Importance of the latex-clearing protein (Lcp) for poly(cis-1,4-isoprene) rubber cleavage in Streptomyces sp. K30

Streptomyces sp. strain K30 induces the formation of an extracellular Lcp (latex-clearing protein) during poly(cis-1,4-isoprene) degradation. To investigate the function of this enzyme in Streptomyces sp. strain K30, the lcp gene was disrupted. This was the first time that the screening for a knock out lcp mutant of Streptomyces sp. strain K30 was successful. The resulting mutant Streptomyces sp. K30_lcpΩKm exhibited reduced growth in liquid mineral salts media containing poly(cis-1,4-isoprene) as the sole carbon and energy source. Additionally, there was no detectable Lcp activity on latex overlay agar plates. When Lcp from Streptomyces sp. strain K30 was heterologously expressed in strains TK23 and TK24 of Streptomyces lividans and a strain of S. erythraea with plasmid pIJ6021::lcp, the recombinant strains acquired the ability to cleave synthetic poly(cis-1,4-isoprene), confirming the involvement of Lcp in initial polymer cleavage. Specific anti-LcpK30 IgGs were employed in Western blot analysis to detect the secretion of Lcp in the supernatant. We have conducted an important experiment to demonstrate Lcp activity using the supernatant of these Lcp-expressing strains in vitro. All three strains obviously secreted a functional Lcp, as indicated by the formation of halo. Functional testing of Lcp with different plasmids in Escherichia coli strains and Pseudomonas strains was, however, not successful.


Introduction
Actinomycetes play a major role in the degradation of natural rubber (NR), while some other bacteria and fungi are also known to attack rubber (Kumar et al. 1983). Microorganisms capable of degrading NR cannot degrade synthetic rubbers other than synthetic isoprene rubber (Linos and Steinbüchel 1998). The latex-clearing protein (Lcp) from the rubberdegrading bacterium Streptomyces sp. strain K30 is involved in the initial cleavage of poly(cis-1,4-isoprene), yielding isoprenoid aldehydes and ketones . Lcp homologues have so far been detected in all investigated clear zone forming rubber-degrading bacteria.
The microbial degradation of natural and synthetic poly(cis-1,4-isoprene) rubber is currently being intensively investigated Rose and Steinbüchel 2005), and two different strategies for the degradation of isoprene rubber have been unraveled thereby distinguishing two differ-ent groups of rubber-degrading bacteria (Peczynska-Czoch and Mordarski 1988).
Members of the first group form translucent halos when cultivated on solid media containing dispersed latex particles, indicating the excretion of rubber-cleaving enzymes. Mycelium-forming actinomycetes such as Actinoplanes, Micromonospora, and Streptomyces species belong to this group. The second group comprises mycolic acid containing Actinobacteria belonging to the genera Gordonia, Mycobacterium, and Nocardia. These bacteria do not form translucent halos, but they grow adhesively on the surface of rubber particles in liquid culture, and they represent the most potent rubberdegrading bacterial strains (Arenskötter et al. 2004). Xanthomonas sp. strain 35Y is the only known rubber-degrading bacterium that does not belong to the actinomycetes but is a Gram-negative bacterium (Jendrossek and Reinhardt 2003).
A rubber oxygenase RoxA, which is synthesized during growth on NR latex by Xanthomonas sp. 35Y, was identified (Jendrossek and Reinhardt 2003;Braaz et al. 2005). This bacterium is strictly aerobic and produces insoluble yellow pigments in the cell. Xanthomonas species belong to the phylum Proteobacteria and stain Gram-negative. However, regarding the strategy of rubber degradation, it belongs to the first group and forms halos on rubber-containing agar plates.
In a hypothetical pathway supposed for rubber degradation, Bode et al. (2000) postulated a not further characterized oxidation of the degradation product acetonyldiprenylacetoaldehyde to the corresponding acid. This aldehyde compound was previously also identified by Tsuchii and Takeda (1990) after incubation of NR with Xanthomonas sp. 35Y and subsequent ether extraction. This oxidation step converting the aldehyde to the corresponding acid could possibly be performed by an enzyme similar to OxiAB whereas Lcp is responsible for the first step in this pathway, the oxidative cleavage of the polyisoprene backbone. These aldehyde and ketones with low molecular weights, which are then possibly further oxidized by OxiAB to the corresponding acids, are activated and metabolized via the β-oxidation pathway in Streptomyces sp. K30 (Fig. 1).  identified the lcp gene encoding a latex clearing protein from Streptomyces sp. strain K30. The clear zone forming phenotype was used to identify clones harboring the lcp gene from Streptomyces sp. strain K30 by phenotypic complementation of a clear zone negative mutant. The 1191-bp structural gene was preceded by a putative signal sequence and restored the capability of forming clear zones on NR latex agar plates in the mutant. Like RoxA, also Lcp is secreted into the extracellular medium leading to the formation of translucent halos on NR latex. However, both proteins share no sequence homologies. The putative translation product of lcp exhibited strong homologies (50% aa identity) to a putative secreted protein from S. coelicolor strain A3 (Bagdasarian and Timmis 1982), which is another clear zone forming strain . Sequence analysis of Lcp and characterization of mutants of Streptomyces sp. strain K30 showed secretion of Lcp via the twin-arginine translocation (Tat) pathway (Yikmis et al. 2008;Thomas et al. 2001).
Because expression of functional Lcp in recombinant Escherichia coli strains or in recombinant γ -Proteobacteria such as Pseudomonas putida was not successful, expression of 14 recombinant Lcp in other bacteria belonging to the genus Streptomyces sp., was performed. In this study, we show a system optimized for the expression of recombinant Lcp and the microbial degradation of rubber by these strains. Three actinomycetes strains, S. lividans TK23, TK24, and Saccharopolyspora erythraea, were able to produce clear zones on rubber overlay agar plates upon transfer of the wild-type lcp gene to these strains. Furthermore, we have conducted an important experiment to demonstrate Lcp activity using the supernatant of these Lcp-expressing strains in vitro. All three strains obviously secreted a functional Lcp, as indicated by the formation of a halo. We also generated a knock out lcp mutant from Streptomyces sp. strain K30 to characterize the role of Lcp with regard to poly(cis-1,4-isoprene) rubber degradation. By isolating and investigating the knock out lcp mutant, we have now confirmed evidence that Lcp is responsible for the initial rubber degradation.

