Improvement in Hyperexpression with a Combination of Truncated Scmp Promoter and Streptomyces Lividans

We have previously reported a powerful promoter from the Streptomyces cinnamoneus TH-2 strain named 'scmp' and created an expression vector of pTONA5a for expression using S. lividans. The full-length scmp promoter sequence consists of 424 bp upstream of a metalloendoprotease gene in the S. cinnamoneus TH-2 genome. The promoter works in the presence of inorganic phosphate and glucose. In this study, we present the essential region of the scmp promoter (promoter C), which lacks 358 bp of the 5' region of the full-length promoter. Promoter C was very short and contained only 63 bp. Using promoter C, we succeeded in the extracellular production of the Streptomyces enzymes of leucine aminopeptidase, ferulic acid esterase, and transglutaminase, which possessed signal peptides for secretion via the type II secretion pathway, at high levels.


Introduction
Actinomycetes are gram-positive bacteria that are useful tools for the biological production of several secondary metabolites, including antibiotics. Actinomycetes are also known to produce various enzymes, such as proteases (Morihara et al. 1969;Hatanaka et al. 2005; Uesugi et al. 2011), cellulase, and hemicellulase (Kumar et al. 2020). In recent years, genome analysis technology has been developed, and there are many opportunities to select and express these useful proteins from genomic information.
However, the production of recombinant proteins from actinomycetes using Escherichia coli as a host sometimes results in the failure to form inclusion bodies. One of the reasons for this is GC-rich (ca. 70%) in their genomes. Actinomycetes have a good ability to secrete proteins into the culture medium because they do not have an outer membrane. Thus, Actinomyces are a suitable host for the production of secreted proteins from actinomycetes (Anné et al. 1993; Anné et al. 2014). We have previously reported a metalloendoprotease (SCMP) from Streptomyces cinnamoneus (previously named S. septatus) TH-2 strain (2). SCMP was highly expressed and secreted using a medium that was rich in glucose and inorganic phosphate, and the yield was 0.3 g /L (2). To utilize this hyperproduction, we cloned the upstream region of the scmp gene as a promoter and created an expression vector of pTONA5a, which was based on pIJ702, for expression in S. lividans. The vector harbors ori and rep for streptomycetes and thiostrepton-resistant genes ). By combining this vector with the S. lividans 1326 strain, we succeeded in the extracellular production of several Streptomyces enzymes Uraji et al. 2018;Wan et al. 2019). In this study, we attempted to identify an essential region of the scmp promoter to enhance its expression. We con rmed that the truncated promoter showed higher protein expression than the full-length promoter. Furthermore, we succeeded in protein expression by using the truncated promoter in medium that was not rich in glucose and phosphate and in commercial tryptic soy broth (TSB).

Materials And Methods
Culture conditions and transformation of S. lividans Escherichia coli JM109 was used as the host cell for vector construction. S. cinnamoneus TH-2 was used as the source of the promoter. We used a secreted leucine aminopeptidase (LAP) from S. griseus as a reporter gene and the S. lividans 1326 strain as an expression host. The PG medium contained 2.0% glucose, 0.8% K 2 HPO 4 , 0.05% MgSO 4 ·7H 2 O, 0.5% polypeptone, and 0.5% yeast extract. In the 0.1% Pglu medium, the K 2 HPO 4 concentration was changed to 0.1% in PG medium, and glucose was replaced with glycerol in PG medium in the Pgly medium. Tryptic soy broth was purchased from Japan Becton, Dickinson, and Company (Tokyo, Japan). Protoplast preparation and transformation were performed according to the method described by Hopwood et al. (Hopwood et al. 1985). The constructed plasmids were transformed into protoplasts of the S. lividans 1326 strain, and transformants were selected by thiostrepton resistance. The obtained transformants were inoculated in LB medium containing 1.0% trypton, 0.5% yeast extract, and 1.0% NaCl and cultured until the stationary phase. The cells were then collected and washed with 0.9% NaCl, and the cells were inoculated into the medium.

