Efficient ilamycins production utilizing Enteromorpha prolifera by metabolically engineered Streptomyces atratus

doi:10.21203/rs.3.rs-3103958/v1

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Research Article

Efficient ilamycins production utilizing Enteromorpha prolifera by metabolically engineered Streptomyces atratus

https://doi.org/10.21203/rs.3.rs-3103958/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 05 Oct, 2023

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Background

With the invasion of green tides and the increase of urban green areas worldwide, multimillion tons of Enteromorpha need to be reutilized. Ilamycins, novel cyclic heptapeptides with significant anti-TB activities, are isolated from Streptomyces atratus SCSIO ZH16, a deep-sea-derived strain. It is crucial to develop an efficient production process that can utilize inexpensive nitrogen sources to achieve massive production of ilamycins.

Results

In this study, Enteromorpha prolifera powder is considered a promising biomass resource for the production of commercial chemical products production. By using E. prolifera powder as a nitrogen source, the production of ilamycins reached 709.97 mg/L through optimization of the nitrogen source using the engineered strain S. atratus SCSIO ZH16 ΔR. After mutant strain constructions and tests, S. atratus SCSIO ZH16 ΔR::bldD was found to be a more suitable strain for utilizing E. prolifera powder. Furthermore, the production titer of ilamycins and ilamycin E reached 1561.77 mg/L and 745.44 mg/L, respectively, in a 5-L bioreactor.

Conclusions

This study suggests that E. prolifera is a promising and eco-friendly nitrogen source for the production of ilamycins. Engineered strain S. atratus SCSIO ZH16 ΔR::bldD was found to be a more suitable strain for utilizing E. prolifera powder.

Ilamycins

Anti-tuberculosis

Streptomyces atratus SCSIO ZH16

Entromorpha prolifera

BldD

In the past ten years, severe eutrophication of seawater has resulted in the occurrence of Enteromorpha prolifera (EP) green tides, which are harmful algal blooms causing significant damage to the economy and environment of coastal cities in Asian [1]. Due to its high organic matter content and abundant production, however, EP has the potential to be used as raw materials for digestion [2]. Carbohydrates in EP exist in the polymer forms of hexose and pentose, which can be subsequently converted into bioethanol or biohydrogen [3]. EP proteins serve as a source of N-containing valuable products and nutrients for humans or animals. They can also be hydrolyzed into amino acids and subsequently converted into commercial chemical products [4]. Therefore, the efficient reutilization and disposal of EP as a source of energy and materials can help mitigating greenhouse gas emissions. However, only a few studies have reported on the application of EP [4]. Actinomycetes species are renowned for the capacity to produce a multitude of high-valued product, even using waste biomass [5–6]. Previous studies have demonstrated that S. atratus SCSIO ZH16, isolated from deep-sea sediment in the South China Sea, exhibits advantages including rapid growth, ease of cultivation, and stable metabolism, for the production of high-value products [7–8]. Among the natural products from S. atratus SCSIO ZH16, nonribosomal peptides ilamycins, especially ilamycin E, show brilliant anti-tuberculosis properties [9], and require yield improvement for the realization of industrial. By Plackett–Burman design model, single factor optimization, pH coordinated feeding and continuous pulse feeding, achieved 415.7 ± 29.2 mg/L ilamycin E yield in a 300-L bioreactor [10]. Our previous research also improved the production of ilamycin E from 13.51 to 762.50 mg/L in a 5-L bioreactor, and found the crucial role of nitrogen source on cellular growth of ZH16-ΔilaR and the production of ilamycins [11]. Soya soybean powder, the preferable nitrogen source identified, has obvious shortcomings such as difficult to sterilize and high cost. Therefore, it is crucial to develop an efficient production process that can utilize inexpensive nitrogen sources to achieve massive production of ilamycin E.

Beside optimization of fermentation conditions, genetic modification is another powerful tool for the improvement of antibiotic-producing strains [12]. Commonly used targets in Streptomyces genetic modification include metabolism pathways that supply or shunt the precursors, resistance proteins that protect the host, modifying factors that determine the activity of enzymes, and regulators that promote or repress secondary metabolism, etc [13]. Among them, genetic operation towards regulatory genes is a promising approach. For an example, global regulator BldD dominates the process of morphology and secondary metabolism in multiple Actinomyces, and overexpression of bldD has boosted the production of daptomycin, avermectin and moenomycin [14–16]. Therefore, this study combined both fermentation condition optimization and strain genetic modification to enhance the yield of ilamycins.

