Precursor supply for erythromycin biosynthesis: engineering of

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Precursor supply for erythromycin biosynthesis: engineering of propionate assimilation pathway based on propionylation modification Di You, Miao-Miao Wang, Bin-Cheng Yin, and Bang-Ce Ye ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00396 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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Precursor supply for erythromycin biosynthesis: engineering of propionate assimilation pathway based on propionylation modification Di You1, Miao-Miao Wang1, Bin-Cheng Yin1, Bang-Ce Ye1,2,3* 1Laboratory

of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China. 2Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China 3School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, 832000, China

Corresponding author Bang-Ce Ye Professor, Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China Tel/Fax: 0086-21-64252094 Email: [email protected]

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HIGHLIGHTS

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ABSTRACT

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Erythromycin is necessary in the medical treatment and known to be biosynthesized with propionyl-CoA as direct precursor. Over-supply of propionyl-CoA induced hyper propionylation, which was demonstrated harmful for erythromycin synthesis in Saccharopolyspora erythraea. Herein, we identified three propionyl-CoA synthetases regulated by propionylation and one propionyl-CoA synthetase SACE_1780 revealed resistance to propionylation. A practical strategy for raising the precursor (propionylCoA) supply bypassing the feedback inhibition caused by propionylation was developed through two approaches: deletion of the propionyltransferase AcuA and SACE_1780 overexpression. The constructed ΔacuA strain presented a 10 % increase in erythromycin yield, SACE_1780 overexpression strain produced 33 % higher erythromycin yield than the wildtype strain NRRL2338 and 22% higher erythromycin yield than the industrial high yield Ab strain. These findings uncover the role of protein acylation in precursor supply for antibiotics biosynthesis, and provide efficient posttranslational modification-metabolic engineering strategy (named as PTM-ME) in synthetic biology for improvement of secondary metabolites.

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A PTM-ME strategy was developed for antibiotics production. AcuA-catalyzed propionylation repressed the synthesis of propionyl-CoA. SACE_1780 is propionyl-CoA synthetase which is insensitive to propionylation. Engineering of PTM system eliminated the feedback inhibition of propionylation, and improved erythromycin yield.

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Key words: precursor supply, propionate assimilation, propionylation, propionyl-

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CoA synthetase, erythromycin biosynthesis

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Introduction

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Bacteria are widely used in commercial production of multiple important drugs including antibiotics, anticancer agents, and immunosuppressants. Genetical improvement of the production capacity is a critical process before the microorganism used commercially (1). Hence, the adequate availability of precursors supply is efficient target for genetical engineering. Enlargement of precursors pool usually leads to notable increase in production. The metabolic engineering of precursor supply pathways was employed to improve the antibiotics production successfully (2, 3). The main strategies used include carbon flux redirection, overexpression of pathwayspecific regulator, overexpression/deletion of pathway-specific regulator and suppression of competing pathway. As was reported, engineering of the malonyl-CoA supply through carbon flux redirection led to significant increase of the mithramycin yield in Streptomyces argillaceus (4). Similarly, in Streptomyces venezuelae, the manipulation of (2S)-ethylmalonyl-CoA resulted in 10-fold enhancement of tylactone production(2). Other natural products such as flavonoids, alkaloids and polyketides utilize precursors derived from the shikimate pathway. Enhancing the certain flux has been used to increase 2.5-fold production of balhimycin (5). Moreover, introduction or inactivation of the proper gene to tune of the precursor supply is also valid approach for the products yield optimization. Introduction of dptJ encoding for tryptophan 2,3-dioxygenase yielded a 1.1-fold increase in daptomycin production(6). Inactivation of the bafilomycin gene cluster and genes from competing pathways in primary metabolism made a 4-fold increased valinomycin production (7). Saccharopolyspora erythraea (S. erythraea) was widely used for the commercial erythromycin production and erythromycin biosynthesis (8-11). Erythromycin and its derivatives are high-demand in medical treatment. Therefore, the production improved strains are still strongly sought after. The parent macrolide aglycone of erythromycin is 6-deoxyerythronolide B (6-dEB) composed of six condensation steps with a starter propionyl-CoA and six methylmalonyl-CoA extender units (12, 13). Metabolic routes leading to propionyl-CoA and methylmalonyl-CoA in streptomycetes have been the subject of many studies which demonstrate that valine catabolism and propionate metabolism play an important role in providing these acyl-CoA precursors for polyketide biosynthesis. Propionyl-CoA, a metabolite of propionate metabolism, is critical regulatory molecule and metabolic intermediate during erythromycin production in S. erythraea. Cells generate propionyl-CoA from several different processes, including the catabolism of odd chain fatty acids, the decarboxylation of succinate, the catabolism of amino acids and the activation of propionate(14-16). Addition of n-propanol or propionate is usually used to improve the erythromycin

