Decreased lincomycin B content by regulating osmotic pressure in fermentation of Streptomyces lincolnensis

doi:10.21203/rs.3.rs-2082602/v1

Download PDF

Research Article

Decreased lincomycin B content by regulating osmotic pressure in fermentation of Streptomyces lincolnensis

https://doi.org/10.21203/rs.3.rs-2082602/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Lincomycin, a clinically important antibiotic against gram-positive bacteria, is a sulfur-containing metabolite produced by Streptomyces lincolnensis. The content of lincomycin B is an important evaluation index of fermentation quality. In this study, we investigated the effects of NaCl as an osmotic regulator on lincomycin production. Based on the flask experiment, the optimal initial osmotic pressure for lincomycin fermentation was in the range between 0.860–1.355 Osmol/kg. The effect of osmotic pressure on the fermentation of lincomycin in 15 L bioreactor showed that the content of lincomycin B in the broth was decreased to 5.78% when adding 10 g/L NaCl while the content of lincomycin B was 8.20% in the control. During the fermentation process, the intracellular homocysteine and S-adenosylmethionine was higher when adding 10 g/L NaCl, which could provide more methyl donor for lincomycin production, causing the reduction of lincomycin B. Besides, high-throughput RNA sequencing was applied to study the mechanism of osmotic regulation on lincomycin fermentation. Under osmotic stimulation, the transcription of 23 genes in lincomycin biosynthesis cluster were significantly up-regulated at 41 h and the transcription of genes in HMP and EMP was up-regulated while the transcription of genes in TCA cycle were decreased. Notably, the genes involved in sulfur assimilation, mycothiol, ergothioneine and S-adenosylmethionine biosynthesis were up-regulated, which can provide more methyl donor (S-adenosylmethionine) and sulfur donor (mycothiol and ergothioneine) during lincomycin fermentation. This might be the reason of increased lincomycin A production and decreased lincomycin B content. Our study provides new insight into the mechanisms underlying osmotic pressure regulation on lincomycin fermentation and may be applied to improve secondary metabolite production.

Streptomyces lincolnensis

Lincomycin

Osmotic pressure

Sulfur metabolite

Transcriptome analysis

Lincomycin A is a chemically important lincosamide antibiotics. It is produced by Streptomyces lincolnensis and has potent antibacterial activity against many Gram-positive microorganisms and few Gram-negative microorganisms such as Mycoplasma (Mason et al. 1964). During the fermentation process, lincomycin A is produced along with lincomycin B, which has lower bioactivity and higher toxicity than lincomycin A (Kucers et al. 1997). The content of lincomycin B is an important parameter to evaluate the fermentation process. According to the pharmacopoeia of China, the content of lincomycin B cannot exceed 5%.

Lincomycin A is composed of two moieties, an amino acids moiety propyl-L-proline (PPL) and C₈ backbone glycosyl moiety methythiolincosaminide (MTL) (Horton et al. 1974). The two moieties are biosynthesized separately and condensed to form N-demethyl-lincomycin, which is then converted to lincomycin A by methylation (Spíek et al. 2004). Both lincomycin A and lincomycin B have the same C₈ backbone glycosyl moiety, but they are different in their amino acids moieties: lincomycin A has a PPL moiety while lincomycin B has an ethyl-L-proline (EPL) one.

Given the high clinical importance of lincomycin, enhancement of lincomycin A production or reduction of lincomycin B production in S. lincolnensis have been performed in many studies by strain screening (Ye et al. 2009; Meng et al. 2013; Huang et al. 2017), fermentation engineering (Xue et al. 2009; Lee et al. 2014; Zhuang et al. 2019) or genetic manipulation (Pang et al. 2015; Xu et al. 2018). All the above studies focused on optimization of medium composition and process conditions or screening strains with more productivity. But no one considered the culture stress condition and its influence on the metabolism of S. lincolnensis. Environmental stress conditions, such as osmotic stress, cause major changes in the metabolism of microorganisms. Osmotic regulation has been mostly used in yeasts fermentation to improve the production of bioproduct (Tian et al. 2012). Increasing osmotic pressure of fermentation broth with sodium chloride could promote erythritol production by Yersinia lipolytica (Yang et al.2013). The effect of osmotic pressure on the synthesis of L-lactic acid by Lactobacillus casei was also enhanced when adding proline as a protective agent (Zhang et al. 2014). Streptomyces caelestis produced compatible solutes such as glucosyl, glycerate and trehalose (Pospíil et al. 2007), ectoine and 5-hydroxyectoine (Bursy et al. 2008), and amino acids, such as proline, leucine and methionine in response to salt stress (Thiebaut et al. 2015). A master regulator ơ^B governed osmotic response in Streptomyces coelicolor A3(2) and accelerated cysteine and mycothiol production upon osmotic shock (Lee et al. 2005).

