Introduction

In the past 10 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 Asia [22]. Due to its high organic matter content and abundant production, however, EP has the potential to be used as raw materials for digestion [13]. 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 [11]. 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 been reported on the application of EP [11]. Actinomycetes species are renowned for the capacity to produce a multitude of high-valued product, even using waste biomass [5, 12]. 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 [19, 20]. 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 production or process. By Plackett–Burman design model, single factor optimization, pH coordinated feeding and continuous pulse feeding, [7] achieved 415.7 ± 29.2 mg/L ilamycin E yield in a 300 L bioreactor. 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 [24]. 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 [2]. 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. [6]. Among them, genetic operation towards regulatory genes is a promising approach. For 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 [4, 10, 17]. 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, environment friendly 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.

Results and discussion

Nitrogen sources screenings 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. Previous studies indicated the significant effect of the selection of organic nitrogen source on the yield of ilamycins in S. atratus SCSIO ZH16 ΔilaR [7, 24]. In view of this, this study started with the systematic screening of composite 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 E1/E2, 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. The 14 alternative nitrogen sources including EP powder, corn steep liquor, two types of yeast extract powder (Angel and OXOID), polypeptone, peptone, yeast extract paste, malt extract, beef extract, bacterial peptone, fish peptone, tryptone, acid-hydrolyzed casein and soybean flour 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.

Fig. 1
figure 1

Utilization of EP powder for the ilamycins production in S. atratus SCSIO ZH16 ΔR. A Yield of ilamycins in media with different complex nitrogen sources. 1, Angel yeast extract powder; 2, acid-hydrolyzed casein; 3, tryptone; 4, corn steep liquor; 5, fish peptone; 6, bacterial peptone; 7, OXOID yeast extract; 8, yeast extract paste; 9, polypeptone; 10, peptone; 11, malt extract; 12, beef extract; 13, Enteromorpha prolifera; 14, bean flour; 15, soybean powder. B Effect of different combinations of carbon and nitrogen sources on the yield of ilamycins. 1, soluble starch; 2, soybean powder; 3, EP powder, ‘(Acid)’ means after acid hydrolysis; 4, corn steep liquor, NaNO3 and (NH4)2SO4. Error bars represent the standard deviations from three independent biological replicates. **: P < 0.01 (Student’s t test). C Weight ratio and D molality of N, C, H in EP powder

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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 nor EP powder produced only 117.73 mg/L ilamycins. Take the shortcomings of soybean powder into account, the yield reduction of replacing soybean powder with EP powder can be acceptable. In addition, when S. atratus was cultured in media containing both soybean powder (N source) and EP powder but lack of soluble starch (C source) (named M3 and M6), the production of ilamycins were extremely low, which means that EP powder cannot replace soluble starch as a carbon source, regardless of whether or not it has been subjected to acid hydrolysis. Besides, the ilamycins yield of M2 is 42.7-fold higher than that of original medium Am3 (Additional file 1: 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 (Additional file 1: Fig. S2), primary and secondary metabolism flux regulation, and ilamycins biosynthesis of ΔR. In addition, macroalgal blooms result from eutrophication, and the consequent oxygen consumption, which threats the survival of other marine plants and animals while not release any toxic compounds [21]. Therefore, E. prolifera is non-toxic and can be utilized as sources of protein and bioactive compounds in formulated feeds, or processed into snacks and condiments [11]. Then, the results also showed that the yield in M8 definitely decreased compared to M2, which is main due to the absence of corn steep flour, because the trace element provided by corn steep liquor, including a variety of amino acids, inorganic salts and vitamins, are significant for the production of natural products [16]. What is more, the results of M5 and M7 add to the fact that the combination of the major organic nitrogen source EP powder, the carbon source soluble starch, the easy-utilized nitrogen sources corn steep flour, NaNO3 and (NH4)2SO4 are the most basic important requirements for production of ilamycins.

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 (Fig. 1C, D). The reason for the inability of EP powder to be utilized as a carbon source may due to that the main component of carbon source in EP powder is algal polysaccharides, which is not suitable for Streptomyces. In a word, when goes to industrial scale production, EP powder is a considerable environment friendly nitrogen source, and the leftover waste after product recovery may be treated as fertilizer or innocuous solid waste. As soluble starch is not considered as an ideal carbon source for industrial fermentation, we are currently attempting to screen alternative carbon sources and hope this issue would be solved in future.

Construction of ilamycins high-yield strains

Given that the substitution of nitrogen source with EP powder caused decrease in ilamycins production to some extent, we turned to construct mutant stains that can increase ilamycins productions of S. atratus in EP powder-containing media EP powder. A screening on genes belonging to four function categories were performed:

  1. (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 (Additional file 1: 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. (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, atr23 (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 [14], 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 ΔRΔatr23 (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. (3)

    Regulator Manipulation towards regulator is a well-validated approach for yield enhancement of natural products [15, 23], 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. (4)

    Protein modification As nonribosomal peptides, efficiently biosynthesis of ilamycins requires a protein modification executed by phosphopantetheinyl transferase (PPtase) to activate carrier proteins [1]. Therefore, overexpression of PPtase has successfully improved the production of multiple antibiotics [18]. 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.

Fig. 2
figure 2

Ilamycins yields of engineered strains in EP powder culture medium after overexpression/knockout of candidate genes. ΔR is abbreviation of S. atratus SCSIO ZH16 ΔR; Δ and – denote knockout, overexpression, and co-expression, respectively

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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 first 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 [1: 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 4 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 to 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, soybean powder and calcium carbonate were insoluble in water, it is 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 h 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 [24]. The dried extracts of the fermentation products were dissolved with accurate amount of methanol and centrifuged for 5 min at 10,000 rpm followed by filtration of 0.22 μm membranes. Reversed phase column Agilent ZORBAX Eclipse XDB-C18 (4.6 mm × 150 mm, 5 μm) with UV detection at 210, 280, 354 nm was used to detect ilamycins under the following program: solvent system (solvent A, water supplemented with 0.1% acetic acid; solvent B, acetonitrile); 15% B to 71% B (linear gradient, 0–10 min), 71% B to 85% B (linear gradient, 10.0–11.5 min), 85% B (11.5–20.0 min), 85% B to 90% B (linear gradient, 20.0–20.1 min), 90% B (20.1–25.1 min), 90% B to 15% B (linear gradient, 25.1–26.0 min), 15% B (26.0–30.0 min); the flow rate was set as 1 mL/min.

The concentration of residual sugar in the broth were determined by a Total Carbohydrate Content Assay Kit (Solarbio Science & Technology Co., Ltd., Bei**g, 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.