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production by increasing the precursor (propionyl-CoA) supply. This propionate assimilation is mainly catalyzed by acyl-CoA synthetases. Acyl-CoA synthetases are regulated independently and complexly, the intracellular carbon flux, multiple regulators and transcription factors all influence the acyl-CoA synthetases expression (17-19). Furthermore, we found that activities of acyl-CoA synthetases were also regulated at the post-translational level (post-translational modification, PTM) by reversible lysine acetylation in S. erythraea (20). Acylation diversity was wellrecognized as closely associated with multiple physiological and pathological processes. The major donor of acylation, acyl-CoA, is also served as key precursor of various natural product. Acyl-CoA pool and its homeostasis was influenced by nutrient availability, growth stage, energy state, environmental condition, and signal stimulation (21, 22). Acyl-CoA induced acylation is thought as result of carbon overflow caused by imbalance of formation and utilization of acyl-CoA. The acylationmediated inhibition of enzyme activities is a reflection of the oversupply of carbon precursors and is directly related with the decreased yield of natural products (23). The lysine acylation added the potential complexity of regulatory function on metabolic enzymatic activities and metabolic flux distribution. In this study, we mainly investigated the propionylation effects on precursors supply during propionate addition. The supply of propionyl-CoA was beneficial to precursor pools, meanwhile caused propionylation of propionyl-CoA synthetases participated in propionate assimilation pathway, and consequently inhibited erythromycin productivity. Using the PTM_ME strategy, we tried to optimize the propionate assimilation and propionylCoA supply through two approaches: knockout the propionyltransferase AcuA and overexpressed propionylation-insensitive propionyl-CoA synthetase SACE_1780. The results demonstrated that these recombinant strains succeeded in eliminating the feedback inhibition of propionylation and strengthening the synthesis of erythromycin.

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Results and Discussion

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Characterization of the key propionyl-CoA synthetases (Pcs) in response to propionate addition In S. erythraea, propionate assimilation is closely related to erythromycin synthesis. The two precursors of erythromycin synthesis, propionyl-CoA and methylmalonyl-CoA, are the metabolites of propionate utilization. Propionate is activated to propionyl-CoA by acyl-CoA synthetases, genetic analysis showed that S. erythraea possessed eight genes (SACE_0337, SACE_2375, SACE_2408, SACE_2851, SACE_3902, SACE_4729, SACE_3848, SACE_1780) coding for acyl-CoA synthetases involved in propionate assimilation (KEGG database, http://www.kegg.jp) (Fig. 1A). To investigate the

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optimized concentration of propionate addition, growth behavior and erythromycin production in the presence of varying amounts of propionate were determined. The result showed that the growth inhibition raised along with the increasing propionate concentration, while erythromycin production first reached the maximum in 20 mM propionate and then decreased with increase of propionate concentration (Fig. 1B and C).

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Figure 1. Response of S. erythraea to propionate addition. (A) The pathway of propionate catabolism in S. erythraea. (B) Growth curve and Inhibition tests of S. erythraea wild type(WT) strain grown in TSB or TSB supplemented with 20 mM propionate (C) Quantitative analysis of erythromycin production of S. erythraea WT strain grown in TBS medium supplemented with varying amounts of propionate by HPLC determination. Error bars show standard deviation from three independent experiments. **P < 0.01, ***P < 0.001.