By utilizing EP powder as a cost-effective, environmental nitrogen source, the engineered strain S. atratus SCSIO ZH16 ΔilaR::bldD with extreme environment tolerance achieved a production of 1561.77 mg/L of ilamycins and 745.44 mg/L of ilamycin E in a 5-L bioreactor.

Nitrogen sources Screening for the production of ilamycins

Nutrients so called nitrogen sources are required for forming nitrogenous cell components or metabolites, selection of which is essential for fermentation optimization. Due to the specific impact of any kind of organic nitrogen source on the growth of bacteria and synthesis of natural products, one bacterium or its genetic mutant has unique preference for organic nitrogen sources. In view of this, this study started with the screening of nitrogen sources on S. atratus SCSIO ZH16 ΔilaR (ΔR for short), an engineering strain that accumulates highly anti-tuberculosis activity component ilamycin F and ilamycin E, for the production of ilamycins.

Given the shortcomings of soybean powder, currently used nitrogen source for ilamycins fermentation, such as difficult to sterilize, foaming properties, complicated production process and high cost, we attempted to seek a superior alternative. 14 alternative nitrogen sources including EP powder, corn steep liquor, yeast extract powder, polypeptone, peptone, yeast extact paste, malt extract, beef extract, bacterial peptone, fish peptone, tryptone, acid-hydrolyzed casein, bean flour and soybean powder were selected for screening. The results showed that soybean powder, bean flour and EP powder were the top three high-yielding nitrogen sources (Fig. 1A). Compared with the other two materials, EP powder has significant advantages in terms of environmental friendliness and cost.

To validate whether EP powder mainly function as the nitrogen source in fermentation, eight media varying in carbon and nitrogen sources (bottom of Fig. 1B, named M1-M8) were designed and tested. As shown in Fig. 1B, compared to M1 containing soybean powder, ilamycins yield of M2 containing EP powder decreased slightly (from 1044.97 to 709.97 mg/L), while M4 containing neither soybean powder or EP powder produced only 117.73 mg/L ilamycins. Besides, the ilamycins yield of M2 is 42.7-fold higher than that of original medium Am3 (Fig. S1). These results demonstrate that selection of nitrogen source is crucial for the production of ilamycins, and EP powder can be used as a good nitrogen source substitute for cellular growth (Fig. S2), primary and secondary metabolism flux regulation, and ilamycins biosynthesis of ΔR. The analysis of element components on EP powder shows that the weight ratio of N, C and H elements are 3.69%, 29.58% and 5.46%, respectively, which converted to the molality ratio is 1:9:21 (Figs. 1C and 1D).

Construction of ilamycins high-yield strains

After the substitution of nitrogen source with EP powder, which potentially affects the flux of metabolism, we set about constructing mutant strains that efficiently utilize EP powder to produce more ilamycins. A screening on genes belonging to four function categories were performed:

1) Self-resistance. Ilamycins resistance genes ilaJ and ilaK inside of the biosynthesis gene cluster and putative resistance gene bla (RS37870) were overexpressed by separately integrating them into the ΦC31 attB site. None of these overexpression strains led to enhancement in ilamycins production (Fig. 2). To be noticed, it has been found that overexpression of ilaJ, ilaK and bla promoted the production of ilamycin D and ilamycins C₁/C₂ in wild type (WT) strain (Fig. S4), but these compounds have low anti-tuberculosis activity. Therefore, the cause of failure in ilamycins production improvement through ilaJ, ilaK and bla overexpression might be the specific transport towards ilamycin D and ilamycins C₁/C₂.

2) Biosynthesis pathway. Two key enzymes from shikimate pathway, chorismate synthase (CS) and shikimate dehydrogenase (SKD), were selected for overexpression, since they participate in the supply of ilamycins biosynthesis precursor tryptophan and tyrosine. Beside precursor supply consolidation, atra (encoding nonribosomal peptide synthetase) from the biosynthesis gene cluster of atratumycin was deleted by CRISPR-Cas9 to weaken the competitive pathway. Atratumycin is a decapeptide composed of amino acids including tryptophan and tyrosine [17], its biosynthesis therefore theoretically inhibits the biosynthesis of ilamycins through competition of precursors. However, no enhancement of ilamycins yield occurred in strains ΔR::CS, ΔR::SKD and ΔRΔatra (Fig. 2). It can be speculated that the supply of tryptophan and tyrosine in strain ΔR is enough for ilamycins biosynthesis, and the shunt pathway for atratumycin biosynthesis is relative weak.