Bacterial strains and culture conditions
Bacteria and plasmids used in this study are listed in Table 1. If not otherwise mentioned, cells of Streptomyces sp. were grown in tryptic soy broth (TSB) medium at 30 • C (Merck, Darmstadt, Germany), whereas cells of E. coli were cultivated at 37 • C in Luria Bertani broth (LB) (Sambrook et al. 1989), mineral salts medium (MSM) (Schlegel et al. 1961), or in standard I (St-I) medium (Merck). Antibiotics were applied according to Sambrook et al. (1989) and as indicated in the text. For growth experiments with natural and synthetic polyisoprene, cells were cultivated in MSM (Schlegel et al. 1961). The following carbon sources were added to liquid MSM: 0.5% (v/v) natural latex concentrate (Neotex Latz; Weber & Schaer, Hamburg, Germany) or 0.3% (w/v) synthetic poly(cis-1,4-isoprene) with an average molecular mass of 800 kDa. Liquid cultures were grown in Erlenmeyer flasks, which were incubated on a horizontal rotary shaker. Solid media were prepared by addition of agar-agar (18 g/L). Purified NR latex from Hevea brasiliensis was a gift from Weber & Schaer and was used for the preparation of overlay plates as described previously (Jendrossek et al. 1997). Latex overlay agar plates were used for growth of clear zone forming strains. For this, MSM agar plates were covered with an overlay of MSM agar containing 0.2% (v/v) disperged latex concentrate.