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The concentration of 20 mM propionate was then chosen to examine the genes response to propionate addition and the following experiments. We analyzed transcription levels of the former described eight genes by qRT-PCR with the 16S rRNA gene as an internal control. As shown in Figure 2A, the propionate addition strongly induced four gene-expression: SACE_0337 had a 7-fold increase, SACE_1780 had a 30fold increase, SACE_4729 had a 45-fold increase and SACE_3848 had a 3-fold increase. While the other genes had no obvious transcription changes. This result suggested

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that SACE_0337, 4729, 3848 and 1780 were selected to be crucial during the propionate addition. SACE_0337 and SACE_4729 encoded AMP-forming acetyl-CoA synthetases AcsA1 and AcsA3 which were demonstrated to have similar catalytic activity for acetyl-CoA and propionyl-CoA synthesis (20). SACE_1780 and SACE_3848 are predicted acyl-CoA synthetase and propionyl-CoA synthetase, respectively. To identify their catalytic activity of propionyl-CoA synthesis, the activity was expressed in mol NADH min-1 mg-1 of protein using a coupled NADH-consuming spectrophotometric assay as described (24). The result implied that SACE_0337, 4729 and SACE_1780 had similar Pcs activity, and SACE_3848 exhibited the highest specific activity about 2-fold higher than the other three (Fig. 2B).

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Figure 2. Response of Pcs to propionate addition. (A)The transcriptional analysis of the eight genes involved in propionate assimilation of S. erythraea WT strain grown to the middle exponential phase in TSB or TSB supplemented with 20 mM propionate at 30 °C. Fold changes represent the level of expression compared to the expression of SACE_0337 in TSB. Error bars show standard deviation from three independent experiments. Propionylation level of total protein from WT strain grown in TSB medium with or without propionate. Equal amount of total protein (100 mg) was added for each lane. (B) The activity of propionate-induced acyl-CoA synthetases in propionyl-CoA synthesis. Error bars show standard deviation from three independent experiments. **P < 0.01, ***P < 0.001.

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Propionate addition caused significantly hyper propionylation of Pcs enzymes. Our recent studies had shown that excessive accumulation of propionyl-CoA in S. erythraea led to hyper propionylation of enzymes involved in propionate metabolism, which repressed erythromycin synthesis (23). Using the anti-propionylated lysine (anti-PrK) antibody, total propionyl-lysine levels in response to propionate addition were detected. The results showed that propionylation level of total protein was obviously increased during the propionate addition (Fig. 3A). We then examined the propionylation level of the selected four Pcs in vivo. The four enzymes were overexpressed and purified from S. erythraea grown in TSB media with or without propionate, respectively. Equal amount of purified protein was used for the Western blotting (WB) analyses. As shown in Figure 3B, SACE_0337, 4729 and SACE_3848 were

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all propionylated in vivo and propionate addition strongly increased the propionylation levels, while SACE_1780 was little propionylated in vivo and its propionylation presented little response to propionate addition. The overexpressed endogenous three propionylated enzymes were then analyzed by mass spectrometry for identification of propionylated sites. Data were listed in Table 1, the propionylated sites included K372, K497, K501, K511, K603, K620 of SACE_0337; and K196, K367, K492, K506, K598, K615 of SACE_4729; and K177, K247, K352, K533, K553, K583, K588 of SACE_3848.

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Figure 3. Propionate addition resulted in an increase of propionylation level. (A) Propionylation level of total protein from WT strain grown in TSB medium with or without 20 mM propionate. Equal amount of total protein (100 mg) was added for each lane. (B) Propionylation level of overexpressed four propionyl-CoA synthetases from WT strain grown in TSB medium with or without 20 mM propionate. Equal amount of each protein was added for each lane.

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Table 1. The propionylated sites of propionyl-CoA synthetases enzyme

condition

SACE_0337

In vitro

propionylated sites enzymatic (AcuA)

K229, K277, K588, K603, K620

non-enzymatic

K201, K229, K277, K372, K497, K501, K511,

(propionyl-CoA)

K588, K603

In vivo SACE_4729

In vitro

K372, K497, K501, K511, K603, K620 enzymatic (AcuA)

K272, K367, K492, K496, K506, K583, K615

non-enzymatic

K196, K224, K272, K367, K492, K496,

(propionyl-CoA)

K506, K583, K598, K610

In vivo SACE_3848

In vitro

K196, K367, K492, K506, K598, K615 enzymatic (AcuA)

K328, K533, K553, K588

non-enzymatic

K159, K177, K247, K328, K352, K533, K553, 7


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(propionyl-CoA)

K583

In vivo SACE_1780

In vitro

K177, K247, K352, K533, K553, K583, K588 non-enzymatic

K81, K291, K434

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The propionylated sites identified both in vivo and in vitro are indicated in bold, the propionylated sites containing conserved P-X4-GK motif were shown in bold with underline.