3) Regulator. Manipulation towards regulator is a well-validated approach for yield enhancement of natural products [18–19], and also adopted in this study. Well-known regulatory genes related to morphology development and secondary metabolism, such as bldD, arpA and rok7B7 etc., were overexpressed in strain ΔR, to explore the possibility of ilamycins yield increasement. Among the nine overexpression strains, ΔR::bldD showed an obvious enhancement in ilamycins yield by 58.13% to 900.04 mg/L (Fig. 2). It is noteworthy that, the effect evaluations of these regulatory genes were all carried out through gene overexpression, because of the extremely low efficiency of CRISPR/Cas9 method in S. atratus. Further assessment on regulatory genes can be performed after optimization of the CRISPR/Cas9 method.

4) Protein modification. As nonribosomal peptides, efficiently biosynthesis of ilamycins requires a protein modification executed by phosphopantetheinyl transferase (PPtase) to activate carrier proteins [20]. Therefore, overexpression of PPtase has successfully improved the production of multiple antibiotics [21]. In this study, two commonly used heterologous PPtase genes from Bacillus subtilis (sfp) and Streptomyces verticillus (svp), and three native PPtase genes (RS01025, RS31655 and RS38670), were expressed in strain ΔR. None of the mutant strains based on PPtase genes led to enhancement of ilamycins yield (Fig. 2), indicating that the modification level of carrier proteins in strain ΔR is not a limiting factor for ilamycins biosynthesis.

In total, as the only high-yield strain screened, strain ΔR::bldD is used for the subsequent mechanism analysis and fermentation optimization.

Mechanism analysis of BldD regulating ilamycins biosynthesis

To understand the regulatory mechanism of BldD on the biosynthesis of ilamycins, the transcription level of biosynthetic genes of ilamycins were firstly examined. Genes inside ilamycins biosynthesis cluster form six operons: ilaAB, ilaC, ilaDEFGHIJKLMNOP, ilaQ, ilaRS and ilaT (Fig. 3A), and the leading gene of each operon were selected for qRT-PCR detection. Since regulatory genes ilaA and ilaB have opposite functions, their transcriptional levels were both tested. RNA from strains ΔR and ΔR::bldD cultured after 48 and 72 h was extracted and detected by qRT-PCR. As shown in Fig. 3B, the transcription level of tested genes was decreased at 48 h, whereas up-regulated at 72 h. According to growth curve and ilamycins yield curve analyzed previously [11] (Zhu et al., 2022), the transformation of strain ΔR from primary to secondary metabolism occurs between 24 and 72 h. Therefore, we speculated that overexpression of bldD delayed the expression initiation of ilamycins biosynthetic genes at early stage, while strengthened the expression level of them at mid-late stage. In addition, both the commonly used internal reference gene hrdB and the novel internal reference gene RS08190 identified by in our lab (encoding protein disulfide isomerase, unpublished data) were used in the qRT-PCR analyses. The almost identical results shown in Fig. 3B indicated that RS08190 is transcript-stable and suitable for qRT-PCR analysis towards S. atratus.

Given the well-known role of BldD in morphology development, morphological observations on ΔR::bldD were performed. Compared to strain ΔR, formation of aerial mycelium and spores in strain ΔR::bldD on YMS medium is more prosperous, which was further proven by scanning electron microscopy (Fig. S5).

Optimization of fermentation conditions

The fermentation process parameters are important factors affecting secondary metabolites. Thus, systematical optimization towards the key process parameters (pH, temperature, inoculation proportion, inoculation time, addition amount of EP powder, rotational speed, liquid volume and Zn²⁺ concentration) were performed on strain ΔR::bldD, to further improve the production efficiency of ilamycins (Figs. 4 and S6).

After optimization, it was found that pH had no significant effect on the production of ilamycins. When pH = 3, the production of ilamycin E and total ilamycins were slightly higher. According to the investigation on the inoculum of seed liquid, 10% was the best inoculum proportion. As for key nitrogen source EP powder, 30g/L was the best addition amount. To be noticed, excessive addition of EP powder will change the viscosity of the fermentation medium, thereby affecting the cellular growth and reducing the yield of ilamycins. Other process parameters (temperature, liquid volume, rotational speed, etc.) optimized were consistent with our previous work [11] (Zhu et al., 2022). Another research in our lab found that addition of Zn²⁺ can further improve the yield of ilamycins, and we found in this work that 1 mmol/L is the best Zn²⁺ concentration for EP powder-containing medium.

