The correlation between the polyhydroxybutyrate content and ascomycin production
Various microorganisms are known to accumulated PHB , but its presence in S. hygroscopicus var. ascomyceticus was uncertain. Here, the parent strain FS35 was fermented for 8 days to investigate the change trends of PHB contents and residual sugars during the entire fermentation (Figure 1b). In strain FS35, the PHB content gradually increased along with the decrease of residual sugars during the first four days. This suggested that when carbon sources are abundant in the environment, PHB is accumulated as intracellular carbon storage granules. This was consistent with the study of PHB in a typical PHB-producing strain Ralstonia eutropha H16 . However, in strain FS35, the PHB content was gradually reduced when residual sugar was already at low level during the last four days (Figure 1b). This implied that when residual sugar was insufficient at the stationary phase, PHB was depolymerized as an alternate carbon source. This change trend was different from that in R. eutropha H16, PHB content in R. eutropha H16 gradually increased, and then remained at a high level until the end of fermentation . So it is speculated that in S. hygroscopicus var. ascomyceticus, the decrease of PHB content during the later fermentation stages might have other use.
To understand the temporal correlation between the PHB content, FK520 yield and strain biomass, fermentation characters of FS35 were recorded and analysis. As can be seen in Figure 1c, when the PHB content increased rapidly during the exponential phase, the biomass also increased correspondingly. Because both PHB accumulation and biomass increase require the consumption of carbon sources , so the synthesis of PHB might be associated with the growth of strain. Excitingly, FK520 yield rapidly increased when the PHB content gradually decreased during the stationary phase (Figure 1c). This indicated that the decomposition of PHB during the stationary phase might be conducive to the synthesis of FK520.
The promoting effect of polyhydroxybutyrate metabolism on strain growth and ascomycin production
In order to explore the influence of PHB metabolism on FK520 production, the PHB synthesis gene should theoretically be knocked out to observe the changes of fermentation characteristics. However, previous studies had shown that the deletion of PHB synthesis gene might cause a series of problems such as low growth rate, high rate of reverse mutation and deficient phenotype of sporulation [22, 32]. These indicate that PHB metabolism may be not a useless cycle, but plays an indispensable role in maintaining the strain growth. Moreover, the sequence of PHB synthesis gene is rarely revealed in Streptomyces, and it is unknown in S. hygroscopicus var. ascomyceticus. Therefore, this paper did not consider inactivating the PHB synthesis gene, but overexpressed the PHB degradation gene fkbU in the parent strain FS35 to construct the overexpressed strain OfkbU. However, the FK520 production in the strain OfkbU was not significantly improved (Additional file 1: Figure S2). This might because that in the strain OfkbU, the amount of PHB synthesized in the exponential phase was not increased, so there were no more PHB available for degradation during stationary phase. Previous studies reported that the synthase and depolymerase of PHB are simultaneously expressed in most PHB-accumulating strains, but their activities are stringently regulated to avoid ineffective circulation [33, 34]. Therefore, the combined overexpression of the PHB synthesis gene and decomposition gene in strain FS35 was deemed a reasonable approach. The PHB synthesis gene (phaC) in R. eutropha H16 had been widely studied and used . Therefore, the exogenous PHB synthesis gene (phaC) and the native PHB decomposition gene (fkbU) were co-overexpressed in the parent strain FS35 to construct the co-overexpression strain OphaCfkbU.
The parent strain FS35 and co-overexpressed strain OphaCfkbU were simultaneously fermented for 192 h to observe the changes of fermentation parameters caused by the co-overexpression (Figure 2a-d). Compared to the parent strain FS35, strain OphaCfkbU consumed more sugar and accumulated more PHB during the exponential phase (Figure 2a). This indicated that the increase of PHB biosynthesis promoted the consumption of carbon sources. Surprisingly, strain OphaCfkbU accumulated more biomass during the exponential phase than strain FS35 (Figure 2b). And the mycelia of strain OphaCfkbU in the fermentation broth at 96h and on plate culture at 20d were all stronger than that of strain FS35 (Figure 2d). This indicated that the PHB biosynthesis was beneficial to the growth of strain. A previous report had shown that in Rhodospirillum rubrum S1, PHB could promote the conversion of acetate into biomass . Accordingly, PHB accumulation might promote the conversion of starch and dextrin into biomass in S. hygroscopicus var. ascomyceticus.