Lincomycin is a unique sulfur-containing secondary metabolite (Zhao et al. 2015) found that two small-molecule thiols, ergothioneine (EGT) and mycothiol (MSH), played an important role in the synthesis of lincomycin through two rare S-glycosylation reactions: EGT mediated the activation, transfer and modification of the C₈ glycosyl moiety while MSH provided sulfur element for lincomycin through thiol exchange (Zhao et al. 2015). EGT and MSH as the major thiols in a number of actinomycetes (Sakuda et al 1994), played a key role in responses to cellular stress, and thiol-disulfide redox balance (Paget 2003, Lee et al. 2005, Emani et al 2019). In actinomycetes, sulfur exists in the form of sulfur-containing amino acids and small molecule thiols. Among them there are cysteine (Cys) and methionine (Met), the biosynthesis of which is linked through transsulfuration pathway via homocysteine (Hcys). (Kulkarni et al. 2015). Also, there is S-adenosyl methionine (SAM) which is synthesized from Met and ATP through methionine adenosyltransferase, serving as an important methyl donor in both primary and secondary metabolism (Chiang et al. 1996). The biosynthesis of small-molecule thiols, EGT and MSH, depends on Cys as a major precursor (Jothivasan 2008, Seebeck,2010).

In this article, we examined sulfur-containing intermediate metabolites and applied RNA sequencing (RNA-seq) to further study the function of osmotic regulation in lincomycin fermentation.

Strain and media

S. lincolnensis ZLW0306 was used as fermentation strain throughout this work. The seed medium was as follows: 10 g/L glucose, 20 g/L starch, 10 g/L soybean meal, 30 g/L corn steep liquor, 1.5 g/L (NH₄)₂SO₄, and 5 g/L CaCO₃ (pH 7.0). The fermentation medium in flask contained: 100 g/L glucose, 25 g/L soybean meal, 2 g/L corn steep liquor, 8 g/L (NH₄)₂SO₄, 8 g/L NaNO₃, 0.3 g/L KH₂PO₄ and 8 g/L CaCO₃ (pH 7.0). The fermentation medium in bioreactor contained: 30 g/L glucose, 5 g/L starch, 15 g/L corn steep liquor, 20 g/L soybean meal, 8.5 g/L NaCl, 8.5 g/L (NH₄)₂SO₄, 0.5 g/L K₂HPO₄, 6 g/L CaCO₃ (pH 8.6). The feeding medium contained: 300 g/L glucose, 20 g/L (NH₄)₂SO₄, 60 g/L soybean meal, 90 g/L corn steep liquor and 1.5 g/L CaCO₃ (pH 7.0).

Culture condition

One milliliter glycerol stock of S. lincolnensis was inoculated into 25 mL seed medium and grown at 30 °C in a 250-mL flask with 220 rpm for 48 h. Then, 2.5 mL seed culture was inoculated into 25 mL fermentation medium. The fermentation process was conducted for 7 days at 30 °C. Batch cultivation was conducted in a 15 L bioreactor (Guoqiang Bioengineering Equipment Co., Ltd, Shanghai, China) with an initial working volume of 10 L. The inoculation volume was 10 % (v/v) of the medium volume. The fermentation temperature and agitation speed were maintained at 30 °C and 450 rpm, respectively, and the aeration rate was 1.0 vvm. Samples were taken at 8 h intervals for off-line analysis.

Analytical methods

The culture samples taken at different times were centrifuged at 4 °C, 10000 rpm for 10 min to collect the mycelia. The precipitated mycelia were washed three times and dried to constant weight at 105 °C to measure the dry cell weight (DCW). The bacterial cell concentration is determined by packed mycelia volume (PMV) to quantify cell growth condition in a quick way. (Yao et al. 2017, Neves et al. 2000).

The pH, lincomycin titer, osmotic pressure, concentration of reducing sugars and ammonia nitrogen were determined after collecting the supernatant. All parameters were determined in triplicate. The osmotic pressure was determined with a freezing point osmometer (Yida Instrument Co., Ltd, Shanghai, China). The reducing sugar concentration was determined by the Fehling method and ammonia nitrogen concentration was determined by formaldehyde titration method (Yao et al. 2017). The lincomycin A titer was assayed by high-performance liquid chromatography (HPLC) (Agilent 1260, Agilent Technologies Co., Ltd, Beijing, China) using a Diamonsil plus C18 column (250 × 4.6 mm, 5 μm, Dikma Corporation, Beijing, China). The column temperature was maintained at 30 °C, and UV detection was set at 214 nm. A mobile phase containing 50 mmol/L ammonium acetate:methanol (3:2, v/v) was used at a flow rate of 0.4 mL/min (Li et al. 2007).

Determination of intracellular thiol amino acids concentration

The culture samples were centrifuged at 10000 rpm for 10 min at 4 °C to collect the mycelia. The precipitated mycelia were immediately frozen in liquid nitrogen and stored at -80 °C until extraction.

