Establishment of an efficient expression and regulation system in Streptomyces for economical and high-level production of the natural blue pigment indigoidine

doi:10.21203/rs.3.rs-3113434/v1

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Establishment of an efficient expression and regulation system in Streptomyces for economical and high-level production of the natural blue pigment indigoidine

https://doi.org/10.21203/rs.3.rs-3113434/v1

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Background Indigoidine, as a kind of natural blue pigment, is widely used in textiles, food, and pharmaceutical and mainly synthesized from L-glutamine via a condensation reaction by indigoidine synthetases, most of which originated from Streptomyces species. Although Streptomyces possesses strong secondary metabolism, the switches of which from the primary metabolism are complex and thus a challenge to holistically optimize their productivities. Therefore, most of the researchers choose to over-express indigoidine synthetases in the heterologous host to achieve high-level production of indigoidine.

Results Considering the advantages of low-cost culture medium and simple culture conditions during the large-scale culture of Streptomyces, here, an updated regulation system derived from Streptomyces self-sustaining system, constructed in our previous study, was established for the highly efficient production of indigoidine in Streptomyces lividans TK24. The updated system was constructed via promoter mining and σ^hrdB expression optimization, and this system was applied to precisely and continuously regulate the expression of indigoidine synthetase IndC derived from Streptomyces albus J1704. Finally, the engineered strain was cultured in the cheap industrial medium, and 14.3 g/L and 46.27 g/L indigoidine could be achieved in flask and 4 L fermentor, respectively, reaching the highest level of microbial synthesis of indigoidine.

Conclusions This study will lay a foundation for the industrial application of Streptomyces cell factories to produce indigoidine.

Streptomyces

σhrdB

indigoidine

metabolic regulation

Natural blue pigment is a crucial chemical used in various industries, including textiles and food [1 − 3]. Indigo, which is one of the oldest blue dyes, is extensively used in the industry [4]. Indigo is an indole compound that can be extracted from several plants, like Indigofera tinctoria and Polygonum tinctorium [5, 6]. However, the extraction process of indigo is complicated and requires a strong alkaline solution [7, 8]. This process can seriously pollute the environment and limit the production of natural blue pigment. To address this problem, the chemical synthesis of indigo has been developed and is gradually replacing the extraction technique. The manufacturing cost of indigo via chemical synthesis is much lower [9]. However, with the increasing awareness of ecological safety and human health, more and more people are returning to natural products. Therefore, an efficient biosynthesis technique for natural blue pigment needs to be rapidly developed.

Recently, researchers have discovered another natural blue pigment, indigoidine, produced by microorganisms. Indigoidine is a diphenyl quinone that offers several advantages over indigo, such as deeper color, stronger stability, and antioxidant and antibacterial activities [10 − 12]. Indigoidine synthetase IndC, which is mainly derived from Streptomyces, such as Streptomyces chromofuscus, Streptomyces lavendulae, and Streptomyces hygroscopicus, can efficiently convert two molecules of L-glutamine into one molecule of indigoidine [1, 13 − 17]. However, producing distinct metabolites during a specific physiological phase in Streptomyces is challenging due to the complex metabolic regulation network and distinct metabolic stages, especially an unclear switch phase between a transient primary metabolism phase and a lengthy secondary metabolism phase [18 − 20]. Additionally, the lack of efficient gene expression systems makes high-level expression of IndC and production of indigoidine in Streptomyces a challenge.

In order to improve the efficiency of indigoidine biosynthesis, researchers have developed microbial cell factories such as engineered Escherichia coli, Corynebacterium glutamicum, yeast, and other hosts through heterologous expression of IndC [21 − 25]. However, the use of expensive media and high concentrations of glucose during large-scale fermentation have limited the industrial production of indigoidine, which was a cheap but demanding chemical. Considering the advantages of homologous gene expression in enzyme activity and the economic cost of Streptomyces cell factory for large-scale fermentation, therefore, if we can design an efficient IndC expression and regulation system in Streptomyces, it is significantly important for the industrial application of indigoidine biosynthesis technique.

