Insights into the mechanism of mycelium transformation of Streptomyces toxytricini into pellet

Abstract Formation of the mycelial pellet in submerged cultivation of Streptomycetes is unwanted in industrial fermentation processes as it imposes mass transfer limitations, changes in the rheology of a medium, and affects the production of secondary metabolites. Though detailed information is not available about the factors involved in regulating mycelial morphology, it is studied that culture conditions and the genetic information of strain play a crucial role. Moreover, the proteomic study has revealed the involvement of low molecular weight proteins such as; DivIVA, FilP, ParA, Scy, and SsgA proteins in apical growth and branching of hyphae, which results in the establishment of the mycelial network. The present study proposes the mechanism of pellet formation of Streptomyces toxytricini (NRRL B-5426) with the help of microscopic and proteomic analysis. The microscopic analysis revealed that growing hyphae contain a bud-like structure behind the apical tip, which follows a certain organized path of growth and branching, which was further converted into the pellet when shake flask to the shake flask inoculation was performed. Proteomic analysis revealed the production of low molecular weight proteins ranging between 20 and 95 kDa, which are involved in apical growth and hyphae branching and can possibly participate in the regulation of pellet morphology.


Introduction
Streptom yces (Gr am-positiv e bacteria) is one of the most explored micr oor ganisms at the commercial scale for synthesizing natural pr oducts.During submer ged fermentation, filamentous microorganisms exhibit morphologies between diffused mycelia and pellets as per the culture conditions and type of microbial strain (Hobbs et al. 1989 ). Streptomyces toxytricini is recognized for producing lipase-inhibitory natural product lipstatin, used in an FDAa ppr ov ed anti-obesity drug (Kumar andDube y 2015 , 2016 ).Lik e other species of the genus, Streptomyces toxytricini also exhibits pellet morphology in submerged fermentation (Kumar and Dubey 2017 ).Mycelial mor phologies ar e associated with pr oducing the desir ed natur al pr oduct.Mor eov er, the pellet mor phologies affect the rheology of a medium, heterogeneous mass transfer, and downstr eam pr ocessing (Rioser as et al. 2014, Wang et al. 2017 ).
Ther efor e, r egulation of pellet morphology is necessary for industrial processes.
Studies conducted at the molecular le v el suggested the involv ement of man y genes and pr oteins that contr ol the mycelium transformation or morphological differentiation of Streptomyces (Vassallo et al. 2020, Wu et al. 2020 ).Streptom yces' gr owth occurs from the spore into vegetative hyphae, regulated by the AMP receptor pr otein Cr p (Der ouaux et al. 2004 ).Further growth of the hyphae takes place by tip extension and br anc hing, wher e polarity protein DivIVA, the first molecular marker of hyphal tips, plays a k e y role (Flardh et al. 2012 ).It was suggested that during the growth of hyphae, the development of cross-walls also takes place, whic h pr otects hyphae fr om fission and forms a m ultinucleated compartment (J akimo wicz and van Wezel 2012 ).Mor eov er, man y pr otein complexes of Streptom yces like Scy, Tat secr etion system, SsgA, and CslA are also associated with the apical tip growth of Streptomyces .In Streptomyces , cellulose synthase-like proteins, whic h ar e r esponsible for the synthesis of beta-glucan-containing pol ysacc harides , pla y an essential role in tissue morphogenesis, hyphal tip growth, and morphological differentiation (Noens et al. 2007, Hempel et al. 2012, Willemse et al. 2012, Holmes et al. 2013 ).It was reported that SsgA is involved in identifying a location for de v eloping the septum and germination site (Noens et al. 2007 ).Inter estingl y , Scy , P arA, and FilP pr oteins ar e assumed to inter act with DivIVA to control the apical growth of mycelia (Ditkowski et al. 2013, Holmes et al. 2013 ).Scy is a scaffold protein that functions as an apical dominance regulator, while FilP is a cytoskeletal protein that affects the hyphal shape (Khushboo et al. 2023 ).One mor e pr otein HyaS has been found conserv ed in str e ptom ycetes and associated with the regulation of pellet morphology by maintaining hyphal contacts (Koebsch et al. 2009 ).
Mor eov er, it was also suggested that some extracellular materials (proteins , sugars , and DN A) w ork as adhesiv es and ar e involved in forming pellet-like structures and providing protection.Extracellular DN A, hy aluronic acid, teichoic acids, and CslA are associated with Streptomyces ' pellet morphology (Kim andKim 2004 , Ultee et al. 2020 ).It was found that cellulose synthase-like protein CslA was also identified near the hyphal tip, which was involv ed in gr owth and structur al tr ansformation.It was also studied that it interacts with DivIVA and r egulates biopol ymer formation with glycosidic bonds near hyphal tips .T he physical phenomenon, such as oxygen transfer and shear rate, also influence the morphology of Streptomyces (Ribeiro et al. 2021 ).Directive and quic k-acting a ppr oac hes, lik e the ad dition of microparticles and macroparticles , ha ve also been used to regulate mycelial morphology (Böl et al. 2020, Yue et al. 2021 ).
In the pr e vious r eport, authors r eported that cultur e conditions (pH of culture medium, medium composition, agitation rate, and inoculum size) influence the pellet size of S. toxytricini (Kumar and Dubey 2017 ).Still, the mechanism of mycelial transformation into a pellet has not been elaborated.Though pellet size was reduced with changes in culture conditions, biomass formation was also reduced.T hus , the mechanism of pellet formation must be studied to reduce the pellet size or maintain dispersed mycelial morphology without affecting the biomass.In the present study, authors attempted microscopic analysis of hyphae growth and br anc hing, whic h further transformed into the pellet.Additionally, a partial analysis of proteins (produced by bacteria in pellet and culture medium) was performed to understand the role of proteins in pellet formation.