The major intracellular thiols were extracted by a modification of a previously reported method (Newton et al. 1996). 200 mg frozen cells were transferred to 1.5 mL microcentrifuge cubes. A 1-mL quantity of warm (60 °C) 50% (W/V) aqueous acetonitrile containing 20 mmol/L HEPES (pH 8.0) and 2 mmol/L monobromobimane (mBBr) was added to each tube, and the samples were sonicated by 200 W until no obvious mycelia suspended. Extracts were maintained in a 60 °C water bath for 15 min in the dark and then added 50 µL 5 mol/L methanesulfonic acid. The tube was centrifuged at 10000 rpm for 10 min at 4 °C. The supernatants were transferred to a new tube and stored at -80 °C until analysis. Negative control and standard sample were prepared based on Fahey’s reported method (Fahey et al 1987).

HPLC was performed using a Shimadzu Prominence system that included a fluorescence detector (RF-20A). The HPLC system was equipped with a Diamonsil plus C18 column (250 × 4.6 mm, 5 μm, Dikma Corporation, Beijing, China), and the flow rate was maintained at 1.2 mL/min. The gradient program from solvent A (40 mmol/L ammonium acetate, pH 3.5) to solvent B (methanol) was 15 % B in 5 min, 15 % to 25 % in 10 min, 25 % to 75 % in 15 min, 75 % to 100 % in 2 min, 100 % to 15 % in 3 min, and 15 % B in 10 min.

Determination of intracellular SAM concentration

Intracellular SAM was extracted and detected as described (Xu et al. 2018). In briefly, 200 mg frozen cells were transferred to 1.5 mL microcentrifuge cubes and extracted with 1 mL of 1 mol/L formic acid at 4 °C for 2 h. The samples were centrifuged at 10000 rpm for 10 min at 4 °C to collect the supernatant for determination. The SAM concentration was assayed by HPLC using a Diamonsil plus C18 column (250 × 4.6 mm, 5 μm, Dikma Corporation, Beijing, China). The column temperature was maintained at 25 °C, and UV detection was set at 280 nm. A mobile phase containing 100 mmol/L phosphate buffer:methanol (4:1, v/v) was used at a flow rate of 0.5 mL/min.

RNA-seq and gene expression analysis

Cell samples were collected at 41 h (initial production stage) and 89 h ( production stage) to investigate the changes of transcriptome profile. The culture samples were centrifuged at 10000 rpm for 10 min at 4 °C to collect the mycelia and immediately frozen in liquid nitrogen and stored at -80 °C for subsequent RNA isolation. Total RNA was isolated using an RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech Co., Ltd., Beijing, China). All RNA preparations were treated with RNase-free DNase (TaKaRa) to eliminate genomic DNA contamination. The purity, integrity and concentration of RNA was determined using a Bioanalyzer 2100 (Agilent Technologies Co., Ltd, Beijing, China). A cDNA library was constructed and sequenced by Illumina HiSeq 2500 platform (Personal Gene Technology Co., LTD, Shanghai, China) using the standard Illumina RNA-Seq protocol.

The raw sequencing reads were trimmed for low-quality ends by removing adapter sequences and ambiguous nucleotides, using the software Cutadapt 1.14 (Personal Gene Technology Co., LTD, Shanghai, China). All sequencing reads with quality scores less than 20 were excluded. The remaining high-quality reads were used in subsequent alignments.

The filtered RNA-Seq reads were mapped to the S. lincolnensis NRRL 2936 reference genome sequence (accession number CP016438) using Bowtie29 (Langmead and Salzberg 2012). The fragments per kilo bases per million fragments (FPKM) was calculated as expression values for each gene. The P value was used to identify differentially expressed genes (DEGs). In this study, the DEGs between each of two samples were screened according to log2|fold change|>1 and P<0.05.

Effect of osmotic pressure regulation on lincomycin production in flask

Most microorganisms have their own preferred osmotic pressure suitable for growth. In this research, 5, 10, 15, 25 and 50 g/L of NaCl was added at the beginning of fermentation to examine the optimal osmotic pressure for lincomycin A production and the fermentation without NaCl addition was used as control. The osmotic pressure corresponded to the concentration of NaCl, which was between 0.86 and 2.49 Osmol/kg in flasks. The results showed that the appropriate initial osmotic pressure for lincomycin fermentation was between 0.860 and 1.355 Osmol/kg, equal to 0-15g/L NaCl (Table 1). When osmotic pressure was over 1.355 Osmol/kg, the cell growth was greatly inhibited and the titer of lincomycin decreased significantly (Fig. 1). The results also showed that lincomycin B decreased with the rise of osmotic pressure in the range between 0% and 4.04%. The content of lincomycin B with 10 g/L NaCl was 0.75% and the titer of lincomycin A titer reached 4465 U/ml, which was an 80% decrease of lincomycin B content and a 26% increase of lincomycin A titer compared to the control (P<0.01).