In our previous work, a Streptomyces self-sustained system (StSS) was designed and constructed for efficient expression of genes and biosynthetic gene clusters (BGCs) in Streptomyces [26]. StSS comprises two modules, a primary metabolism module (PM) included gene expression cassette of target gene or BGC under the control of σ^hrdB-dependent promoter P_{12 − 16} (PkasO* mutant) and a secondary metabolism module (SM) consisted of the expression cassette of σ^hrdB under the control of a secondary metabolism promoter, which maintained continuous activity of P_{12 − 16} in the secondary metabolism phase. As a proof-of-concept, StSS was applied to regulate the expression of IndC in Streptomyces lividans TK24 resulting in achieving 5.2 g/L of indigoidine in flask fermentation. While this result demonstrated the highest production level of indigoidine in Streptomyces thus far, the productivity of this engineered Streptomyces strain was insufficient compared to other reported microbial cell factories [23, 24]. Thus, the optimization of StSS is crucial to improving the productivity of engineered Streptomyces for high-level biosynthesis of indigoidine.

σ^hrdB is the most important housekeeping sigma factor in Streptomyces, and it works for the global regulation of the great majority of genes, especially the genes relating to primary metabolite [27 − 29]. Researchers have reported that the ability of the Streptomyces cell factory could be significantly increased through over-expressing the σ^hrdB in the primary metabolism stage [30]. Similarly, we found that the metabolites productivity of StSS regulated strains in the primary metabolism stage were much lower than those in the secondary metabolism stage, even though the target genes were expressed under the strength constitutive promoters, which mainly worked during the primary metabolism stage. Because of that the strong constitutive promoters worked in the StSS were σ^hrdB-dependent promoters, these strong promoters must compete with σ^hrdB, which was also indispensable for the global regulation of the primary metabolism. Consequently, the expression of σ^hrdB was far from enough in the StSS-regulated Streptomyces cells. Therefore, in this study, the σ^hrdB over-expressed module in the StSS was optimized to increase the expression level of σ^hrdB via promoter engineering strategy in the primary metabolism stage. Finally, the updated StSS was used to regulate the expression of IndC for the high-level production of indigoidine in S. lividans TK24. The results indicated that the optimized regulatory system was a simpler and more powerful regulation system to boost the productivity of indigoidine and other specific metabolites in Streptomyces species.

Screening multifunctional promoters for optimizing the expression of σ ^hrdB

In our previous study, we designed a Streptomyces regulation system StSS based on the Streptomyces constitutive promoter, housekeeping sigma factor σ^hrdB, and Streptomyces secondary metabolic promoter, which were assembled into two modules, primary metabolism module (PM) and secondary metabolism module (SM) [26]. The PM included endogenous σ^hrdB and σ^hrdB-dependent constitutive promoter P_{12 − 16} (PkasO* mutant), which was used to express target genes in the primary metabolism phase. SM consisted of the expression cassette of σ^hrdB under the control of a secondary metabolism promoter (P_RVR2353), which maintained continuous activity of the σ^hrdB-dependent promoter in the secondary metabolism phase to continuously accumulate target metabolites, such as indigoidine (5.2 g/L in shake flask). However, we found that the productivity of target metabolites during the primary metabolism phase was much lower than that during the secondary metabolism phase, which would seriously restrict the high-level production target metabolites in Streptomyces.

Undeniably, σ^hrdB is the most important housekeeping sigma factor and works for regulating the majority of genes working for the primary metabolism in Streptomyces. Researchers have verified that the intensity of Streptomyces constitutive promoters could be strengthened via over-expressing σ^hrdB in the primary metabolism stage [31, 32]. These results indicated that the expression of σ^hrdB was far from being satisfied for the regulation of both endogenous and heterologous elements during the primary metabolism stage. Considering that σ^hrdB should also work for regulating the heterologous element P_{12 − 16}, a strength constitutive promoter, in StSS, the inadequate expression of σ^hrdB would be the reason that caused low efficient biosynthesis of target metabolites during the primary metabolism stage. Therefore, in this study, we would optimize the expression module of σ^hrdB in StSS to increase the productivity of indigoidine during the primary metabolism stage via the promoter engineering strategy (Fig. 1). Whereas the over-expression of σ^hrdB would be occurred during both primary and secondary metabolism stage, the optimized Streptomyces regulatory system was named StreptomycesGlobal Enhanced System (StGES).