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Enzyme-catalyzed propionylation inhibited the activities of SACE_0337, 4729 and

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SACE_3848 except for SACE_1780

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Previous studies indicated that acetylation occurred through enzymatic or non-

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enzymatic reaction, the two approaches revealed different influences on acetyl-CoA

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synthetase activity due to the different ability of acetylating the conserved P-X4-GK

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site (20). To investigate the effect of propionylation on Pcs activity, the activities of

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non-propionylated and propionylated enzymes were determined. We characterized

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in vitro enzymatic propionylation using S. erythrae AcuA and non-enzymatic

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propionylation using high concentrations of propionyl-CoA with recombinant four Pcs

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proteins as substrates respectively. WB analyses were conducted to directly test the

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propionylation status and the Pcs activity was also analyzed. As shown in Figure 4A,

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after incubation with AcuA and propionyl-CoA, SACE_0337, 4729 and SACE_3848 were

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propionylated while SACE_1780 could hardly propionylated by AcuA at this condition.

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Propionylation caused strong activity decrease (>70%) of SACE_0337, 4729 and

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SACE_3848. While SACE_1780 activity showed no significant changes, which was

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consisted with its WB results. The non-enzymatic propionylation was also

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characterized and WB analysis demonstrated that high concentrations of propionyl-

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CoA led to obvious propionylation of SACE_0337, 4729 and SACE_3848 and slight

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propionylation of SACE_1780 (Fig. 4B). Compared with enzymatic propionylation

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catalyzed by AcuA, the non-enzymatic propionylation had a lot less impact on the

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enzyme activity ( 5-fold increase compared with WT stain (Fig. 7A). The growth behavior of O1780 and WT strain in 20 mM propionate was then detected, unlike WT strain, O1780 strain presented significantly better growth even in the presence of propionate (Fig. 7B), suggesting that SACE_1780 overexpression improved cell tolerance to propionate. The intracellular propionyl-CoA concentration was then analyzed by HPLC. As shown in Figure 7C, propionate was shown to directly affect intracellular propionyl-CoA level. In addition, the O1780 strain presented

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relatively higher propionyl-CoA concentration compared with WT strain when grown in same condition, and propionyl-CoA accumulation in O1780 strain had an earlier fast-growth phase. The higher propionyl-CoA level was resulted from overexpression of SACE_1780 whose activity was not repressed by feedback inhibition of propionylation. Intracellular propionyl-CoA was thought to be a rate-limiting step for erythromycin synthesis in S. erythraea, and we speculated whether the erythromycin synthesis increased in O1780. As expected, a notable increment was observed when propionate existed, erythromycin production had a significant 33% increase in O1780 strain (18 mg/L) compared to WT strain (13.5 mg/L) (Fig. 7D). The following global propionylation levels showed that O1780 strain presented lower propionylation level than WT strain in propionate-supplemented medium (Fig. 7E). The reason and mechanism of SACE_1780-overexpression-induced significant growth differences and the lower propionylation level is not clear, we speculated that the concentration of intracellular propionyl-CoA usually maintains low level in which nonenzymatic propionylation could hardly occur. It was reported that the intracellular propionyl-CoA concentration is 5.3 μM, only 0.8% of the acetyl-CoA concentration (610 μM), 2.3% of the succinyl-CoA concentration (230 μM) and 15% of the malonylCoA concentration (35 μM) in E.coli (30). Although there was no systematic report of the intracellular acyl-CoA concentration in actinomycetes yet, our results showed propionyl-CoA concentration of O1780 had only a ~0.06 nmol mg-1DCW increase than the WT strain when grown in the same condition. We thought the changes were too small to affect the propionylation level and the lower propionylation level might due to other uncertain reasons. Furthermore, to validate that the effects of Pcs resistance to propionylation is the key mechanism enhancing erythromycin production in O1780 strain, we investigated erythromycin yield in strain overexpressing SACE_0337. The results in Figure S1 indicated that the growth behavior, propionylation level and erythromycin production of O0337 were similar to WT strain. This result supported that O1780 improved erythromycin production by bypassing inhibition of propionylation. Taken together, compared with ΔacuA, SACE_1780 overexpression could eliminate the feedback inhibition of propionylation and much more efficiently improve the synthesis of erythromycin. We finally estimated the feasibility of this strategy applicated in industrial production, SACE_1780 was overexpressed in the industrial high yield Ab strain and erythromycin production was detected. After 7 days fermentation in 50 ml ABPM8 production medium(31) supplemented with propionate, HPLC tests revealed that a 14% increase of erythromycin produced in AbO1780 strain than AbWT under ABPM8 media (Fig. 7F) and a more sizable 22% increase in AbO1780 strain over AbWT when supplemented with 12



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propionate (Fig. 7F). This result further confirmed that the PTM-ME strategy presented an achievable mechanism for improving natural product biosynthesis.

