicZnONPs were also used as a coating for textile cottonsurfaces, displaying good UV-blocking, self-cleaning,and antimicrobial properties (Kundu et al. 2014).Finally, these NPs resulted to be efficacious against coloncarcinoma cells (HT-29), without showing cytotoxicityagainst normal peripheral blood mononuclear cells(Kundu et al. 2014).

To date, R. aetherivorans BCP1 represents one of the mostversatile and well-known strain belonging to this genus forNM biosynthesis. This fundamental technological trait relies,for the most part, on the ability of this bacterium to handlevery high concentrations of the toxic oxyanions TeO3

2− andSeO3

2−, biotically converting them in their elemental forms(Te0 and Se0) generating Te or Se NSs, such as NRs or NPs(Figs. 8 and 9). Moreover, by playing with the physiologicalstate of BCP1 cells, as well as the amount of precursor con-centration supplied to the culture medium, this strain couldimprove the average length of the intracellularly producedTeNRs, which featured a regular single-crystalline structure(Fig. 8) (Presentato et al. 2018c). These TeNRs also possessedan electrical conductivity comparable to chemogenic TeNMs,further supporting the technological value of Te-basednanoproducts synthesized by BCP1 cells (Presentato et al.2018c). Curiously, exposing the same strain to SeO3

2−, theproduction of SeNPs and NRs was associated with the cellmembrane (Fig. 9) (Presentato et al. 2018b). Another

distinctive case is represented by BCP1 incubated with in-creasing concentrations of arsenate [AsO4

3-, also referred toas As(V)] in the presence of glucose as the sole carbon andenergy source. Here, some preliminary TEM analyses showedthe presence of electron-dense hexagonal nanoplates (possiblyassociated to metal(loid) biosorption) that deserve furtheranalysis (Firrincieli et al. 2019).

The physical–chemical characterization of the metal(loid)NMs produced by Rhodococcus spp. also highlighted the ab-sence of NS aggregation phenomena under diverse condi-tions, indicating their high thermodynamic stability andallowing to avoid post-production treatments of these biogen-ic NMs to ensure their high quality and applicability. Thiscrucial feature seemed to rely on the presence of organic mol-ecules derived from bacterial cells, which may simultaneouslyact as both electrostatic and steric stabilizers for the NMs insuspension (Piacenza et al. 2018). This aspect was deeplyinvestigated for the ZnO NPs produced by R. pyridinivoransNT2 (Kundu et al. 2014), as well as biogenic Se or Te NMssynthesized by R. aetherivoransBCP1 (Presentato et al. 2016,2018b, 2018c). Proteins and peptides were found to be impor-tant components of the biogenic NM extracts recovered fromboth rhodococci strains, as tyrosine (Kundu et al. 2014) ortryptophan residues (Presentato et al. 2016, 2018b, 2018c)were detected within the organic material recovered fromNT2 or BCP1 cells, respectively. Additionally, intra and/orextracellular heterocyclic metabolites (in NT2 strain; Ku

biotechnology of rhodococcus for the production of valuable … · 2020. 9. 12. · mini-review...

Documents