Isolation, analysis, and manipulation of DNA
Plasmid DNA was prepared from crude cell lysates by the alkaline extraction method (Kieser et al. 2000). Cells of Streptomyces were incubated at 37 • C for lysis in presence of lysozyme (2 mg/mL) for at least 2 h. Recombinant DNA techniques in Streptomyces were performed as described by Kieser et al. (2000). Total DNA from Streptomyces was isolated by the versatile quick-prep method for Gram-positive bacteria according to Pospiech and Neumann (1995). DNA was restricted with endonucleases (Gibco/BRL, Gaithersburg, MD) as mentioned in the text under the conditions recommended by the manufacturer. All other genetic procedures and manipulations were conducted as described by Sambrook et al. (1989).

Aldehyde staining of poly(cis-1,4-isoprene) and degradation products
Aldehyde groups resulting from poly(cis-1,4-isoprene) cleavage during clear zone formation on NR latex overlay agar plates were stained for 20 min with Schiff 's reagent. Afterwards, the staining reagent was removed, and the slides were washed with sulfite solution. The composition of the staining solution was as follows: 2 g of fuchsin dissolved in 50 mL of glacial acetic acid, 10 g Na 2 S 2 O 5 , 100 mL of 0.1 N HCl, and 50 mL H 2 O. The composition of the sulfite solution was 5 g of Na 2 S 2 O 5 plus 5 mL of concentrated HCl (37-38%, v/v) in a 100-mL aqueous solution.

Cloning and expression of Lcp
The coding region of lcp from Streptomyces sp. K30 was amplified by PCR by applying primers Lcp EcoRI 6021 and Lcp NdeI 6021. The amplified PCR product was then cloned into the pGEM-T Easy vector, excised by restriction with EcoRI and NdeI, and ligated to EcoRI-NdeI-linearized plasmid pIJ6021 DNA. For expression analyses, the resulting plasmid, pIJ6021::lcp, was transferred to Streptomyces strains via protoplast transformation (Hidalgo et al. 2004). These strains  were cultivated in LB medium containing antibiotics, which were applied according to Sambrook et al. (1989), at 30 • C on a rotary shaker at 180 rpm. After 48 h of incubation, the cells were harvested by centrifugation (20 min, 4 • C, 4000 rpm; Megafuge 1.0R, HERAEUS SEPATECH GMBH, Osterode, Germany). The resulting supernatant was used for further characterization by SDS-polyacrylamide gel electrophoresis (PAGE).

Expression of 6xHis-tagged Lcp in E. coli strain BL21(DE3), isolation of inclusion bodies, and generation of anti-LcpK30 antibodies
Escherichia coli strain BL21(DE3) harboring plasmid pET-23a::lcp His was cultivated in LB medium at 37 • C to an OD600 of 0.5, and then expression was induced by addition of IPTG to a final concentration of 1 mM for 3 h yielding cells with inclusion bodies (IBs). For isolation of IBs, the cells of a 100-mL culture were harvested, resuspended in 4 mL 20 mM Tris-HCl (pH 8.0) buffer, and disrupted by a twofold French press passage at 1000 MPa. The disrupted cells were centrifuged at 25,000 g for 15 min at 4 • C. The obtained pellet was resuspended in 3 mL cold IB wash buffer (2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0) by sonication (1 min/mL with an amplitude of 40 μm) with a Bandelin Sonopuls GM200 ultrasonic disintegrator. After 15 min centrifugation at 4 • C and 25,000 g, treatment with IB wash buffer, resuspension by sonication, and centrifugation were repeated for three times. The purified IBs were dissolved in SDS denaturation buffer (Laemmli 1970). A sample, consisting of the dissolved IBs containing the extracted Lcp protein, was separated by SDS-PAGE, excised from the gel, and its identity was confirmed by MALDI-TOF analysis (Bröker et al. 2008), before it was used for generation of polyclonal antibodies in rabbits in custom by "Eurogentec" (Seraing, Belgium). Purified polyclonal rabbit anti-LcpK30 IgGs were obtained from the serum by chromatography on Protein A-Sepharose (Hjelm et al. 1972).