Microorganism and growth conditions
Streptomyces toxytricini NRRL B-5426 strain was obtained from the Agricultur al Researc h Service (NRRL), Department of Agricultur e, USA.The bacterium was grown in a shake flask as per the conditions mentioned in Kumar and Dubey ( 2017 ).The incubation temper atur e was maintained at 27.5 • C till the a ppear ance of visible colonies.Well-grown colonies of S. toxytricini were pink in color, ele v ated, circular in shape, and had a specific odor.

Analysis of pellet morphology
For pellet morphology analysis, an inoculum of S. toxytricini was pr epar ed by tr ansferring a loopful of bacteria ( S. toxytricini ) fr om a Petri plate into a shake flask, modified from Kumar and Dubey ( 2017 ).
After incubation, pellets were settled down and centrifuged at 5000 rpm for 10 min to remove the culture medium.Aggregated pellets were washed thrice with 0.1 M sodium phosphate buffer of pH 7.2 ± 0.1 and stored for further processing.The Gram staining and methylene blue staining of pellets were performed to analyze mor phology and sha pe.Visualization was done in a compound microscope.For scanning electron microscopy (SEM) of pellets, primary fixation of pellets was performed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.2 ± 0.1.Further dehydration, fixation and coating, and SEM anal ysis wer e done at Advanced Instrumentation Research Facility, J aw aharlal Nehru Univ ersity, Ne w Delhi (India).For biomass anal ysis, pellets wer e k e pt for drying at 50 • C in a hot air oven till the a ppear ance of constant weight.

Proteomic analysis of samples
Intracellular and extracellular proteins produced by S. toxytricini during gro wth w ere extracted separately from the growth medium and pellets .T he acetone precipitation method was used for protein extr action fr om cultur e br oth, and for pr otein isolation fr om pellets, pellets were processed in lysis buffer at pH 7.0 (Hobbs et al. 1989, van Veluw et al. 2012 ).Isolated proteins were analyzed by 10% SDS PAGE.