Influence of osmotic pressure regulation on lincomycin production in 15 L bioreactor

Based on the result of flask fermentation, osmotic pressure had a positive effect on reducing lincomycin B content and improving lincomycin A production. Thus, fermentation was carried out in 15 L bioreactors to study the osmotic influence on fermentation process (Fig. 2). The result showed that the osmotic pressure in bioreactors was lower compared to that in flask fermentation for the difference of culture medium between flask and bioreactor in composition and concentration. The initial osmotic pressure in bioreactor was 0.561 Osmol/kg without NaCl addition while adding 10 g/L NaCl the osmotic pressure increased to 0.976 Osmol/kg. Osmotic pressure dropped rapidly for the reason of substrate consumption during the first 40 h of fermentation process. After that, the osmotic pressure became stable at about 0.6 Osm/kg compared to 0.3 Osm/kg of the control. The titer reached 7267 U/mL with 10 g/l NaCl addition, whereas the control’s titer was 7099 U/ml. NaCl addition kept a positive effect on lincomycin A until 90 h, but the promotion tendency almost ceased after 90 h. The content of lincomycin B with NaCl addition was 5.78%, decreased 29.51% compared to the control (p<0.01). The result of bioreactor fermentation was consistent with the result in flask, which indicated that adding NaCl to increase osmotic pressure was beneficial to reducing the content of lincomycin B.

Influence of osmotic regulation on fermentation process parameters

The trend of lincomycin fermentation with or without NaCl addition in 15 L bioreactor was studied (Fig 3.). The cell kept growing until 50 h (without NaCl) reaching the maximum PMV of 50%, while the cell grew to 42% at 60 h (with NaCl). Addition of NaCl greatly influenced the growth, a 16% reduction compared to the control. The pH with NaCl addition was slightly higher than that of the control, which suggested that osmotic pressure caused the change of cell metabolism such as the utilization of amnio acids which may produce more alkaline substances. The glucose consumption rate with or without NaCl addition were similar during cell growth phase before 40 h. However, the rate without NaCl addition was higher at about 70 h probably because of better cell growth. Amino nitrogen concentration indicated that addition of NaCl promoted the utilization of amino nitrogen.

Influence of osmotic pressure regulation on the intracellular accumulation of sulfur-containing metabolites

Sulfur is critical to microorganism and it is everywhere in life system. In microorganisms sulfur exists mainly in the form of cysteine, methionine, SAM and small- molecular thiols (Slobodkin et al. 2019). Lincomycin is a sulfur containing substance, of which sulfur moiety is derived from small-molecule thiols. Both EGT and MSH directly participate in the biosynthesis of lincomycin, acting as carrier and sulfur donor, respectively (Zhao et al. 2015). Lincomycin has three methyl groups, which are N-, C-, S- methylation. Among them, the transfer of methyl group is from SAM, which is synthesized from methionine and ATP. In order to study the influence of osmotic regulation on intracellular sulfur-containing metabolites, we studied the change of cysteine (Cys), homocysteine (Hcys), ergothioneine (EGT) and S-adenosylmethionine (SAM) concentration during the fermentation process. (Fig. 4)

The concentration of Cys increased gradually during the growth phase (20-40 h), with similar trend with or without NaCl addition. During the production phase, the difference of the Cys concentration was apparent: it was much lower with NaCl addition than that without NaCl addition. But the sample was 41% higher (1.320 μmol/gDCW) than that of the control (0.935μmol/gDCW) at 140h, meaning the less utilization of Cys at late fermentation phase.

Hcys is an intermediate metabolite of cysteine to methionine. It was found that Hcys concentration increased from 20 to 40 h and then maintained at a constant concentration in both conditions. The final concentration was 1.074 μmol/gDCW when adding NaCl, whereas 0.565 μmol/gDCW without NaCl, almost doubling the amount. The ratio of Hcys to Cys reflects the level of methylation. The results showed that the ratio of Hcys: Cys in NaCl addition experiment was always higher than that of the control, except for the early fermentation period. These results suggested that high osmotic pressure resulted more Hcys synthesis and the increased Hcys concentration might sustaine more SAM synthesis from methionine.

SAM is the methyl donor of many transmethylation reactions, which are involved in many important biochemical processes. In the process of lincomycin biosynthesis, SAM is the direct methyl donor of lincomycin, including C-methylation, N-methylation and S-methylation. Among the three methylation, C-methylation determines whether the product is lincomycin A or lincomycin B and sufficient SAM supply can lower the content of lincomycin B (Pang et al. 2015). The results showed that adding NaCl caused higher intracellular SAM concentration, which may be the reason of reduced lincomycin B production.