Determination of mineralization
Evidence for biodegradation of the poly(cis-1,4-isoprene) hydrocarbon chain to CO 2 was obtained by determination of CO 2 evolution during aerobic cultivation of cells in presence of poly(cis-1,4-isoprene) as the sole carbon source. Determination was carried out in tightly closed Erlenmeyer flasks by using the property of Ba(OH) 2 to precipitate CO 2 as BaCO 3 . The flasks, containing 50-mL MSM, the rubber substrate [latex concentrate or poly(cis-1,4-isoprene)], and a test tube containing 15 mL of a 0.2 M Ba(OH) 2 solution, were inoculated with 0.3% (v/v) of a well-grown culture. At each measurement point, the flasks were aerated, and the test tubes were replaced by new tubes containing fresh Ba(OH) 2 solution. Consumption of carbonate by precipitation of CO 3 2− as BaCO 3 was determined for each period by titration with HCl and was compared to that of a noninoculated control. The mineralization rate was calculated as follows: mineralization (% CO 2 ) = (required amount HCl [mL] × 0.252 M)/(C content of applied amount of cis-1,4-polyisoprene [mmol]) × 2.

Heterologous expression of lcp in E. coli
We previously identified Lcp as an important gene required for rubber degradation by Streptomyces sp. strain K30 ) and aimed to characterize the heterologous expression of the gene product in the present study. For functionally and detailed characterization of the secretion-expression of lcp from Streptomyces sp. K30, the gene lcp was amplified employing the primers Hya FW XbaI and Hya RW NcoI (Table  1), and the PCR product was subsequently cloned into the XbaI and NcoI site of pET23a yielding pET23a::lcp. Additionally, lcp was subcloned into plasmids pUC19 and pET19b. However, the expression of all these different recombinant plasmids in several E. coli strains resulted in an overproduction of an inactive Lcp protein. However, despite of applying various experimental conditions such as cultivating the cells in LB medium or MSM, the protein was not active. In addition, different incubation temperatures (37 • C, 28 • C, or 20 • C) with high or slow shaking rates of the culture vessels were tested, however, also here, no E. coli transformant showed an active Lcp protein, which allowed further analysis (data not shown). Escherichia coli was therefore not suitable to study the expression of Lcp.

Heterologous expression of lcp in Pseudomonas
After due consideration, we constructed hybrid plasmids for gene cloning in the metabolically versatile bacterial genus Pseudomonas (Regenhardt et al. 2002). Pseudomonas putida KT2440, a saprophytic soil bacterium, which colonizes plant roots, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes (Bagdasarian and Timmis 1982;Moreno et al. 1988;Iwasaki et al. 1994;Jimenez et al. 2002). The lcp gene from Streptomyces sp. K30 was amplified employing the primers pqspBBR-for: Sal and pqspBBR-rev: Sac, and also the primers pqspJB-for: Sbf and pqspJB-rev: Sac (Table 1). Both PCR products were cloned into the SalI/SacI site of pBBR (Table 1) and the Sbf/SacI site of pJB, yielding pBBR1MCS2::Lcp His6 and pJB653::Lcp His6, respectively. Although all experiments with conditions optimized for Pseudomonas strains resulted in the overproduction of Lcp in the supernatant and successful purification by nickel chromatography of the His-tagged protein, Lcp was inactive.
High-level expression and secretion of proteins in the native form has been proven to be difficult in both hosts, E. coli and in γ -Proteobacteria such as P. putida. Escherichia coli cells are the most commonly used host cells for large-scale production of recombinant proteins, but some proteins are difficult to express in E. coli. This includes proteins with low stability (Bertani 1951), proteins that are toxic to the host, and proteins that tend to form IBs. Due to the low content of lcp in Streptomyces sp. K30, it is difficult to isolate the overproduced protein from the original producer. Therefore, we applied a new strategy.