Growth of S. toxytricini
The growth of S. toxytricini in submerged fermentation sho w ed that if inoculum was tr ansferr ed fr om the Petri plate to the shake flask, it produced diffused mycelia and pellets of smaller size, while the transfer of inoculum from the shake flask to the shake flask produced visible pellets in the culture broth.Thus it can be assumed that in the Petri plate, bacterial mycelium was not pr ogr ammed for pellet formation, but in the culture broth, S. toxytricini produces some chemical compound (intracellular or extr acellular) that dir ects the formation of pellets at the submerged le v el.Micr oscopic examination of S. toxytricini, which was grown using inoculum from a Petri plate, revealed that mycelial aggregation took place, but the majority of these a ggr egations wer e clumps, loose mycelia, and smaller pellets (up to 20 μm).While shake flask to shake flask inoculation produced pellets of larger size (30 μm-2 mm) as major morphological form and very less loose mycelia.T hus , it ma y be assumed that culture conditions influence the transformation of mycelia into pellets, and when S. toxytricini is grown in submerged conditions, it produces some bioc hemicals or pr oteins that contr ol pellet formation.According to a pr e viousl y r e ported stud y, this heterogeneity of mycelial forms (loose, clump, and pellet) is maintained in a broth medium (Kumar and Dubey 2017 ).Similarly, some studies have reported that variation in culture conditions directly affected the pellet morphology of bacterial strains, but such mycelial morphology is unwanted for industrial processes (Flardh et al. 2012, van Veluw et al. 2012, Rioseras et al. 2014 ).

Morphology of pellets
For a better insight into pellet structure, the morphology of pellets was analyzed by scanning electron microscope (Fig. 1 ).In this study, it was observed that pellet formation takes place by interwoven hyphae (Fig. 1 A), where hyphae are joined with each other and form a compact network (Kim andKim 2004 , Koebsch et al. 2009 ).The surface of the pellet has loose mycelia, which are sites of growth, and these mycelia undergo fragmentation during agitation to form a new pellet (Fig. 1 B) (Koebsch et al. 2009 ).Further magnification (2 μm scale) revealed that hyphae are attached to each other and form tight junctions (Fig. 1 C).Microscopic analysis r e v ealed that growing hyphae bear bud-like structures behind the apical tip, and these bud-like structures form branches that were de v eloped at regular intervals (Fig. 1 D) (Hempel et al. 2012 ).Thus it may be assumed that br anc hing behind the apical tip and tight junctions assist in compact pellet formation.As reported earlier, such types of association protect hyphae from fission and produce m ulti-nucleoid structur es (J akimo wicz and van Wezel 2012 ).The microscopic analysis strongly supported the observations of pr e vious r esearc hers, suc h as; the gener ation of m ultiple polarity centers (Holmes et al. 2013 ), growth and development of pellet by tip extension, br anc hing, and cr oss-w all formation (Flar dh et al. 2012, Hempel et al. 2012, Khushboo and Dubey 2023 ).

Proteomics analysis
Researc hers hav e r eported man y pr oteins (DivIVA, Scy , FilP , SsgA, ParA, Tat, CslA, Afsk, CRP, HyaS, TrpM, and Mat complex, etc.) involved with apical growth, formation of septa, br anc hing in growing hyphae (Noens et al. 2007, Hempel et al. 2012 , Willemse et al. 2012, Ditkowski et al. 2013, Holmes et al. 2013, van Dissel et al. 2018, Vassallo et al. 2020, Zhang et al. 2023 ).To further elucidate the involvement of different proteins behind the pellet formation of S. toxytricini , secreted proteins in the culture medium and intracellular pr oteins wer e anal yzed by electr ophor esis.In this study, the partial analysis of proteins (intracellular and extracellular) was conducted.It was observed that large numbers of proteins of different molecular weights were produced at the intracellular and extracellular levels ranging from 95 to < 20 kDa (Fig. 2 ).It is assumed that many of these proteins are involved in pellet formation.It was observed in Fig. 1 D that growing hyphae form branches behind the apical tip.Studies have reported the expression of Di-vIVA (21.5 kDA) at the gr owing tip, whic h is suggested to interact with Scy, ParA, and FilP to regulate pellet formation (Ditkowski et al. 2013, Holmes et al. 2013 ).It has been suggested that Scy regulates a number of polarity centers by associating with DivIVA for new tip construction during br anc hing (Holmes et al. 2013 ).Including this, Sc y w as found to sequester DivIVA and initiate the formation of new polarity centers (Holmes et al. 2013 ).The protein Scy is also assumed to associate with ParA to hyphal tips and contr ol pol ymerization of P arA.The Scy-P arA association is assumed to be involved in the transition of hyphal elongation into sporulation (Ditkowski et al. 2013 ).Thus these proteins need further analysis for their involvement in pellet formation.Including this, the presence of other proteins involved in the growth and br anc hing of hyphae into pellet formation needs to be analyzed before making any final conclusion.