As expected, in strain OphaCfkbU, the more abundant supply of PHB during the exponential phase ensured the more decomposition of PHB during stationary phase (Figure 2c). This confirmed that the co-overexpression operation in strain OphaCfkbU did not cause an ineffective circulation, but instead promoted the accumulation of PHB during the exponential phase as well as its decomposition during the stationary phase. And this resulted in an increased of FK520 yield to 511.50 mg/L, 1.73-fold higher than that of strain FS35 (296.29 mg/L). This was the first time that PHB was found to promote the biosynthesis of antibiotics. And this means that the accumulation and depolymerization of PHB may be a very valuable cycle for the production of the secondary metabolites.
Transcriptomic evidences of the stimulating effect of polyhydroxybutyrate on strain growth
In order to fully verify the function of PHB as a carbon reservoir, the different patterns of PHB metabolism during the exponential phase and stationary phase were proved firstly through qRT-PCR. In Streptomyces hygroscopicus var. ascomyceticus, gene hcd is responsible for the synthesis of PHB monomer, and gene fkbE is responsible for the dehydration of PHB monomer (Figure 1a). Considering that the genes phaC and fkbU were co-overexpressed in this study, the different patterns of PHB metabolism during the exponential phase and stationary phase were testified by the comparative transcription analysis of genes hcd and fkbE (Figure 3a). During the exponential phase, the expression level of gene hcd in strain OphaCfkbU was higher than that in strain FS35, while the expression level of gene fkbE was lower. This indicated that 3-hydroxybutyryl-CoA was used more to synthesize PHB than dehydrate to crotonyl-CoA during the exponential phase. On the contrary, during the stationary phase, the expression level of gene hcd in strain OphaCfkbU was lower than that in strain FS35, while the expression level of gene fkbE was higher. This reflected that during the stationary phase, the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA was mainly dependent on the depolymerization of PHB rather than the supply of acetoacetyl-CoA. These results demonstrated that PHB was indeed stored during the exponential phase and decomposed for reuse during the stationary phase. And the time difference between the accumulation and degradation ensured the double promotion of PHB on strain growth and FK520 production.
To provide further evidence of the stimulating effect of PHB metabolism on strain growth, comparative transcriptomic analysis between strain FS35 and OphaCfkbU was carried out using RNA samples drawn at 50h. Differential expression analysis revealed 285 up and 218 down-regulated genes in strain OphaCfkbU compared to strain FS35 (Figure 3b). These differentially expressed genes were clustered using GO enrichment analysis into three categories (biological process, cellular component and molecular function) (Figure 3c and 3d). The up-regulated genes were mostly related to the biosynthesis and metabolism of organic substances, which are necessary for cell growth (Figure 3c). The down-regulated genes were mainly involved in the biosynthesis of heterocyclic organics, which is unfavorable for biomass accumulation (Figure 3d). These results provided evidences for the stimulating effect of PHB on strain growth in both positive and negative aspects.
Furthermore, the significantly up-regulated genes in strain OphaCfkbU were mainly mapped to six metabolic pathways according to the Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis (Table 1). The up-regulation of genes in the starch and sucrose metabolism confirmed the promoting effect of PHB accumulation on the utilization of sugar. The up-regulation of genes in the ABC transport system reflected that PHB could not only accelerate the transport of sugar sources to provide more raw material for intracellular biosynthesis, but also provide more metal ions as cofactors for various enzymes. The up-regulation of genes in the ribosome biosynthesis pathway confirmed that PHB could increase the generation of ribosomes, which are key organelles responsible for translation. The up-regulation of genes in the pantothenate and coenzyme A biosynthesis pathway proved that PHB could promote the biosynthesis of coenzyme A and acyl-carrier protein (ACP), which participate in the metabolism of carbohydrates, fatty acids and energy . The up-regulation of genes in the nicotinate and nicotinamide metabolism indicated that PHB could increase the biosynthesis of cofactors (NADH, NAD+, NADPH, NADP+), which are the important components of redox enzymes in the glycolysis pathway (EMP), citrate cycle (TCA) and electron transport chain . The up-regulation of genes in sulfur metabolism could improve the generation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and sulfide. PAPS is a sulfate donor for many sulfating reactions and sulfide is an important material for the biosynthesis of cysteine . This indicated that the synthesis of PHB could promote the creation of sulfur-containing compounds and cysteine. All these data proved that PHB could stimulate the growth of strain by promoting the utilization of carbon sources for biosynthesis processes. As an intracellular carbon reservoir, PHB not only stored carbon sources in the form of polymer, but also drove the flow of carbon sources to biomass.