Overview of changes in the transcriptome of Streptomyces lincolnensis in response to osmotic stress

RNA-Seq data analysis was performed to provide a better understanding of the response of S. lincolnensis to osmotic pressure. We got 44.41-50.15 MB of raw data from four libraries which are fermentation in bioreactor with or without NaCl at 41 h and 89 h. After filtering of low-quality sequences and removing the redundant reads, we obtained 41.17-48.31 MB clean reads. The ratio of total mapped reads in four libraries were over 97 % and the ratio of coverage of genome were over 93 %, indicating a full statistic coverage and good quality of the result. After differential expression genes analysis, 60 and 103 genes were up-regulated at 41 h and 89 h, respectively, while 124 and 325 genes were down-regulated at 41 h and 89 h, respectively (Fig. 5).

The significantly enriched Gene Ontology (GO) terms were associated with differential expressed genes. The GO term analysis detected a total of 112 GO terms significantly overrepresented at 41 h, with a p-value <0.05, 75 terms referring to molecular function, 32 referring to biological processes and only 5 referring to cellular components. At 89 h, the total number was 101. Among them 50, 39, and 12 referred to molecular function, biological processes, and cellular components, respectively.

Among the most significant terms, we selected the first 20 terms (Fig. 6). The up-regulated genes, which include genes involved in oxidation-reduction process（6,7-dihyfropteridine reductase activity, nitric oxide dioxygenase activity、oxygen carrier activity）, oxidoreductase activity, cofactor binding , sulfate metabolism and ectoine metabolic & biosynthetic process, molybdenum ion binding. At 89h, cation binding, metal ion binding, carbohydrate metabolic process, carbohydrate binding, and intracellular part were significantly enriched, other enriched terms included biological adhesion, intracellular part, intracellular organelle, sulfate assimilation, triglyceride lipase activity, oxidoreductase activity, GTPase activity, hydrolase activity(acting on glycosyl bonds, hydrolyzing O-glycosyl compound) and so on.

Differential expressed genes involved in central carbon metabolism

Although lincomycin is the product of secondary metabolism, its synthesis is closely related to primary metabolism in which glucose is the main carbon source in the lincomycin fermentation. The differential expression genes in central carbon metabolism and sulfur metabolism as well as some important precursors supplement were studied (Fig. 7).

Embden-Meyerhof pathway (EMP), Hexose Monophosphate Pathway (HMP), and Tricarboxylic acid cycle (TCA) are the main pathways that cells utilize glucose and synthesize abundant precursors for cell growth, sulfur assimilation and lincomycin synthesis. As Fig. 7 showed, glk which encodes glucokinase was up-regulated and so was pfk. Because these are the key enzymes, the activity of these enzymes has a large impact on the activity of EMP. gap which encodes phosphoglycerate mutase responsible for the synthesis of glyceraldehyde-3-P, was down-regulated, inferring that the osmotic stress might lead to more carbon flux to HMP. At 89 h, the expression of gap decreased by 13 folds in the EMP pathway. The number of differentially expressed genes in EMP was less at 89 h than that at 41 h, indicating that the cells might have adapted to the osmotic change.

HMP provides important precursors for the synthesis of lincomycin. Among them are areribose-5-P and sedoheptulose-7-P for MTL, NADPH for reduction, erythrose 4-P for tyrosine and PPL. Thus, the HMP was very important for lincomycin production. Zhuang et al. has showed that addition of calcium gluconate improved lincomycin production (Zhuang et al. 2019). At 41 h, tktA2 and rpe were up-regulated while tktB was down-regulated. Both tktA2 and tktB are the encoding genes for transketolase. The genes gntk and zwf, encoding the proteins for synthesizing glucose -6-P, were down-regulated. At 89 h, gene rpe was up-regulated and tktB was down-regulated, while zwf was down-regulated, which catalyzes the first rate-limiting step of HMP, causing the decrease of HMP rate. These results indicated that the precursors and reducing power may not be sufficient at the late fermentation period, causing the inhabitation of lincomycin production.

TCA is the source of precursors for many metabolism pathways such as amnio acids, glycerol and fatty acid. At 41 h, the expression of genes encoding the synthesis of acetyl coenzyme A was upregulated. This meant more pyruvate were produced to enter TCA cycle. Gene cs encoding citrate synthase which is the rate-limiting enzyme for TCA was down-regulated at 41 h and 89 h, indicating that high osmotic pressure could decrease TCA flux. In contrast, pckA upregulated 1.86 folds and 1.97 folds at 41 h and 89 h, respectively, indicating that more oxaloacetic acid were used to synthesize PEP, which is the precursor of tyrosine, the component of amino acid moiety for lincomycin biosynthesis.