Mechanism of pellet formation
In the pr e vious study, the authors discussed the pr ocess of pellet formation (Kumar and Dubey 2017 ) (Fig. 3 ).DivIVA is found to be associated with cell wall synthesis, genetic competence, and c hr omosome segr egation during sporulation (Labajov a et al.

2021
).It has also been suggested that DivIVA, with some other pr oteins (Scy, P arA, and FilP), regulates the formation of polarisome and forms br anc hes in hyphae .T hough continuous agitation of culture medium enables clumping of mycelia, some external biopolymers may assist the association of hyphae with each other, resulting in compact pellets.To analyze this process, micr oscopic observ ation was conducted to understand the pattern of pellet formation.It was r e v ealed that hyphae perform growth and br anc hing behind the a pical tip during gr owth.The gr owing hyphae come in close proximity to form a network of hyphae, which forms a reaction center-like structure .T his structure further grows and associates with other hyphae and finally converts into a compact structure (Fig. 3 ).This hypothesis is also supported by the study that a pical gr owth r egulates cell polarity, whic h determines the morphology of the pellet (Hempel et al. 2012, Holmes et al. 2013 ).Including this, r esearc hers hav e demonstr ated cr osswall formation during the growth of stre ptom ycetes, which compact the structure into pellets (Flardh et al. 2012, Zhang et al. 2023 ).T hus it ma y be suggested that pellet formation in volves the process of hyphae growth, development of polarity, branching, and formation of cr oss-walls, whic h r esults in the tr ansformation of hyphae into compact pellets.

Conclusions
Stre ptom ycetes are filamentous microorganisms reported for producing man y v aluable natur al pr oducts, including antibiotics and enzymes .T he present study has led to the following conclusions: 1.The hyphae of these microbes represent a distinct morphological form, i.e. pellet during submerged cultivation and undesirable for industrial processes .T hough studies hav e r eported the pr oduction of antibiotics using pellet mor phology.Inv estigators hav e r eported the involv ement of  culture conditions and biomolecules controlling the morphology of hyphae.2. In this study, microscopic analysis of morphological forms of S. toxytricini at different times revealed that during submer ged gr owth, the gr owth of hyphae took place by tip extension, br anc hing behind the apical tip. 3. The growing hyphae possibly attached to each other and formed cr oss-walls, whic h further tr ansformed into compact pellets (Flardh et al. 2012, Hempel et al. 2012 ). 4. The partial proteomic analysis to understand the role of proteins in pellet formation r e v ealed that ther e is the pr esence of lo w-w eight proteins in the pellet and culture medium, whic h ar e possibl y involv ed in pellet formation.

Ac kno wledgments
Authors sincer el y ac knowledge the United States Department of Agriculture for providing S. toxytricini NRRL B-5426 and the Adv anced Instrumentation Researc h Facility, J aw aharlal Nehru Univ ersity, Ne w Delhi (India), for scanning electr on micr oscopy of pellets.

Figure 1 .
Figure 1.Analysis of pellet morphology and mycelial structure by SEM.(A) Morphology of pellet at 10 μM scale.(B) Surface of pellet showing free mycelia, whic h show gr owth and br anc hing at 10 μM scale.(C) Structure of hyphae showing association with each other and br anc hing at gr owing tips (2 μM scale).(D) Apical growth and branching of hyphae (2 μM scale).

Figure 2 .
Figure 2. Analysis of extracellular and intracellular proteins of S. toxytricini by SDS-PAGE.Lane 1 r epr esents total intr acellular pr oteins, and lanes 2 and 3 r epr esent total extr acellular pr oteins isolated fr om the cultur e medium.The mark er lane is a molecular-weight mark er.

Figure 3 .
Figure 3. Pr oposed mec hanism for pellet formation.(A) Diffused mycelia, (B) mycelia are coming together, (C) mycelia are clumping, (D) mycelial clumps are more visible, and a reaction center-type structure is formed, which possibly directs pellet formation, (E) -(G) mycelial clumps are more condensed, and (H) pellet formed and periphery having mycelia.