The mechanism underlying the promoting effect of polyhydroxybutyrate on ascomycin production
In order to explore the influence mechanism of PHB depolymerization on FK520 biosynthesis, crucial genes in the primary metabolic pathways and all genes in the FK520 gene cluster were selected for the comparative analysis of transcription levels between FS35 and OphaCfkbU at 112 h (Figure 4). Compared to the parent strain FS35, the expression of fkbU in strain OphaCfkbU was up-regulated significantly as expected (Figure 4a). Moreover, the expression of all the selected genes in EMCP was also up-regulated significantly in this strain. This indicated that the degradation of PHB regulated the metabolic flow of EMCP to increase the biosynthesis of precursor ethylmalonyl-CoA and methylmalonyl- CoA. The mut and aceB genes encode the enzymes connecting EMCP with TCA. The up-regulation of their expression in strain OphaCfkbU promoted the conversion of metabolites between EMCP and TCA. The genes gltA, idh and korB encode key enzymes of TCA . The up-regulation of their expression in strain OphaCfkbU indicated that the TCA was strengthened on account of the enhancement of PHB metabolism. Oxaloacetate in the TCA is not only the source of the precursor pipecolate for FK520, but also the junction between the TCA and EMP. The up-regulated expression of fkbL and pkcA in strain OphaCfkbU indicated that the enhancement of the TCA promoted the conversion of oxaloacetate into precursor pipecolate and intermediate phosphoenolpyruvate. The latter increased the synthesis of precursor 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC), which was reflected by the up-regulated expression of the gene fkbO in strain OphaCfkbU. The up and down-regulated expression of selected genes in the EMP suggested that the enhancement of interconversion between the metabolites in TCA and EMP also regulated the metabolic flow of EMP. Furthermore, the up-regulated expression of the genes fkbG, fkbH, fkbI, fkbJ and fkbK in strain OphaCfkbU indicated that the synthesis of the precursor methoxymalonyl-ACP was also increased. All the data above illustrated that PHB could increase the biosynthesis of FK520 precursors by regulating the metabolic flows of the EMCP, TCA and EMP. On this basis, the expression of genes responsible for different functions in the FK520 gene cluster was also up-regulated accordingly to different degrees (Figure 4b). This confirmed that the enhancement of PHB metabolism increased the production of FK520 by promoting the biosynthesis of precursors.
FK523 is the main impurity in the production of FK520, resulting from the assembly of methylmalonyl-CoA onto the C21 position of macrolide skeleton instead of the specific precursor ethylmalonyl-CoA . To rule out competitive use of these precursors by by-products, the yield of FK523 and the ratio of FK523/FK520 in the strain FS35 and OphaCfkbU was recorded as shown in Figure 4c. Although the yield of FK523 in the strain OphaCfkbU was higher than that in the parent strain FS35, the ratio of FK523/FK520 was decreased slightly. This indicated that the increase of precursors was mainly beneficial to the synthesis of FK520 but not FK523. This might because that the degradation of PHB promoted the synthesis of ethylmalonyl-CoA more than methylmalonyl-CoA, which could be demonstrated by the change folds of genes fkbE (3.98), fkbS (4.12), ecm (3.35) and bccA (3.27) (Figure 4a). Previous study also showed that the overexpression of fkbS could promote the yield of FK520 but decrease the production of FK523 by increasing the synthesis of ethylmalonyl-CoA . Here, the priority supply of PHB to the specific precursor ethylmalonyl-CoA guaranteed the increase of FK520 production was not affected by the by-products.
The influence of cofactor concentration on polyhydroxybutyrate metabolism
To further develop the value of PHB as the intracellular carbon reservoir, the factors influencing the synthesis of PHB deserved to be explored. For the biosynthesis of PHB, the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA is an important step, which catalyzed by the NADH-dependent 3-hydroxybutyryl-CoA dehydrogenase (encoding by hcd) or the NAD(P)H-dependent acetoacetyl-CoA reductase (encoding by phaB) (Figure 5a). The NAD(P)H pool and the ratio of NAD(P)H/NAD(P)+ could significantly influence the content of PHB [40, 41]. The sequence alignment showed that there is a copy of hcd gene in the genome of S. hygroscopicus var. ascomyceticus. (Additional file 1: Figure S3), but no copy of phaB gene. Thus it was inferred that the synthesis of PHB in S. hygroscopicus var. ascomyceticus might be influenced by the pool of NADH and the ratio of NADH/NAD + (Figure 5b). To test this inference, the concentrations of NADH, NAD+, NADPH, NADP+ and the ratio of NAD +/NADH, NADPH/NADP+ in strain FS35 and OphaCfkbU were measured at the end of the exponential phase.