Differential expressed genes involved in sulfur metabolism and biosynthesis pathway of MSH and EGT

The MSH, SAM, and EGT biosynthesis pathways and the related gene expression were studied (Fig. 8). Cysteine synthesis starts with glycerate-3-phosphate from EMP. Gene serA encoding D-glycerate-3-phosphate dehydrogenase was down-regulated. Gene related to sulfur assimilation, including cysD (encoding adenosine pyrophosphatase), cysC (encoding adenine-5- phosphoric sulfuric kinase) were up-regulated 3.05 and 3.03 folds respectively with NaCl addition at 41 h. This meant osmotic regulation could increase the transcription of sulfur assimilation so as to produce more sulfur containing metabolites.

The genes in the pathway of SAM biosynthesis were also up-regulated, such as metE and metH, encoding Hcys S-methyltransferase and methionine synthase, increased 3.45 folds and 2.55 folds, respectively, at 41 h. At the same time, the expression of metK encoding methionine adenosyltransferase which catalyzes the reaction from methionine to SAM increased almost two folds. Transcriptomics data showed that increased osmotic pressure enhanced the synthesis of SAM. SAM converts to S-Adenosyl-L-homocysteine (SAH) after it transfer methyl group to the receptor. SAH can be recycled by SahH, the adenosylcysteinase. It was showed that gene sahH up-regulated 1.2 folds at 41 h, but downregulated at 89 h, probably because the cells were adaptive to osmotic pressure at the late periods of fermentation.

MSH and EGT are the most important small-molecular thiols in actinomycetes. The functions of MSH are similar to that of GSH in B. subtilis and E. coli, which are protecting cells from osmotic stress, interacting with nucleophile and functioning as a reserve pool of Cys. The RNA-seq data showed that the genes in MSH biosynthesis pathway, such as mshB, mshC, and mshD up-regulated while mshA down-regulated. After the transsulfuration in lincomycin biosynthesis, MSH turns into GlcN-Ins, which could be turned back into MSH through a two-step reaction catalyzed by MshC and MshD. In the EGT biosynthesis pathway, egtA, egtD, egtB, egtC, egtE upregulated correspondingly. This result indicated that more EGT is synthesized at the early stage of fermentation with NaCl addition. The enhancement of MSH and EGT might be the reason for higher lincomycin production with NaCl addition, for producing more precursors.

In conclusion, it can be suggested that the enhancement of sulfur assimilation pathway by NaCl addition is beneficial to the synthesis of Cys, and then to MSH, EGT, and SAM. With more sulfur and methyl donors, the production of lincomycin A was improved and the production of lincomycin B was reduced.

Differential expressed genes involved in biosynthesis pathway of lincomycin A

There are three resistance genes in the lincomycin synthesis gene cluster (lmrA, lmrB, lmrC), two regulator genes(lmbU, lmbQ) and 23 structural genes.

At 41 h, the 23 structural genes significantly increased after NaCl addition compared to that of the control (log₂foldchange>1，p<0.05) (Fig. 9). Among them, the expression of lmbB2 which catalyzes the first step of the PPL synthesis, upregulated 4.7 folds compared to that of the control. Moreover, lmbR which is a transaldolase catalyzing the first step of MTL synthesis was over expressed by 3.3 folds. These results contributed to the fact that the lincomycin synthesis rate with NaCl addition was consistently higher than that of the control. lmbW, which is related to S-methylation, increased 3 folds. This indicated that more methyl donor was supplemented, which may be the reason for the lower content of lincomycin B in the experimental group. The regulator genes, lmbU and lmbQ were upregulated by 2.70 and 3.00 folds, respectively.

At 89 h, the expression of resistance genes, lmrA (encodes transmembrane protein relevant to lincomycin secretion) and lmrC (encodes lincomycin output ABC protein) with NaCl addition upregulated by 2.60 and 2.20 folds compared to that of the control. MTL production related genes, lmbR, lmbN, lmbP, lmbK, lmbO, lmbL, lmbM, lmbS, lmbF, lmbZ, lmbT were down-regulated to varying degrees without NaCl addition. Among them, lmbO which is responsible for encoding guanosine transferase, catalyzing the synthesis of important intermediate metabolite for lincomycin, GDP-octose and lmbS which encodes NDP-hexanose amino acid transferase downregulated the most. This might be the reason for the production of lincomycin A which was not so much promoted with NaCl addition.

This article found that the appropriate initial osmotic stress was between 0.860–1.355 Osm/kg, with the best NaCl addition was 10 g/L for lincomycin in flask fermentation. The titer of lincomycin A could reach 4465 U/mL when adding NaCl of 10 g/L at the beginning of fermentation, an increase of 26% compared to 3540 U/mL without NaCl addition. The content of lincomycin B was only 0.75%, a sharp decrease of 80% compared to 4.04% without NaCl addition. In the scale-up experiment in 15 L bioreactors, the content of lincomycin B was consistently lower when adding 10 g/L NaCl than that of the control during the whole fermentation process. At the end of the fermentation, the content of lincomycin B (5.78%) was remarkably reduced, 29.51% lower than that of the control (8.20%).