Heterologous expression of lcp in S. lividans TK23, TK24, and S. erythraea
The transfer of lcp to and expression of Lcp in the different host strains described above had no effect on the activity of this protein. Alternative methods for the overexpression of Streptomyces proteins in engineered expression hosts of the same or related species of this genus were in the past successfully applied to the overproduction of different enzymes (Kayser and Kilbane 2001;Moreno et al. 2003Moreno et al. , 2005Hidalgo et al. 2004;Torres-Bacete et al. 2007;García-Hidalgo et al. 2011).
Streptomyces lividans TK23, TK24, and S. erythraea were chosen as expression hosts as the expression of Lcp activity in Gram-negative bacteria E. coli and Pseudomonas was not successful. In contrast to S. lividans TK23, the genome of S. lividans TK24 is completely sequenced and the genome has definitely no lcp homologous. Needless to say, we have analyzed TK23 for lcp homologous with PCR without evidence but the expression of Lcp in TK24 cannot be disputed, as there is no lcp homologous in the sequence of the genome.
Therefore, lcp was cloned in the E. coli-Streptomyces shuttle expression vector pIJ6021. The resulting hybrid plasmid pIJ6021::lcp was transferred to other Streptomyces strains by protoplast transformation to study its expression. Bands presenting proteins of the expected size were visible in SDSpolyacrylamide gels after separation of the concentrated supernatants of the recombinant strains of S. lividans TK23 and TK24 as well as S. erythraea (Fig. 2a). Furthermore, Western blot analysis and immunological detection employing the Lcp antibodies raised against the purified Lcp protein of strain K30 confirmed the results and showed that Lcp was indeed synthesized by the recombinant strains (Fig. 2b).
As described previously (Jendrossek and Reinhardt 2003), purified NR latex from H. brasiliensis was used for the preparation of overlay agar plates to analyze the activity of Lcp. For this, MSM agar plates were covered with an overlay of MSM agar containing 0.2% (v/v) dispersed latex concentrate. These latex overlay agar plates were used to demonstrate clear zone formation and also growth of the recombinant strains. After four to seven days cultivation of the recombinant strains of S. lividans TK23, TK24, and S. erythraea harboring the plasmid pIJ6021::lcp on NR latex overlay plates at 30 • C clear zones was observed. Thiostrepton (25 μg/mL) was used for  plasmid maintenance. A recombinant strain harboring only the vector without lcp did not form clear zones. Furthermore, we have conducted an important experiment to demonstrate Lcp activity using the supernatant of these Lcp-expressing strains in vitro (Fig. 3a-c). All three strains obviously secreted a functional Lcp, as indicated by the formation of halo. This is the first time when Lcp activity using the supernatant of Lcp-expressing strains was successful. This is an important result for future works, for example, the difficult purification of Lcp.