In order to improve the production of terpene S. reveromyceticus SN-593, Khalid et al. mined different kinds of promoters via transcriptome analysis[33]. Considering the high compatibility of Streptomyces promoters among different species, we have verified that P_RVR2353 was a classical secondary promoter in other Streptomyces species, which was used for constructing the σ^hrdB expression module of StSS. In this study, three promoters P_RVR2030, P_RVR6682, and P_RVR9017, which probably possess activity in the primary and secondary metabolism stage, were selected to optimize the σ^hrdB expression module of StSS. To analyze the character and performance of these promoters, the reporter GusA (a common reporter that catalyzes 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside to a blue product) [34, 35] and indigoidine biosynthetic pathway were used in S. lividans TK24, respectively. As shown in Fig. 2A, 2B, and Additional file 1: Fig. S1A, compared with P_RVR6682 and P_RVR2353, P_RVR2030 and P_RVR9017 possessed efficient activity for the expression of GusA at the beginning of the primary metabolism stage, which could be used to optimize the σ^hrdB expression module and efficiently over-express σ^hrdB during the primary metabolism stage. In addition, to exhibit the relationship between the properties of these promoters and characteristics of the Streptomyces growth and metabolism, the biosynthesis of indigoidine was used. As shown in Fig. 2C, 2D, and Additional file 1: Fig. S1B, except for P_RVR9017, the other three selected promoters P_RVR6682, P_RVR2353 and P_RVR2030 had activity for the expression of IndC during both primary and secondary metabolism stages in S. lividans TK24. However, it is worth noting that the strength of P_RVR2030 was strongest during the primary metabolism stage, which would work well for the optimization of StSS and adequate expression of σ^hrdB during the primary metabolism stage. Therefore, P_RVR2030 was used to reconstruct StGES to regulate the S. lividans TK24 for the high-level production of indigoidine.

Construction of St GES for the indigoidine production

In our previous work, the Streptomyces regulatory system StSS was designed and constructed to regulate the biosynthesis of indigoidine in S. lividans TK24, and the production of indigoidine could reach 5.2 g/L in shake flask, which was the highest production of indigoidine in Streptomyces sp. but far from that in other microbial cells [26]. Therefore, here, we should reconstruct a more efficient Streptomyces expression and regulation system. Considering that the productivity of indigoidine during the primary metabolism phase was much higher than that during the secondary metabolism phase, and we affirmed that it was due to the inadequate expression of σ^hrdB for regulation of both endogenous and heterologous elements during the primary metabolism stage. Therefore, P_RVR2030, which had the activity during the whole metabolism stage and more efficient strength during the primary metabolism stage, was selected to optimize the expression of σ^hrdB and reconstruct the StGES for regulating the production of indigoidine. As shown in Fig. 3A and 3B, compared with the signal module regulated strain PM_P12−16-IndC and StSS regulated strain StSS-IndC, the indigoidine production of engineered strain StGES-IndC was significantly increased during the primary metabolism stage, which illustrated that the reconstructed regulation system StGES could efficiently enhance the biosynthesis of indigoidine via increase the expression of σ^hrdB. In addition, the indigoidine could be continuously produced under the regulatory of StGES during the secondary metabolism stage and the indigoidine yield of engineering strain StGES-IndC was reached 12.39 g/L at 96 h in shake flask, which was 10.50 and 2.88 times to that of engineered strains PM_P12−16-IndC and StSS-IndC, respectively (Fig. 3B). Besides that, we found the growth properties, such as biomass accumulation and sporification, of StGES-IndC had been enhanced (Fig. 3C and 3D). These results indicated that the reconstructed regulation system StGES had no effect on the bound between primary and secondary metabolism but was being beneficial to the metabolic ability of Streptomyces. More importantly, the sporification of StGES-IndC was occurred at 72 h, which was 24 h and 48 h earlier than that of StSS-IndC and PM_P12−16-IndC, respectively. Just because of the optimized expression of σ^hrdB, the metabolic performances of StGES-IndC during the primary metabolism stage were significantly improved, so that the indigoidine production of StGES regulatory strain was efficiently increased to the highest level via microbial cell factories.