Intracellular Cys and Hcys quantification result showed that the ratio of Hcys/Cys was persistently higher with NaCl addition, indicating a stronger flux of Hcys, which means a promoted methylation to produce Met and SAM. In the process of lincomycin synthesis, SAM is the important methyl donor of lincomycin methylation. Regulation of osmotic stress resulted in the increase of intracellular SAM, which may be the reason of the reduction of lincomycin B production because of enough SAM supply.

The comparison of transcriptomics analysis by RNA-seq at 41 h and 89 h with or without NaCl addition were studied to better understand the influence of osmotic regulation on lincomycin synthesis. 41 h was the beginning of lincomycin production, while 89 h was in the middle production period. According to the transcriptomics analysis, the genes involved in central carbon metabolism, sulfur metabolism, synthesis of low-molecular thiols, lincomycin synthesis gene cluster and oxidation/osmotic stress were the most differentially regulated pathways by osmotic change. The differential expression of carbon metabolism genes showed that the key enzyme changed its expression under osmotic stress. On average, the genes in EMP and HMP were up-regulated while TCA was down-regulated which was good for sulfur metabolism and precursor supplement. The differential transcription research on sulfur metabolism and synthesis of sulfur molecules, such as Cys, SAM, MSH and EGT was studied. And the results showed that genes related tosulfur assimilation pathway were upregulated, as well as the genes in the synthesis of SAM, MSH and EGT. This indicated that osmatic regulation enhanced sulfur assimilation and synthesized more Cys related substances. Cys is the precursor of SAM, MSH and EGT which has a positive influence on the production of lincomycin A and reduction of lincomycin B. The differential expressed genes in the lincomycin synthesis were studied. At 41 h, the 23 structural genes with NaCl addition were up-regulated compared to that of the control, which was consistent with the faster lincomycin production rate at the early stage of fermentation. However, at 89 h, a series of MTL synthesis genes were down-regulated, which might be the reason why the lincomycin production with NaCl addition failed to be further improved at the late stage of antibiotic producing period.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have read and approved the final manuscript.

Availability of data and materials

All data and materials are available in the main text and additional file.

Acknowledgements

We thank State Key Laboratory of Bioreactor Engineering and East China University of Science and Technology.

Author contribution

SL was responsible for data analysis and the draft of this article, ZW was responsible for the design and implement of the experiments, HG was responsible for the funding and revise of this work.

Funding

This work was supported by National Key Research and Development Program of China (2020YFA0907800) and National Natural Science Foundation of China (No. 21506056).

Competing interests

The authors have no conflicts of interests to declare.