To sum up, the optimal fermentation conditions of strain ΔR::bldD in shake-flask stage are as follows. The inoculation time is 96 h, the inoculation proportion is 10%, the initial pH = 3, the addition amounts of EP powder and Zn²⁺ are 30 g/L and 1 mM respectively, the liquid content is 25 mL (in 250 mL shake-flask), the fermentation temperature is 26 ℃, the rotational speed is 175 rpm, and the fermentation time is 216 h.

Scale-up culture in a 5-L reactor

Bioreactor fermentation is the vital step to achieve scale-up of the target molecule production, and the fermentation scale-up process from shake-flask level to 5-L reactor level was studied in this work (Fig. 5). The key parameters during the fermentation of strain ΔR::bldD were optimized as follows: the inoculation time is 96 h, the inoculation amount is 10%, the initial pH value is 3, the addition amount of EP powder is 30 g/L, the fermentation temperature is 26 ℃, the rotational speed is 175 rpm, the fermentation time is 216 h, the liquid content is 3 L. Production of ilamycin E in a 5-L reactor continued to rise and reached a peak of 745.44 mg/L on the 10th day, which is better than that of shake-flask culture (449.46 mg/L).

Impact of seawater on production

As a marine-origin algae, E. prolifera contains a large amount of sediment and metal ions from seawater before processing. In order to further reduce the amount of labor and cost, the feasibility of S. atratus strains to use fresh E. prolifera obtained from the sea directly was tested by adding artificial seawater into the medium. As shown in Fig. 6, both strains ΔR and ΔR::bldD can produce ilamycins after addition of artificial seawater, though the yield were decreased to some extent. This result indicates that S. atratus SCSIO ZH16 is capable of producing ilamycins under artificial seawater conditions.

In summary, waste biomass E. prolifera was utilized as a complex nitrogen source for the production of ilamycins in this work. The production related genes of ilamycins were screened and engineered from the perspectives of antibiotic resistance, protein modification, regulatory factors and precursor supply. Fermentation results indicate that the bldD overexpression strain S atratus SCSIO ZH16 ΔR::bldD is more conducive to produce ilamycins by using E. prolifera. Combining optimization of fermentation parameters and submerged fermentation, the production of ilamycins was increased to 1561.77 mg/L in a 5-L bioreactor. Therefore, the strategy of using EP powder to produce ilamyins is economy and high effectiveness, which meets the needs of industrial production and benefits for protecting the ecological environment.

Strains, plasmids and primers

The bacterial strains and plasmids used in this study are listed in Table 1. All primers used in this study are listed in Table S1 in Supplementary data, and synthesized by Azenta (Suzhou, China). S. atratus SCSIO ZH16 ΔilaR, a genetically engineered mutant strain of Streptomyces atratus SCSIO ZH16 (NCBI Reference Sequence:NZ_CP027306.1), was given by Ju’s laboratory(South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China).