Compared with strain FS35, there were higher concentrations of NADH + NAD+ and NADPH + NADP+ in strain OphaCfkbU (Figure 5d). This reflected that the increase of PHB biosynthesis during the exponential phase promoted the biosynthesis of these cofactors. This was consistent with the transcriptome data above (Table. 1). The concentration of NAD+ in strain OphaCfkbU was higher than that in strain FS35 at the end of the exponential phase, and the concentration of NADH in strain OphaCfkbU was lower (Figure 5c). So, the ratio of NAD+/NADH in strain OphaCfkbU was significantly higher than that in strain FS35 (Figure 5d). This indicated that in OphaCfkbU, the increase of PHB biosynthesis during the exponential phase accelerated the consumption rate of NADH. On the contrary, the concentration of NADPH in strain OphaCfkbU was higher than that in strain FS35, but the concentration of NADP+ in strain OphaCfkbU was lower at the end of the exponential phase (Figure 5c). So, the ratio of NADPH/NADP+ in strain OphaCfkbU was significantly higher than that in strain FS35 (Figure 5d). This indicated that the production rate of NADPH in OphaCfkbU was increased during the exponential phase. This might because the increase of PHB synthesis in the exponential phase stimulated the carbon flux of pentose phosphate pathway (PPP) (Figure 5b). Since the NADPH is an essential reducing power in the assembly of FK520 precursors [42, 43], so the high production rate of NADPH also gave a reasonable explanation for the high yield of FK520 in the strain OphaCfkbU. On the whole, all the data above mean that the synthesis of PHB during the exponential phase depended on the conversion of NADH to NAD+. Therefore, increasing the intracellular concentration of NADH might be an effective strategy to further enhance the PHB metabolism.
Further increase of the ascomycin yield by strengthening the polyhydroxybutyrate metabolism
Although PHB was proved could increase the synthesis of cofactors (NADH, NAD+, NADPH, NADP+) by stimulating the utilization of carbon sources (Table 1), the total concentrations of these cofactors in the strain OphaCfkbU was only slightly higher than that in strain FS35 (Figure 5d). This might be caused by the insufficient of residual sugar at the later stage of exponential phase (Figure 2a). So the optimization of carbon source addition was carried out to explore its effects on NADH pool, the PHB accumulation and the FK520 production. As the main carbon sources in the fermentation medium, the addition of starch and dextrin was first optimized in single-factor experiments (Figure 6a and 6b). When the addition of starch increased to 24 g/L, the FK520 yield reached up to 576.14 mg/L, increased by 64.64 mg/L than that in the initial fermentation medium (511.50 mg/L) (Figure 6a). When the addition of dextrin increased to 56 g/L, the FK520 yield reached up to 602.73 mg/L, increased by 91.23 mg/L than that in the initial fermentation medium (511.50 mg/L) (Figure 6b). Then, the mixed addition of starch and dextrin were optimized in two-factor experiments. When the addition of starch and dextrin increased to 22 g/L and 52 g/L respectively, the FK520 production reached up to 626.30 mg/L, 1.22-fold higher than that in the initial medium (511.50 mg/L) and 2.11-fold higher than that of the parent strain FS35 in the initial medium (296.29 mg/L) (Figure 6c). Thus, 22 g/L starch and 52 g/L dextrin were determined as the optimal concentrations of carbon sources for the strain OphaCfkbU to produce FK520.
In the optimal fermentation medium, strain OphaCfkbU possessed a higher concentration of cofactor pool at the end of the exponential phase (Figure 6f). And the amount of NADH converted to NAD+ was increased during the exponential phase, even though a slightly decrease of conversion rate (Figure 6e and 6f). Furthermore, strain OphaCfkbU also possessed the more PHB content in the optimal fermentation medium (Figure 6d). These reflected that the addition of carbon sources enhanced the NADH-dependent PHB accumulation. Meanwhile, the amount of NADP+ converted to NADPH was also increased during the exponential phase (Figure 6e), providing more reducing power for the synthesis of FK520 during the stationary phase. On the whole, the addition of carbon sources leaded to more accumulation of PHB and more production of FK520, playing the role of carbon reservoir to a greater extent.