Considering that the precursor of indigoidine was derived from the glycolysis pathway and TCA cycle, and the glucose in the medium was exhausted at just about 24 h after the fermentation, therefore, glucose and glycerol were supplied into the medium at 24 h, respectively. As shown in Fig. 4A and 4B, the glycerol used as a feed supplement was better than glucose, and the indigoidine yield of engineered strain StGES-IndC was reached 14.95 g/L at 96 h after adding 1% (w/v) glycerol in medium, further improving the ability of the engineered strain StGES-IndC to produce indigoidine and establishing a foundation for the industrial production of indigoidine via Streptomyces cell factory. Interestingly, the biomass of engineered strain StGES-IndC was significantly increased after supplementing glycerol into the medium. Considering that no metabolic pathway of StGES-IndC was further reconstructed, therefore, the improvement of biomass was probably the reason for the increase in indigoidine yield.

Transcriptomic analysis of genes related to indigoidine biosynthesis

In our previous work, we found that the cell metabolism regulated under the StSS had minimal changes to that under PM in global transcription profiles. The reason was probably that the expression of σ^hrdB was almost not increased during the primary metabolism stage, in which most of σ^hrdB regulated genes worked. However, under the regulation of StGES, the biomass of engineering strain was increased by 25% and the sporification of engineered strain was advanced from 120 h to 72 h. Since the metabolic pathway of strain StGES-IndC for the indigoidine biosynthesis was not remodeled, undoubtedly, the transcript level of the genome in engineered strain StGES-IndC was significantly changed. To confirm this hypothesis, transcriptomic profiles in engineered strain StGES-IndC were evaluated in comparison with PM_P12−16-IndC at the end of the exponential growth phase with the same growth status (72 h). In stark contrast, the transcriptional patterns of engineered strain StGES-IndC exhibited considerable differences from that of strain PM_P12−16-IndC, with 351 up-regulated genes and 1016 down-regulated genes (|log2FoldChange|> 1; p < 0.05), among about 7232 total genes (Fig. 5A). These data clearly demonstrated that the global metabolism of the host underwent tremendous changes under StGES regulation. Further, these observations highlight the significant value of StGES for the deep reconstruction of cellular metabolism in the high-level production of indigoidine.

Except that, the KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analysis was used to identify the metabolic differences between the engineered strain StGES-IndC and the control strain PM_P12−16-IndC, and the results exhibited that most of the genes working for the transcription, translation, and cell growth and death were up-regulated, which would efficiently accelerate the performances of cells. However, most of the genes working for the secondary metabolism were down-regulated, suggesting that the improvement of σ^hrdB expression during the primary metabolism stage would hinder the secondary metabolism in Streptomyces (Fig. 5B). It is worth noting that significant changes also took place in the glycolysis and TCA cycle pathways. Interestingly, some key genes of the glycolysis pathway and TCA cycle were up-regulated, indicating that the precursors, acetyl-CoA and α-ketoglutaric acid, could be available for indigoidine biosynthesis in StGES regulatory strain. In addition, most of the genes for the L-glutamine metabolism were significantly up-regulated, while key genes for pyruvate, valine, leucine, tryptophan, and phenylalanine metabolisms were significantly down-regulated (Additional file 1: Fig. S2). Thus, these results pointed to a specific pathway to increase the biosynthesis of indigoidine.

Obviously, the differentiation of Streptomyces cells could be regulated by StGES. To explore the reasons for the acceleration of sporulation of StGES regulatory strain, the relative genes should be analyzed. The differentiation of Streptomyces cells could be divided into four stages (Fig. 5C): substrate mycelium formation, aerial hypha formation, sporotrichial formation, and spore maturation. WhiAB and WhiGHI are the most important regulatory pathways for the sporulation in Streptomyces, and the genes regulation between these two pathways is mutual [36 − 40]. During the aerial hypha stage, whiA binds to σ^whiG (encoded by whiG) and inhibits the transcription of σ^whiG-dependent genes, which hinders the beginning of sporulation. WhiB, encoded by whiB, is also an important regulator, which could combine with WhiA to form a complex WhiAB to activate the expression of key genes required for sporulation, such as bldB, fliP, ftsZ, and ftsK [41 − 44]. In addition, WhiD and SigmaF, indirectly regulated by σ^whiG, are two important members of WhiGHI for controlling the probability of spore deformity. Therefore, the expression level of WhiD and SigF would not affect the sporulation. As shown in Fig. 5D, seven genes (bldB, whiB, whiA, whiE, flip, ftsZ, ftsK) were significantly up-regulated and three genes (whiG, whiD, sigF) were significantly down-regulated. The results clearly demonstrated that the process of sporulation was efficiently enhanced under StGES regulation, and the key points of WhiAB and WhiGHI pathways could contribute to further investigation of sporulation by reverse engineering in Streptomyces.