Table 1

Bacterial strains and plasmids used or constructed in this work
Strains and plasmids	Relevant phenotype
Strains
E. coli
DH5α	F-ϕ80lacZΔM15Δ(lacZYA-argF)U169recA1endA1hsdR17(rk-mk+)phoAsupE44λ-thi-1gyrA96relA1	From Prof Wu
S17-1	recA pro hsdR RP4-2-Tc::Mu-Km::Tn7	From Prof Wu
S. atratus
SCSIO ZH16	Wild type strain, ilamycins producer	From Prof Ma
ΔR	SCSIO ZH16 with in-frame deletion of ilaR	Lab stock
ΔR::ilaJ	ΔR Φ31 attB::pIBilaJ	This study
ΔR::ilaK	ΔR Φ31 attB::pIBilaK	This study
ΔR::ilaJ-ilaK	ΔR Φ31 attB::pIBilaJK	This study
ΔR::bla	ΔR Φ31 attB::pIBbla	This study
ΔR::PPtase1	ΔR Φ31 attB::pIBPPtase1	This study
ΔR::PPtase2	ΔR Φ31 attB::pIBPPtase2	This study
ΔR::PPtase3	ΔR Φ31 attB::pIBPPtase3	This study
ΔR::sfp-svp	ΔR Φ31 attB::pWHU2449	This study
ΔR::shbA	ΔR Φ31 attB::pIBshbA	This study
ΔR::bldD	ΔR Φ31 attB::pIBbldD	This study
ΔR::arpA	ΔR Φ31 attB::pIBarpA	This study
ΔR::afsR	ΔR Φ31 attB::pIBafsR	This study
ΔR::rok7B7	ΔR Φ31 attB::pIBrok7B7	This study
ΔR::bldA	ΔR Φ31 attB::pIBbldA	This study
ΔR::adpA	ΔR Φ31 attB::pIBadpA	This study
ΔR::sigF	ΔR Φ31 attB::pIBsigF	This study
ΔR::sigN	ΔR Φ31 attB::pIBsigN	This study
ΔR::ilaQ	ΔR Φ31 attB::pIBilaQ	This study
ΔR::CS	ΔR Φ31 attB::pIBCS	This study
ΔR::SKD	ΔR Φ31 attB::pIBSKD	This study
ΔRΔatra	ΔR with in-frame deletion of atr23(RS26165) in BGC13	This study
Plasmids
pKCcas9dO	A CRISPR/Cas9 editing plasmid harboring an actII-ORF4-specific gRNA and two homologous arms for in-frame deletion, aac(3)IV, pSG5	[1]
pKCcas9datr23	A CRISPR/Cas9 editing plasmid harboring an atr23(RS26165)-specific gRNA and two homologous arms for in-frame deletion, aac(3)IV, pSG5	This study
pIB139	Integrative vector based on Φ31 int/attP, aac(3)IV, oriT RK2, PermE*	[2]
pIBilaJ	pIB139 harboring ilaJ under control of PermE*	This study
pIBilaK	pIB139 harboring ilaK under control of PermE*	This study
pIBilaJK	pIB139 harboring ilaJ-ilaK under control of PermE*	This study
pIBbla	pIB139 harboring RS37870 under control of PermE*	This study
pIBPPtase1	pIB139 harboring RS01025 under control of PermE*	This study
pIBPPtase2	pIB139 harboring RS31655 under control of PermE*	This study
pIBPPtase3	pIB139 harboring RS38670 under control of PermE*	This study
pWHU2449	pIB139 harboring sfp-svp under control of PermE*	[3]
pIBshbA	pIB139 harboring RS19455 under control of PermE*	This study
pIBbldD	pIB139 harboring RS07905 under control of PermE*	This study
pIBarpA	pIB139 harboring RS12555 under control of PermE*	This study
pIBafsR	pIB139 harboring RS24115 under control of PermE*	This study
pIBacsR	pIB139 harboring RS33465 under control of PermE*	This study
pIBrok7B7	pIB139 harboring RS32300 under control of PermE*	This study
pIBbldA	pIB139 harboring RS16910 under control of PermE*	This study
pIBadpA	pIB139 harboring RS15155 under control of PermE*	This study
pIBsigF	pIB139 harboring RS21525 under control of PermE*	This study
pIBsigN	pIB139 harboring RS21520 under control of PermE*	This study
pIBilaQ	pIB139 harboring ilaQ under control of PermE*	This study
pIBCS	pIB139 harboring RS07940 under control of PermE*	This study
pIBSKD	pIB139 harboring RS07945 under control of PermE*	This study