Construction of expression plasmids containing the truncated scmp promoters
The pTONA5a plasmid with the leucine aminopeptidase gene (pTONA5a-lap) was constructed as described previously . PCR was performed to obtain genes with truncated promoters. To amplify the fragments of A, C, and D, the forward primers with AseI site (ATTAAT) were constructed at the -277 bp, -66 bp, and -46 bp sites of scmp, and the reverse primers were constructed at the end of the inserted genes with HindIII. The fragment of A was ampli ed with the following primers: 5′-ATTAATCGGCAACCAGCCGCCGACGG-3′ (the underlying indicates an AseI site) and 5′-AAGCTTAGGTGGGCGGTTCGCCGGTG-3′ (the underling indicates a HindIII site). The fragment of C was ampli ed with the following primers: 5′-ATTAATCACCACACCTGCACCACAGG-3′ (the underling indicates an AseI site) and 5′-AAGCTTAGGTGGGCGGTTCGCCGGTG-3′ (the underling indicates a HindIII site). The fragment of D was ampli ed with the following primers: 5′-ATTAATGCGGTCGTACAACCGGCTGC-3′ (the underlying indicates an AseI site) and 5′-AAGCTTAGGTGGGCGGTTCGCCGGTG-3′ (the underlying indicates a HindIII site). The fragment of B was ampli ed with the following primers: 5′-ATTAATCGGCAACCAGCCGCCGACGG-3′ (the underlying indicates an AseI site) and 5′-CATATGTGTCTCCTATGAGGGGGGTTGG-3′ (the underlying indicates an NdeI site). The pTONA5a-lap plasmid was used as the template. The ampli ed fragments were cloned in pCR-Blunt II-TOPO (Thermo Fisher Scienti c, Tokyo, Japan), and the inserted sequences were con rmed. Clones A, C, and D were digested with AseI and HindIII, and the fragments were ligated into the AseI-HindIII gaps of pTONA5. Clone B was digested with AseI and NdeI, and the fragment was ligated into the AseI-NdeI gap of pTONA5.

Construction of expression plasmids for ferulic acid esterase (FAE) and transglutaminase (TG)
The FAE gene from S. cinnamoneus NBRC12852 was cloned and ampli ed with the following primers: 5′-CATATGATCATGATCAGCTCGATGAGGAAGACATC-3′ (the underling indicates an NdeI site) and 5′-AAGCTTCACTTCATGAAGGTGACCTCGGGGT-3′ (the underlying indicates a HindIII site). The PCR product was cloned into pCR-Blunt II-TOPO, and cloning was con rmed by sequencing. The resulting FAE gene was cloned into the gap of NdeI-HindIII of pTONA containing promoter C. TG construction from S. mobaraensis NBRC13864 has already been reported (Tokai et al. 2020).
Assay of LAP activity LAP activity was determined using Leu-pNA (Sigma-Aldrich Japan, Tokyo, Japan) as a substrate. The sample (0.1 mL) and 0.1 mL of the substrate (32 mM) were added to 0.8 mL of a 100 mM Tris-HCl (pH 8.0) and 1 mM CaCl 2 solution. The amount of p-nitroaniline released was determined by measuring the absorbance at 405 nm using a spectrophotometer (UV2800, Hitachi Ltd.). The initial velocity was

LAP activity and expression by full-length scmp promoter in PG medium
We have reported that SCMP, which is a metalloendoprotease from S. cinnamoneus TH-2 strain controlled under the scmp promoter, required 2% glucose and 0.8% phosphate to be secreted into the culture as an active form (Hatanaka et al. 2005). Based on our ndings, we examined four media for secreted LAP expression using the transformant harboring the pTONA5a-lap. After 4 days of culture at 30°C, the LAP activity of the supernatant was analyzed, and protein expression was detected by SDS-PAGE. The supernatant of the culture in PG medium showed LAP activity and protein expression (Fig. 1). In contrast, low phosphate medium (0.1% K 2 HPO 4 in PG), Pgly medium (2% glycerol instead of glucose in PG), and TSB showed lower LAP activity and expression than PG medium (Fig. 1).