Application performance of the St GES regulatory strain

To verify the practical application value of the indigoidine strain StGES-IndC, a 4 L fermentor was used through batch processing. As shown in Fig. 6, the results illustrated that the production titer of indigoidine was rapidly accumulated after 12 h and the productivity of indigoidine during the primary metabolism stage was more efficient than that during the secondary metabolism stage, which would be significantly beneficial for decreasing the energy consumption. At last, after 120 h, the indigoidine titer reached 46.27 g/L via feed batch culture technology, one of the highest titers reported to date (Table 1) [1, 21 − 26, 45]. Considering that the cheap industrial medium and glycerol were used for feed batch culture, therefore, the excellent performance of strain StGES-IndC exhibited the application prospect for indigoidine production.

Table 1

Indigoidine production by microbial cell factories.
Stains	Production (g/L)	Fermentation scale	Supplement	References
E. coli	8.81 g/L	flask	L-glutamine	[1]
S. coelicolor	0.59g/L	flask	No	[21]
E. coli	2.78 g/L	flask	No	[21]
A. oryzae	1.41 g/L	flask	No	[22]
S. cerevisiae	0.98 g/L	flask	No	[25]
S. lividans	5.2 g/L	flask	No	[26]
C. glutamicum	49.30 g/L	bioreactor	glucose	[23]
R. toruloides	86.3 g/L	bioreactor	glucose	[24]
R. toruloides	2.9 g/L	bioreactor	sorghum lignocellulosic hydrolysate	[24]
P. putida	25.6 g/L	bioreactor	glucose	[45]
S. lividans	46.27 g/L	bioreactor	glycerol	This study

Indigo and indigoidine are two kinds of widely used natural blue pigments. Compared to the former, indigoidine is much stabler, and it has antioxidant and antibacterial activities. Therefore, indigoidine possesses huge potential for industrial application. Indigoidine synthetases, such as IndC, derived from Streptomyces could efficiently catalyze L-glutamine to synthesize indigoidine. However, the switches of Streptomyces metabolism are complex and thus a challenge to holistically optimize their productivities, which makes it difficult to produce bulk chemicals with low added value in Streptomyces. Therefore, most of the researchers choose to over-express indigoidine synthetases in the heterologous host, such as E. coli, C. glutamicum et al., to achieve high-level production of indigoidine.

Considering the advantages of a low-cost culture medium and simple culture conditions during the large-scale culture of Streptomyces, in our previous study, we constructed a Streptomyces regulatory system StSS, which could be used to continuously produce metabolites spanning the primary and secondary metabolism stages in Streptomyces. However, due to the inadequate expression of σ^hrdB in the Streptomyces primary metabolism stage, the productivity of metabolites during the primary metabolism stage was much more inefficient than that during the secondary metabolism. Therefore, in this study, the expression module of σ^hrdB in StSS was optimized via promoter engineering, and a novel regulatory system StGER was constructed to over-express the indigoidine synthetase IndC from S. albus J1074 to efficiently produce indigoidine in S. lividans TK24. Finally, the engineering strain StGER-IndC, even without any metabolic reconstruction, could produce 46.27 g/L in a 4 L fermentor, which fully illustrated the applicant potential of indigoidine production of StGER-IndC.

However, there are still shortcomings in this work, such as a lack of work on metabolic reconstruction. Therefore, in the later work, we will further improve the metabolic flow of L-glutamine, the precursor of indigoidine, via metabolic engineering and CRISPRi strategies to further improve the yield of indigoidine. In addition, the industrial medium for Streptomyces culture should also be optimized to be more efficiently applied for indigoidine production.