Standard DNA manipulation, construction of overexpression and knock-out plasmids

Clones of S. atratus SCSIO ZH16 ΔilaR were cultured in YEME medium contaning 5 g/L polypeptone, 3 g/L yeast extract, 3 g/L malt extract, 10 g/L glucose, 5mM MgCl₂ and a natural pH. Genomic DNA was extracted from the cultured clones using the salting-out method. For PCR amplification, 2×Hieff Canace® Plus PCR Master Mix (With Dye) from vectors using T4 DNA ligase (TransGen Biotech, Beijing, China) or the Hieff Clone® Plus One Step Cloning Kit (Yeasen Biotechnology, Shanghai, China). Plasmid MiniPrep Kit (Yeasen Biotechnology, Shanghai, China) was used for plasmid extraction, and the EasyPure® Quick Gel Extraction Kit (Yeasen Biotechnology, Shanghai, China), was used for DNA product purification. DNA sequencing was carried out by Azenta (Suzhou, China). To amplify the complete open reading frame (ORF) of the target gene, primer pair x-F/R was used to amplify the gene from the S. atratus genomic DNA. The resulting PCR product was then digested using NdeI and EcoRI enzymes (Thermo Fisher Scientific, USA). Subsequently, the digested fragment was ligated with the linear site-specific integrative vector pIB139, resulting in plasmid pIB139-x. In the case of the afsR gene, since its sequence contains an EcoRI digest site, the ORF of afsR was ligated into the linear vector using the Hieff Clone® Plus One Step Cloning Kit (Yeasen Biotechnology, Shanghai, China). To delete the NRPS gene atr23 from S. atratus SCSIO ZH16, a previously reported CRISPR-Cas9 system was employed [22]. The construction of the pKCcas9datr23 plasmid involved three steps: (1) Amplification of upstream and downstream homologous arms of atr23: The upstream and downstream homologous arms of atr23 were amplified from S. atratus SCSIO ZH16 genomic DNA using PCR. Two primer pairs, uarm-atr23-F/R and darm-atr23-F/R, were used for this amplification. (2) Ligation of atr23-targeted sgRNA: A specific N20 sequence was incorporated into the sgRNA targeting atr23, and it was ligated upstream of the upstream homologous arm using the primer pair sg-atr23/uarm-atr23-R. This ligation generated sg-up-arm. (3) In-fusion cloning of sg-up-arm and down-arm into pKCcas9dO: The sg-up-arm and downstream homologous arm were then ligated into the pKCcas9dO vector between the SpeI and HindIII restriction sites using in-fusion cloning. This step was performed using the in-fusion cloning kit (Yeasen Biotechnology, Shanghai, China).

Genome editing procedure

The E. coli DH5α was used to construct and amplify vectors and the E. coli S17-1 was used to transfer the recombinant plasmids into S. atratus SCSIO ZH16 ΔilaR by conjugation. The desired kanamycin-resistant overexpression conjugants were checked by PCR and sequencing with primer pair 152-F/R. The desired apramycin-resistant deletion mutants of atr23 were checked by PCR and sequence with primer pair knock-atra-F/darm-atra-R, and the strain knocked-out accurately was named ΔRΔatr23.

Media and growth condition

S. atratus SCSIO ZH16 ΔilaR and its derivatives were grown on YMS agar (4 g/L yeast extract, 10 g/L malt extract, 4 g/L soluble starch, 10 g/L oatmeal, 2 g/L CaCO₃, 20 g/L agar, pH 7.2–7.4) and incubated at 28°C for 5 days to harvest matured spores. Then four agar tablets (5 mm×5 mm) were transferred into a 250-mL shake flask containing 25 mL seed medium (Am2ab medium). Am2ab medium is composed of 20 g/L glucose, 2 g/L peptone, 2 g/L yeast extract, 5 g/L soya soybean powder, 0.5 g/L MgSO₄·7 H₂O, 0.5 g/L KH₂PO₄, 4 g/L NaCl, 2 g/L CaCO₃ and 30 g/L sea salt, pH 7.2–7.4. After shaking at 200 rpm and 28 ℃ for 72 h, 2.5 mL of the preculture medium was seeded into a 250-mL shake flask containing 25 mL fermentation medium (soluble starch 120 g/L, soya soybean powder 23.49 g/L, corn steep liquor 2.4 g/L, NaCl 6 g/L, NaNO₃ 9.6 g/L, KH₂PO₄ 0.24 g/L, (NH₄)₂SO₄ 4.68 g/L, CaCO₃ 17.67 g/L) at 175 rpm, 26℃ for 216 h.

The tested media are listed in Table 2. For nitrogen sources variation, soya soybean powder was substituted with different nitrogen sources (EP powder, corn steep liquor, yeast extract powder, polypeptone, peptone, yeast extract paste, malt extract, beef extract, bacterial peptone, fish peptone, tryptone, acid-hydrolyzed casein, bean flour). All the nutrients were investigated with same quality fraction. To test the influence of EP on ilamycins production, soluble starch and soya soybean powder were substituted with E. prolifera of various concentrations and different pre-processed separately. All common biological and chemical reagents were obtained from standard commercial sources.