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Figure 7. SACE_1780 overexpression strengthens the synthesis of erythromycin. (A) The transcriptional analysis of SACE_1780 of S. erythraea WT and O1780 strains grown to the middle exponential phase in TSB medium at 30 °C. Fold changes represent the level of expression compared to the expression of SACE_1780 in WT. (B) Growth curve of S. erythraea WT and O1780 strains grown in TSB or TSB supplemented with 20 mM propionate. (C) The determination of propionyl-CoA concentration in WT and O1780 strains grown in TSB medium supplemented with propionate or not. (D) Quantitative analysis of erythromycin production of S. erythraea WT and O1780 grown in TBS medium supplemented with propionate or not by HPLC determination. (E) 13


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Propionylation level of total protein from WT and O1780 strains grown in TSB medium with or without propionate. Equal amount of total protein (100 mg) was added for each lane. (F) Quantitative analysis of erythromycin production of S. erythraea high-yield strain AbWT and AbO1780 grown in ABPM8 production medium supplemented with propionate or not by HPLC determination. Error bars show standard deviation from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

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Conclusion. In this study, we found that propionate addition induced hyper-propionylation of three key Pcs (SACE_0337, 4729, and 3848) responsible for propionyl-CoA synthesis. Propionylation inactivated these Pcs enzymes, which resulted in the repression of erythromycin synthesis (feedback inhibition). The detected propionylation occurred through two distinct approaches: enzymatic propionylation catalyzed by AcuA and non-enzymatic propionylation under high concentration of propionyl-CoA. These two mechanisms revealed significant differences in propionylation sites. The conserved sites critical for catalytic activities of Pcs were only propionylated via enzymatic propionylation, indicating the key role of AcuA in Pcs activity regulation. Consequently, knockout of acuA contributed to a 10% increase in erythromycin yields. Differing from propionylation-regulated Pcs, SACE_1780 revealed propionylation resistance. The engineered O1780 strain succeeded in a 33% higher erythromycin production than WT strain NRRL2338 and a 22% higher than the industrial Ab strain. These results set good examples of PTM-ME strategy for enhancing biosynthesis of natural products (Fig. 8). Therefore, further design and optimization about the appropriate fermentation conditions are still necessary. Acylation is considered as consequences of excess acyl-CoA derived from carbon overflow. Our research here reveals that artificial modification of the acylation system could in turn affect intracellular acyl-CoA pool and production of secondary metabolites. This practical PTM-ME strategy is designed for balancing intracellular generation/consumption of acyl-CoA, maintaining sufficient acyl-CoA supply and contributing to the output increases. The engineering methods include regulating the activity of acyltransferase /deacylase, adjusting the acylation level of key sites in metabolic enzymes, replacing or deleting the acylation-sensitive metabolic enzymes. We believed that PTM-ME strategy would have great potential for improving the biosynthesis of natural products.

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Figure 8. PTM-ME model showed propionate assimilation and propionyl-CoA pool in S. erythraea

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WT, ΔacuA, and O1780 strains.

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Methods

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Bacterial strains, growth conditions, and reagents All strains and plasmids used in this work are listed in Table S1. Eight S.erythraea strains, the wild-type NRRL2338 (from DSM 40517), O0337 strain, O4729 strain, O3848 strain, O1780 strain, ΔacuA strain, industrial high yield AbWT strain and AbO1780 strain were used in this study. And the S.erythraea genetic techniques have been described previously(28). S. erythraea strains were grown in TSB medium (20). Aerobic 100 ml batch cultures were grown in 1 L flasks at 30 °C on a rotary shaker at 200 rpm. Cultures were inoculated to an optical density (OD600) of 0.05 units with exponentially growing precultures. Propionyl lysine antibody was from PTM Biolabs, Inc.

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RNA preparation and real-time RT-PCR RNA preparation and RT-PCR were performed as previously described (20). Overproduction and purification of proteins All genes were amplified by PCR from the genomic DNA of S.erythraea. The primers used in this work are listed in Supporting Information Table S2 and (20). After 15


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restriction digest, the genes coding for SACE_3848 and SACE_1780 were cloned into pET-28a. The proteins were expressed using E. coli BL21 (DE3) strain. Purification of proteins was performed as previously described (20).