Deletion of Lcp from Streptomyces sp. strain K30
An additional experiment was necessary to verify the function of lcp in rubber degradation. The construction of a knock out mutant of lcp in Streptomyces sp. K30 was not successful hitherto; unfortunately, only very low transformation and conjugation frequencies were achieved with this newly isolated strain although intensive efforts were made to increase the transfer rates of foreign DNA. In this study, we succeeded in constructing a knock out mutant. The 1224-bp sequence comprising the entire lcp coding region including a unique restriction site for SmaI was located downstream of the putative start codon. For this reason, this fragment was amplified by PCR using the primers N Lcp and C Lcp; subsequently it was cloned into pGEM-T Easy (Table 1), which does not possess a cleavage site for SmaI. The resulting plasmid, pGEM-T::lcp, isolated from E. coli TOP10 could not be digested with SmaI, indicating methylation at its recognition site, it was transferred to E. coli ET12567 lacking the DNA methylase. The plasmid DNA could then be linearized with SmaI, and an approximately 1000-bp SmaI-SmaI kanamycin resistance cassette ( Km) was inserted at position 281 of lcp. The 2.2-kbp lcp Km DNA fragment was amplified by PCR, and the resulting linear DNA fragment was purified, dialyzed, and transferred to Streptomyces sp. lcp produces clear zones stainable with Schiff's reagent (right side). These strains obviously secreted a functional Lcp, as indicated by the formation of a halo. On the left, the negative control, harboring only pIJ6021 and producing no clear zones, is shown. After incubation for two to three days, agar plates were stained with Schiff's reagent to visualize aldehydes resulting from poly(cis -1,4-isoprene) cleavage.
In total, many individual transformation reactions yielded more than 80 kanamycin-resistant colonies. Colony PCR using the primers N Lcp and C Lcp gave only one transformant that did not exhibit the wild-type 1224-bp PCR product, but the 2.2-kbp lcp Km knock out PCR product instead (Fig. 4). All other clones exhibited both the wild-type lcp fragment and the 2.2-kbp lcp Km fragment, indicating an unspecific integration of the 2.2-kbp lcp Km DNA fragment somewhere else in the chromosome.
If Lcp has an essential function for poly(cis-1,4-isoprene) degradation in Streptomyces sp. K30, its absence should have a deleterious effect on the utilization of this polymer. The effect of lcp inactivation on growth of mutant Streptomyces sp. K30 lcp Km in presence of poly(cis-1,4-isoprene) was abundantly clear. Even after two weeks of incubation, Streptomyces sp. K30 lcp Km no clear zone formation was observed; staining with Schiff 's reagent the reaction was negative (Fig. 4b). Based on this result, the effect of Lcp on the utilization of the polymer was obvious. The capability of the lcp knock out mutant to use poly(cis-1,4-isoprene) as carbon source was compared to that of the wild type in mineralization experiments. Highest mineralization of poly(cis-1,4isoprene) was obtained with the wild-type strain Streptomyces sp. strain K30. After 50 days of mineralization, the wild-type K30 had metabolized about 1.63% of the supplied NR cultures to CO 2 . In contrast, the lcp knock out mutant mineralized only about 0.82% of NR to CO 2 in the same period. The experiment to measure the value of metabolized rubber was repeated three times with the wild-type Streptomyces sp. K30 and the lcp disruption mutant Streptomyces sp. K30 lcp Km. The rubber degradation rate of the wild-type Streptomyces sp. K30 is quite slow (seven days to form clear zones on latex overlay agar plates), strains such as TK23 and TK24 show similar results; hence, we consider the difference to be significant.

Complementation of Streptomyces sp. K30 lcp Km
The genetic complementation of the lcp knock out mutant was analyzed in detail. Plasmid pIJ702::lcp 1, harboring the wild-type gene including the native promoter region of lcp from Streptomyces sp. strain K30, was transformed by protoplast transformation into the corresponding mutant. This plasmid restored the wild-type phenotype in the lcp knock out mutant. The recombinant strain was able to produce a clear zone on latex overlay plate and to produce aldehydes as revealed by staining with Schiff 's reagent. These results confirmed the successful complementation of the lcp knock out mutant with the wild-type lcp gene.
This was the first time that an lcp knock out mutant from Streptomyces sp. strain K30 was successfully generated. All previous efforts in our laboratory had failed. In contrast to the parent strain (Fig. 4c), the lcp mutant was unable to form a clear zone (Fig. 4b). Furthermore, it did not form metabolites staining with Schiff 's reagent. Moreover, mineralization experiments clearly revealed that poly(cis-1,4-isoprene) degradation was almost completely diminished in the lcp knock out mutant of Streptomyces sp. strain K30 when compared to the wild-type Streptomyces sp. strain K30. These indisputable findings confirmed that the initial cleavage of poly(cis-1,4isoprene) is solely dependent on Lcp in Streptomyces sp. K30.  Cell material from a single colony of a putative lcp disruption mutant was suspended in 50-μl TE buffer, and the suspension was then boiled for 15 min. After centrifugation, 0.5 μl was applied as template for a PCR employing the primers N Lcp and C Lcp (Table 1) It is therefore unlikely that other proteins than this are additionally involved in rubber cleavage of the poly(cis-1,4isoprene) chain in this bacterium.
This study encourages further studies of rubber degradation in Gram-positive microorganisms. In the future, the latex-clearing protein, Lcp, must be purified to unravel the reaction mechanism of this enzyme acting on polyisoprene and to employ this protein for biotechnological applications, for example, for the conversion of rubber waste material.