LAP activity and expression by truncated scmp promoters
To identify the essential region of the scmp promoter, we constructed expression vectors combined with truncated promoters and the lap gene (Fig. 2). pTONA5a-lap, which included the full-length scmp promoter (FL), and expression vectors with four types of truncated scmp promoters (A, B, C, and D) were transformed into the S. lividans 1326 strain, and the obtained transformants were cultivated in PG medium. As shown in Fig. 3A, the A promoter, which lacks 148 bp of the 5′ region of the scmp promoter, showed three-fold higher LAP activity than FL. In addition, promoter C, which lacked 358 bp of the 5′ region of the scmp promoter, showed four-fold higher activity than that of FL. Promoter D, which harbors 46 bp of the 3′ region in the scmp promoter, also showed two-fold higher LAP activity than FL. Promoter B lacked 206 bp of the 3 region of the scmp promoter. The secreted LAP proteins were detected except for promoter B, and the content of LAP proteins increased in order from A and C to D compared with FL (Fig.  3B).

Truncated promoter C required no glucose and phosphate for lap expression
From the results of the truncated promoter in PG medium (Fig. 3), we expected that promoter C would require no glucose and phosphate and would be easy to use. Thus, we compared the secreted LAP activities using FL and truncated promoters A and C under the conditions of low phosphate concentration (0.1% phosphate), the exchanged carbon source of glucose with glycerol (Pgly), and TSB. The LAP activity using the FL promoter was the highest in the PG medium compared to the other three cultivation conditions (Fig. 4A). The LAP activity of promoter A was remarkably increased in the low-phosphate medium. This value was three times higher than that of FL. Furthermore, the LAP activity of promoter C was higher than that of FL and A under all conditions (Fig. 4A). Moreover, the protein expression levels under the four conditions by the C promoter were also increased compared with those under the FL promoter under PG (Fig. 4B). In particular, the LAP protein was strongly secreted by the combination of the C promoter and Pgly medium (Fig. 4B). In addition, the levels of other secreted proteins were reduced by using promoters C and TSB, although the content of secreted LAP protein was slightly lower than that in PG or Pgly (Fig. 4B).

Expression of other actinomyces proteins by promoter C
We attempted to produce other proteins with promoter C. FAE is an enzyme that releases ferulic acid from plant biomass (Uraji et al. 2013). When the Streptomyces FAE gene (Uraji et al. 2018) was cloned downstream of promoter C and expressed in PG and TSB media, enzyme production was con rmed in both (Fig. 5). To utilize the full-length scmp promoter, it was necessary to use PG medium; however, when cultured in PG medium, the expression of proteins other than the target protein was induced, and multiple proteins were often expressed simultaneously. It was necessary to purify the culture supernatant and extract only the target protein. In the case of FAE expressed by promoter C, the expression of contaminating proteins was suppressed in both PG and TSB medium, and the expression of other proteins was particularly reduced in TSB medium (Fig. 5). TG mainly connects proteins (glutamine side chain) to proteins (lysine side chains) by covalent bonds; by utilizing this property, it can be used in the processing of marine products, modi cation of meat, and processing of dairy products (Tokai et al. 2020). Similar to the FAE, TG was expressed by promoter C in PG or other media. The enzyme is released into the culture's supernatant due to its secretory signal; there is a prosequence downstream of the secretory signal, which is cleaved by another enzyme (peptidase) secreted after TG. By removing the prosequence, the enzyme is converted into an active mature form of TG. When TG was expressed by promoter C in PG medium, a mature protein mixed with the prosequence form was expressed (Fig. 5). On the other hand, when TG was expressed in Pgly medium, conversion to the mature form of TG was completed. These results indicate that using promoter C facilitates the secretion of several types of proteins because the promoter does not restrict the carbon source.