Mason DJ, Dietz A, DeBoer C (1962) Lincomycin, a new antibiotic. I. Discovery and biological properties. Antimicrob Agents Ch 1962:554-559.
Kucers A, Crowe SM, Grayson ML, Hoy JF (1997) The use of antibiotics: a clinical review of antibacterial, antifungal and antiviral drugs. Oxford University Press:5.
Horton D, Wander JD, Foltz RL (1974) Chemical-ionization mass spectrometry of Lincomycin and Clindamycin. Anal Biochem 59(2):452-461.
Spížek J, Řezanka T (2004) Lincomycin, cultivation of producing strains and biosynthesis. Appl Microbiol Biot 63(5):510-519.
Ye RF, Wang Q, Zhou XF (2009) Lincomycin, rational selection of high producing strain and improved fermentation by amino acids supplementation. Bioproc Biosyst Eng 32(4):521-529.
Huang WF, Zhang P, Niu C, Bi GD (2017) Breeding of high yield Streptomyces linconinensis producing strain. Chin J of Antibiotics 42:647-651.
Xue ZL, Zhu Y, Zhang XM, Pan WJ (2009) Optimization of culture medium for lincomycin production by response surface methodology. Chin J Antibiotics 34:277-280.
Lee YR, Lee MJ, Chun GT, Jeong YS (2014) Optimization of cultivation medium and fermentation parameters for lincomycin production by Streptomyces lincolnensis. Biotechnol Bioproc E 19:1014-1021.
Zhuang ZH, Zhang LW, Yang CC, Zhu DY, Mao QG, Wang QY, Gao SH (2019) Enhanced lincomycin A production by calcium gluconate feeding in fermentation of Streptomyces lincolnensis. Bioresour Bioprocess 6(1):1-9.
Pang AP, Du L, Lin CY, Qiao J, Zhao GR (2015) Co-overexpression of lmbW and metK led to increased lincomycin A production and decreased byproduct lincomycin B content in an industrial strain of Streptomyces lincolnensis. J Appl Microbiol 119(4):1064-1074.
Xu YR, Tan GQ, Ke ML, Li J, Tang YQ, Meng ST, Niu JJ, Wang YS, Liu RH, Wu H, Bai LQ, Zhang LX, Zhang BC (2018) Enhanced lincomycin production by co-overexpression of metK1 and metK2 in Streptomyces lincolnensis. J Ind Microbiol Biot 45(5):345-355.
Bursy J, Kuhlmann A U, Pittelkow M, Hartmann H, Jebbar M, Pierik AJ, Bremer E (2008) Synthesis and uptake of the compatible solutes ectoine and 5-hydroxyectoine by Streptomyces coelicolor A3(2) in response to salt and heat stresses. Appl Environ Microb 74(23):7286-96.
Tian L, Zhang T, Jiang B (2012) The effect of salt osmotic pressure on the production of erythritol through fermentation of Candida magnoliae SK25.001. Food Ferment Ind 38.10:44-46.
Yang LB, Zhan ZY, Zhan XB (2013) Glycine and proline improvement the production of erythritol from glycerol by Yarrowia lipolytica under high osmotic pressure. Food Ferment Ind 39.12:1-6.
Zhang N, Tian XW, Wang YH, Chu J, Zhang SL (2014) Influence of osmotic stress on fermentative production of L-lactic acid by Lactobacillus paracasei. Sci Technol Food Ind 10: 98-102.
Pospíšil S, Halada P, Petříček M, Sedmera P (2007) Glucosylglycerate is an osmotic solute and an extracellular metabolite produced by Streptomyces caelestis. Folia Microbiol 52.5:451-456.
Thouvenot L, Deleu C, Berardocco S, Haury J, Thiébaut G (2015) Characterization of the salt stress vulnerability of three invasive freshwater plant species using a metabolic profiling approach. J Plant Physiol 175:113-121.
Lee EJ, Karoonuthaisiri N, Kim HS, Park JH, Cha CJ, Kao CM, Roe JH (2005) A master regulator σB governs osmotic and oxidative response as well as differentiation via a network of sigma factors in Streptomyces coelicolor. Mol Microbiol 57(5):1252-1264.
Zhao QF, Wang M, Xu DX, Zhang QL, Liu W (2015) Metabolic coupling of two small-molecule thiols programs the biosynthesis of lincomycin A. Nature 518(7537):115-119.
Kulkarni A, Zeng Y, Zhou W, Lanen SV, Zhang WW, Chen S (2015) A branch point of Streptomyces sulfur amino acid metabolism controls the production of albomycin. Appl Environ Microb 82(2):467-477.
Chiang PK, Gordon RK, Tal J, Zeng GC, Doctor BP, Pardhasaradhi K, McCann PP (1996) S-Adenosylmethionine and methylation. FASEB J 10.4: 471-480.
Neves AA, Pereira DA, Vieira LM, Menezes JC (2000) Real time monitoring biomass concentration in Streptomyces clavuligerus cultivations with industrial media using a capacitance probe. J Biotechnol 84.1:45-52.
Yao K, Gao S, Wu Y, Zhao Z, Wang W, Mao Q (2017) Influence of dextrins on the production of spiramycin and impurity components by Streptomyces ambofaciens. Folia Microbiol 44:1-8.
Li XB, Zhao GR, Zheng H, Yuan YJ (2007) Improved industrial fermentation of lincomycin by phosphorus feeding. Process Biochem 42.4:662-668.
Newton GL, Arnold K, Price MS, Sherrill C, Declardayre SB, Aharonowitz Y, Cohen G, Davies G, Fahey RC, Davis C (1996) Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J Bacteriol 178.7:1990-1995.
Fahey RC, Newton GL (1987) Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Method Enzymol 143.1:85-96.
Chen G, He LB, Luo LF, Huang R, Liao LJ, Li YM, Zhu ZY, Wang YP (2018) Transcriptomics sequencing provides insights into understanding the mechanism of grass carp reovirus infection. Int J Mol Sci 19.2:488.
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9.4:357-359.
Slobodkin AI, Slobodkina GB (2019) Diversity of sulfur-disproportionating microorganisms. Microbiol 88(5), 509-522.

Graphicalabstract.pdf

Download PDF

Version 1

posted

You are reading this latest preprint version

Decreased lincomycin B content by regulating osmotic pressure in fermentation of Streptomyces lincolnensis

Status:

Version 1

Abstract

Figures

Introduction

Media And Methods

Strain and media

Culture condition

Analytical methods

Determination of intracellular thiol amino acids concentration

Determination of intracellular SAM concentration

RNA-seq and gene expression analysis

Results and discussion

Effect of osmotic pressure regulation on lincomycin production in flask

Influence of osmotic pressure regulation on lincomycin production in 15 L bioreactor

Influence of osmotic regulation on fermentation process parameters

Influence of osmotic pressure regulation on the intracellular accumulation of sulfur-containing metabolites

Overview of changes in the transcriptome of Streptomyces lincolnensis in response to osmotic stress

Differential expressed genes involved in central carbon metabolism

Differential expressed genes involved in sulfur metabolism and biosynthesis pathway of MSH and EGT

Differential expressed genes involved in biosynthesis pathway of lincomycin A

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1