Table 2

The components of Mediums
Medium	Component (g/25 mL)
M1	3g soluble starch, 0.5873g soybean powder, 0.06g corn steep liquor, 0.15g NaCl, 0.24g NaNO₃, 0.006g KH₂PO₄, 0.117g (NH4)₂SO₄, 0.4418g CaCO₃
M2	3g soluble starch, 0.5873g E. prolifera, 0.06g corn steep liquor, 0.15g NaCl, 0.24g NaNO₃, 0.006g KH₂PO₄, 0.117g (NH₄)₂SO₄, 0.4418g CaCO₃
M3	1g E. prolifera, 0.5873g soybean powder, 0.06g corn steep liquor, 0.15g NaCl, 0.24g NaNO3, 0.006g KH₂PO₄, 0.117g (NH₄)₂SO₄, 0.4418g CaCO₃
M4	3g soluble starch, 0.06g corn steep liquor, 0.15g NaCl, 0.24g NaNO₃, 0.006g KH₂PO₄, 0.117g (NH₄)₂SO₄, 0.4418g CaCO₃
M5	0.5873g soybean powder, 0.06g corn steep liquor, 0.15g NaCl, 0.24g NaNO₃, 0.006g KH₂PO₄, 0.117g (NH₄)₂SO₄, 0.4418g CaCO₃
M6	1g Acid-hydrolyzed E. prolifera, 0.5873g soybean powder, 0.06g corn steep liquor, 0.15g NaCl, 0.24g NaNO₃, 0.006g KH₂PO₄, 0.117g (NH₄)₂SO₄, 0.4418g CaCO₃
M7	3g soluble starch, 0.15g NaCl, 0.006g KH₂PO₄, 0.4418g CaCO₃
M8	3g soluble starch, 0.5873g E. prolifera, 0.15g NaCl, 0.006g KH₂PO₄, 0.4418g CaCO3
Am3	0.375g soluble starch, 0.375g bacterial peptone, 0.125g soybean powder, 0.375g glycerin, 0.05g CaCO₃, 0.75g sea salt

RNA isolation and transcript quantification

To verify the transcriptional level of intra-cluster genes of ilamycin BGC, total RNAs were extracted at 48 h and 72 h from different mutants grown in fermentation medium, using BioFlux® SimplyP Total RNA Extraction Kit (BIoer Technology, Hangzhou, China). Reverse transcription was obtained using Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qRT-PCR (gDNA digester plus) (Yeasen Biotechnology, Shanghai, China). Real-time RT-PCR was performed in 96-well plate using the CFX Connect real-time PCR detection system (Bio-Rad), and the reaction solutions were prepared with Hieff UNICON® Universal Blue qRT-PCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China). Housekeeping gene RS08190 was chosen as a loading control. The transcriptional levels were calculated with three replicates using 2^−ΔΔCt value. Sequences of primers used in this study are listed in Table S1.

Fermentation in a stirred-tank 5-L bioreactor

The optimized fermentation medium and parameters were conducted in a 5-L bioreactor (Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd., Shanghai, China), which was equipped with pH, temperature and pO2 probes. 300 mL of seed culture was incubated for four days and then inoculated into the stirred-tank bioreactor containing 2.7 L fermentation media. The aeration was kept at 1.0–2.0 vvm (air/culture volume/min) and the agitation speed was set from 200–600 rpm, linking with the dissolved oxygen level. During the fermentation process, broth was cultivated at 26 ℃ for 240 h and samples were taken every 24 h to detect ilamycins production, dry weight and residual sugar of the broth.

Analytical methods of ilamycins

As the EP powder, soya soybean powder and calcium carbonate were insoluble in water, it’s hard to detect the dry cell weight, dry weight of mixed non-soluble particles and biomass were used instead. The supernatant and solid substances were separated by vacuum filtration. After 24 hours of drying in a 50 ℃ oven, the dry weight was calculated using subtraction method. For the extraction and quantitative analysis of ilamycins by HPLC, detailed steps were performed as described previously [11] (Zhu et al., 2022). The concentration of residual sugar in the broth were determined by a Total Carbohydrate Content Assay Kit (Solarbio Science & Technology Co., Ltd., Beijing, China). The content of carbon and nitrogen elements in the E. prolifera was detected through an Elemental analyzer (ELEMENTAR, German).