Discussion
In this study, we identi ed the essential region (63 bp, promoter C) of the scmp promoter. By using pTONA containing promoter C and S. lividans, we succeeded in the extracellular expression of three Streptomyces enzymes, LAP, FAE, and TG, which possessed signal peptides for secretion via the second pathway.
The full-length scmp promoter consists of 424 bp upstream sequences of the SCMP gene in the S. cinnamoneus TH-2 genome. To date, we have not determined the sequence similarity to the genome information of other organisms or actinomycetes. Because the promoter is powerful, it is used to commercially produce two Streptomyces enzymes. However, there is a restriction on the medium component of enzyme expression using the promoter. This study aimed to remove the restriction on the use of the scmp promoter by identifying the essential region. We presented the essential region of the scmp promoter (promoter C), which lacks 361 bp of the 5′ region of the full-length promoter. Promoter C was very short and consisted only of 63 bp. Moreover, there was no restriction of the medium component by the combination of the promoter and the S. livians 1326 strain. This system will be useful for producing and characterizing streptomycete proteins.  (Kato et al. 2002). First, we estimated that the scmp promoter also controlled the AdpA protein or a similar regulation system of AdpA. We found four AdpA-binding motif-like sequences, -CTGCCCGAAC-, -AGGTGCGTTT-, -ATGCCCGAAC-, and -CGTCCGGTAT-, in approximately 250 bp of the scmp promoter. However, the truncated scmp promoters C and D, which lacked more than 300 bp in the 5′ region of the full-length scmp promoter, were active (Fig. 3). These results suggest that the working mechanism of the scmp promoter is different to that of SGMP by AdpA.
The feature of the scmp promoter is that a large amount of protein is secreted extracellularly. By using PG medium, the content of secreted proteins reached the maximum at least 4 days of culture, this cultivation time resulting in the secretion of many proteins, which might be produced by secondary metabolism (Fig. 3B). Using promoters C and TSB, the content of secreted proteins was more intense in the target protein of LAP than in PG medium (Fig. 4B). It was indicated that promoter C might be regulated differently to be compared with the full-length promoter. Further research is needed to reveal how the promoter functions in streptomycetes.
Because the medium component was not restricted, extracellular production of recombinant proteins using the truncated promoter C was more convenient than the full-length promoter. In Fig. 5, we represented FAE and TG expression using promoter C in PG and other media. By using promoter C and the PG medium, this conversion was insu cient, and zymogen and active TG coexisted in the culture ( Fig. 5(B)). The conversion was complete using promoter C and Pgly media. From these results, it can be inferred that the expression of the host peptidase was active in the Pgly medium, and conversion to the mature form was performed more rapidly than that in the TSB and PG media. This result suggests that it was di cult to select various media with the conventional full-length scmp promoter, but by using the 63base promoter, the medium's components were no longer restricted and the enzyme to be expressed was expanded.
Promoter C is very short and convenient, exerting no restriction on the medium component. A similar short promoter of kasOp* (97 bp) from S. coelicolor has been reported. The promoter was engineered by random mutagenesis between sites -10 and -35, and screened using the Lux reporter. The kasOp * exhibited showed higher activity at the transcription and protein levels than those of known strong promoters of ermEp* and SF14p (Wang et al. 2013). Further work is in progress to compare the activities of promoter C and the kasOp * promoter in Streptomyces protein expression.

Conclusions
In this study, we present the essential region of the scmp promoter (promoter C), which lacks 361 bp of the 5' region of the full-length promoter. Promoter C was very short and only consisted of 63 bp. Moreover, there was no restriction on the medium component by the combination of the promoter and the S. livians 1326 strain. This system will be a useful tool for producing and characterizing streptomycetes proteins.   Schematic representation of pTONA5a plasmid construction, including truncated SCMP promoters and the lap gene; promoter; scmp promoter, terminator; kibilysin terminator, lap; leucin aminopeptidase, FL; full length scmp promoter.  SDS-PAGE analysis of culture supernatants with FL in PG medium and C truncated scmp promoters in different media. The gel used was 10% polyacrylamide and was stained by Coomassie Brilliant Blue. All lanes were loaded with 10 μL of culture supernatant.