Materials

Antibiotic apramycin (Apr) and standards were purchased from Aladdin (Shanghai, China). Restriction enzymes, DNA polymerase, ligase, and other DNA modifying enzymes were obtained from TaKaRa (Dalian, China). A One-step Cloning Kit was acquired from Yesen (Shanghai, China). Other chemicals for this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Bacterial strains, plasmids, and culture conditions

The bacterial strains and plasmids used are shown in Table 2. All of the primers used are listed in Additional file 2: Table S1. The E. coli DH5α was used as the host for cloning. The E. coli was grown aerobically on a rotary shaker (200 rpm) at 37°C in Luria–Bertani (LB) broth or on a plate containing LB supplemented with 1.5% (w/v) agar. All of the plasmids were transformed into S. lividans TK24 by protoplast transformation, and the transformed cells were grown at 28°C on a R2YE plate for 3–5 d. The engineered Streptomyces cells were grown at 28°C on a rotary shaker at 200 rpm in TSB media.

Table 2

Strains and plasmids used in this study.
	Properties	Source
Stains
E. coli DH5α	F⁻, φ80, lacZΣM15, Σ(lacZYA-argF)U169, endA1, recA1, hsdR17(r_K⁻, m_K⁺) supE44, λ⁻, thi-1, gyrA96, relA1, phoA	Tiangen
S. lividans TK24	wild type	Our laboratory
Plasmids
pFQ-GusA	pCRISPomyces-2 derivative with promoterless gusA gene	Our laboratory
pFQ-Promoter-GusA	pFQ-GusA with a promoter in front of gusA gene, promoter contains (P_RVR2030, P_RVR2353, P_RVR6682, P_RVR9107)	This study
pFQ-Promoter-IndC	pFQ-IndC with a promoter in front of indC gene, promoter contains (P_RVR2030, P_RVR2353, P_RVR6682, P_RVR9107)	This study
PM_P12−16-IndC	pFQ-P_{12 − 16}-IndC derivative with promoterless hrdB gene	Our laboratory
StSS-IndC	pFQ-P_{12 − 16}-IndC derivative with hrdB gene under the regulation of P_RVR2353	This study
StGES-IndC	pFQ-P_{12 − 16}-IndC derivative with hrdB gene under the regulation of P_RVR2030	This study

Elements for the vector construction

Because the selected Streptomyces promoters (P_RVR2030, P_RVR2353, P_RVR668, and P_RVR9107) used in this study were over 300 bp long (sequences were listed in Additional file 2: Table S2), we synthesized these templates at Personalbio (Shanghai, China) and amplified them with corresponding forward and reverse primers to yield PCR products, which were then inserted at the forward end of genes with overlap PCR method, respectively. GusA was used as reporter in this study, and the template of the reporter gene gusA [26] was stored in our lab. The hrdB and indC genes (sequences were listed in Additional file 2: Table S3) were amplified from S. lividans TK24 and S. albus J1704 genomes, respectively. pFQ [29] were used as vectors to transfer the complete gene circuits in E. coli and Streptomyces, respectively. The Gibson assembly method was used to cycle the PCR product and linearized plasmid skeleton.

Vector construction for the expression of GusA and IndC in Streptomyces

The pFQ-F and pFQ-R primers were used to amplify the pFQ template to yield the plasmid skeleton. The plasmids for the identification of selected promoters were constructed in the same manner. For example, to construct pFQ-P_RVR2030-GusA, gusA gene was amplified with primers GusA-F and GusA-R and the promoter P_RVR2030 was amplified with primers P_RVR2030-F and P_RVR2030-R. Then, then products were mixed together and amplified with primers P_RVR2030-F and GusA-R to achieve product P_RVR2030-GusA. Finally, the P_RVR2030-GusA was cyclized with plasmid skeleton with Gibson assembly method. In the same manner, P_RVR2353, P_RVR668, and P_RVR9107 were into plasmid skeletons for the expression of the GusA and IndC in Streptomyces (plasmid maps were listed in Additional file 1: Fig. S3).