Scanning Electron Microscope (SEM)

The S. atratus mutant strains were cultured on YMS medium at 28 ℃, and a 2 mm×2 mm area of the strains was harvested from the plate at 3, 4, 5, 6, 7, 8 and 9 d by scalpel. The samples were soaked with 2.5% glutaraldehyde at 4℃ overnight and then dried naturally. Finally, the observation of the dehydrate cells were conducted on a S3400-N scanning electron microscopy (Hitachi, Tokyo, Japan) according to standard procedures.

Supplementary Information

The online version contains supplementary material available at

Additional file 1. Fig. S1 Comparison of ilamycins production of ΔR strain in M2 medium and Am3 medium.

Fig. S2 The effect of each combination of carbon and nitrogen sources on the dry weight of ΔR strain.

Fig. S3 Reducing sugar, total sugar, nitrogen and oil content in E. prolifera before and after sterilization.

Fig. S4 Effect of overexpressing ilaJ and ilaK on the production of ilamycins in wild type strain.

Fig. S5 Macroscopic and microscopic morphological changes of strains ΔR and ΔR::bldD. (A) The macroscopic morphology of the bacteria from day 3 to day 8 (corresponding to a-f, respectively), 1-3 being ΔR::bldD, 4-6 being ΔR. (B-E) Scanning electron microscopy was used to observe the morphological differentiation of strains ΔR and ΔR::bldD at 2300 and 6000 times on day 4,5,7,8. (B) Day 4 (a-b) ΔR, (c-d) bldD; (C) Day 5 (a-b) ΔR, (c-d) bldD; (D) Day 7 (a-b) ΔR, (c-d) bldD; (E) Day 8 (a-b) ΔR, (c-d) bldD) .

Fig. S6 Effect of (A) pH, (B) temperature, (C) inoculation amount, (D) inoculation time, (E) addition amount of E. prolifera, (F) rotational speed, (G) liquid volume and (H) Zn²⁺ concentration on dry weight of ΔR::bldD strain fermentation broth.

Table S1 Primers used in this study.

Table S2 RT-qPCR Primers used in this study.

Author contributions

Yu-Xi Jiang: Investigation, Formal analysis, Data Curation, Writing - Original Draft, Visualization, Methodology. Gao-Fan Zheng: Investigation, Validation, Methodology, Writing - Review & Editing, Visualization. Xiu-Juan Xin: Supervision, Project administration. Hui Wu: Supervision, Project administration. Ming Zhao: Supervision, Project administration. Jun-Ying Ma: Resources. Jian-Hua Ju: Resources. Ruida Wang: Conceptualization, Methodology, Resources, Writing - Review & Editing. Fa-Liang An: Conceptualization, Writing - Review & Editing, Visualization, Supervision, Project administration, Funding acquisition.

Acknowledgments

We thank Professor Hai-Zhen Wu from East China University of Science and Technology for kindly providing us with the strain, E. coli DH5α and E. coli S17-1. We also thank Professor Xudong Qu, from Shanghai Jiao Tong university for kindly providing us with the plasmid pWHU2449.

Funding

This work was funded the National Key R&D Program of China (Nos. 2019YFC0312504, 2018YFC1706206), the Excellent Youth Scholars of Anhui Provincial Education Department (2022AH030097). This work was also supported by the Open Research Fund Program of State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing Technology.

Data availability

Data will be made available on request.

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author details

¹ State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China

² Department of Applied Biology, School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China

³ CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 528225, China

⁴ Marine Biomedical Science and Technology Innovation Platform of Lin-gang Special Area, No.4, Lane 218, Haiji Sixth Road, Shanghai 201306, China

⁵Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, College of Biology and Food Engineering, Anhui Polytechnic University, Wuhu 241000, China

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Efficient ilamycins production utilizing Enteromorpha prolifera by metabolically engineered Streptomyces atratus

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Abstract

Background

Results

Conclusions

Figures

Background

Results and discussion

Nitrogen sources Screening for the production of ilamycins

Construction of ilamycins high-yield strains

Mechanism analysis of BldD regulating ilamycins biosynthesis

Optimization of fermentation conditions

Scale-up culture in a 5-L reactor

Impact of seawater on production

Conclusions

Materials and methods

Strains, plasmids and primers

Standard DNA manipulation, construction of overexpression and knock-out plasmids

Genome editing procedure

Media and growth condition

RNA isolation and transcript quantification

Fermentation in a stirred-tank 5-L bioreactor

Analytical methods of ilamycins

Scanning Electron Microscope (SEM)

Declarations

References

Additional Declarations

Supplementary Files

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