Vector construction for regulating the expression of IndC biosynthesis enzymes in Streptomyces

The hrdB gene was amplified with the primers HrdB-F and HrdB-R. The selected promoter P_RVR2030 was amplified with P_RVR2030-HrdB-F, P_RVR2030-HrdB-R, and PCR products were fused with the hrdB gene via overlap PCR. Then, fused PCR products P_RVR2030-hrdB were inserted into pFQ-P_{12 − 16}-IndC (amplified with pFQ-2-F and pFQ-2-R) to yield plasmid StGES-IndC (plasmid map was listed in Additional file 1: Fig. S4).

Analysis of GusA strength and indigoidine production

S. lividans TK24 containing plasmids for the expression of GusA were grown in fresh TSB media at 28°C. The activity of GusA was analyzed via a standardized protocol.³⁴ The indigoidine in vivo and vitro of Streptomyces was extracted with DMSO, and the production of indigoidine was analyzed via a standardized protocol.⁴⁵

Transcriptional analysis

The engineered strains PM_P12−16-IndC and StGES-IndC were grown in TSB medium to the end of the exponential growth phase (72 h) for transcriptomic analyses. A 30 mL aliquot of each cell type was then centrifuged at 8,000 × g for 10 min at room temperature. After removal of the supernatant, the cell pellets were frozen in liquid nitrogen, and total RNA was extracted using the QIAGEN RNeasy Mini kit. RNA sequencing (results were listed in Additional file 2: Table S4) via next-generation sequencing was performed with Illumina HiSeq at Prsonalbio (Shanghai, China). The KOBAS software package was used to investigate the statistical significance of the enrichment of differentially expressed genes among KEGG pathways (results were listed in Additional file 2: Table S5). Triplicate samples were used for transcriptional analysis.

Cultivation in 4 L of fermentation broth

S. lividans TK24 engineered strain StGES-IndC was inoculated into TSB medium and grown in a shaking flask for 36 h at 28°C. The cheap industrial peptone was used to provide nitrogen source. Glycerol was used as a supplemental carbon source by administering feed boluses. Finally, 300 mL seed culture was inoculated into 4 L of fresh TSB medium in a 4 L lab-scale fermentor. The temperature and agitation speed were maintained at 28°C and 280 rpm throughout the growth. The aeration rate was maintained at 2 L/min. The fermentation broth was withdrawn every 12 h. Cell growth was monitored by measuring DCW. The production of indigoidine was measured via a standardized protocol.

Acknowledgements

Not applicable.

Author contributions

MZ and FQW designed the research. MZ and LBX performed plasmids construction experiments, strains analysis experiments. XSZ and KL performed growth curves, metabolites analysis, and cloning. XFL performed high-density fermentation. YL assisted with cloning and preliminary experiments. MZ and YL analyzed the results. LBX supervised the research. MZ and FQW wrote the manuscript.

Funding

This work was financially supported by the Science Foundation for the Excellent Youth Scholars of Anhui Provincial Education Department (2022AH030097) and the Science and Technology Plan Project of Wuhu (2022jc19).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its additional information files.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors agree to submit the work to the journal.

Competing interests

The authors declare no competing financial interests.

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No competing interests reported.

Additionalfile1.docx
Supplementary Information Additional file 1: Fig. S1. Growth curve of the engineered strains for the expression of GusA and IndC. Fig. S2. Amino acid metabolism analysis of transcripts based on KEGG pathways. Fig. S3. Plasmid mapsfor constructing plasmids pFQ-Promoter-GusA and pFQ-Promoter-IndC. Fig. S4. Plasmid maps for constructing plasmids StSS-IndC and StGES-IndC.
Additionalfile2.xls
Additional file 2: Table S1. Sequences of primers used in this study. Table S2. Sequences of promoters used in this study. Table S3. Sequences of genes used in this study. Table S4. Transcriptomic data for genes expression. Table S5. Transcriptomic data for KEGG analysis.

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Establishment of an efficient expression and regulation system in Streptomyces for economical and high-level production of the natural blue pigment indigoidine

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Abstract

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Background

Results and discussion

Transcriptomic analysis of genes related to indigoidine biosynthesis

Conclusions

Materials and methods

Materials

Bacterial strains, plasmids, and culture conditions

Elements for the vector construction

Analysis of GusA strength and indigoidine production

Transcriptional analysis

Cultivation in 4 L of fermentation broth

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References

Additional Declarations

Supplementary Files

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