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Overexpression of His-1780 from S. erythraea Using S. erythraea genomic DNA as template, the SACE_1780 gene was amplified by PCR. The PCR product was digested with EcoRV and NdeI, then inserted into the corresponding sites of integrative plasmid pIB139(20, 32), yielding pIB1780. By PEGmediated protoplast transformation, plasmids of pIB1780 was introduced into the S. erythraea wild type strain and industrial high yield Ab strain(31). The overexpression strains were obtained by apramycin resistance screening and confirmed by PCR analysis with the primers apr-F and apr-R (20). The purification of overexpressed protein was performed as previously described (20).

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In vitro propionyl-CoA synthetase assays

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The activity of propionyl-CoA synthetase was expressed in mol NADH min-1 mg-1 of protein using a coupled NADH-consuming spectrophotometric assay as described (33).

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In vitro enzymatic protein propionylation In vitro enzymatic propionylation assays were performed as previously described (28, 33, 34). Purified protein (5 μM) was incubated with GST-AcuA (0.2 μM, (28)) and 20 μM propionyl-CoA(Sigma) in HEPES buffer (50 mM, pH 7.5) containing 200 μM tris(2carboxyethyl) phosphine hydrochloride. After 2 h incubation at 37 °C, samples were divided into two portions: one portion was analyzed by SDS-PAGE and Western blot, and the other was used for measurement of the propionyl-CoA synthetase activity. The propionylated protein were isolated by SDS-PAGE and analyzed by LC-MS/MS. In vitro non-enzymatic protein propionylation Purified protein (5 μM) were incubated with 600 μM propionyl-CoA(Sigma) in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl for 6 h at 37 °C (35, 36). After the incubation, samples were divided into two portions: one was analyzed by SDS-PAGE and Western blot, and the other was used for measurement of propionyl-CoA synthetase activity. The propionylated protein were isolated by SDS-PAGE and analyzed by LC-MS/MS. Western blot analysis Western blot analysis was performed as previously described (20). Anti-propionyllysine antibody (hereafter Anti-PrK) diluted 1:5000 in TBST was used. Mass spectrometry peptide fingerprinting Protein digestion was performed according to the FASP procedure described by Wisniewski et al (37). Tryptic digests (approximately 30 μg of pre-digested protein) were solid phase extracted and analyzed by mLC–MS/MS using a Micromass (Waters) Q exactive spectrometer (Thermo Finnigan, San Jose, CA) to locate protein 16



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propionylation sites. The details were described in our previous work (20).

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Fermentation and erythromycin determination Flask fermentation of wild type and mutant strains was cultivated at 30 °C. For bioassay-based erythromycin determination (38), 5 μl fermentation supernatants were added onto LB agar plates sprayed with overnight culture of B. subtilis 168 and incubated at 37 °C for 12 h. The erythromycin titer was evaluated by detecting and measuring the growth-inhibition zones. HPLC analysis for erythromycin production was performed as previously described (39).

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Propionyl-CoA assay For analysis of propionyl-CoA concentration in S. erythraea, intracellular propionylCoA assay was performed as previously described (40-47). Flask fermentation of wild or mutant strains were grown at 30 °C for 48 h, 2 ml culture was collected and centrifuged for 10 min at 4 °C, cells were washed twice with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KHPO4). Cells were resuspended in 800 μl lysis buffer (10% trichloroacetic acid, 90% ddH2O, 2 mM DTT) followed by freeze-thaw cycles until full lysis. The supernatant was then added into Sep-pak column (Waters, 1cc, 50 mg tC18 Cartridge) and washed with 1.2 ml 0.1% TFA/H2O. Propionyl-CoA was eluted by 1.2 ml 0.1% TFA and 40% ACN, freeze-drying (lyophilization) technique was utilized with Speed Vac. Samples were directly analyzed by HPLC after re-dissolving in acetonitrile. Acyl-CoA esters was separated using linear gradient elution, and detection was performed at a wavelength of 254 nm with an injection volume of 10 µl.

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Author Information

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Corresponding Author Bang-Ce Ye Professor, Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China Tel./Fax: 0086-21-64252094. E-mail: [email protected]

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Author Contributions Di You and Bang-Ce Ye designed the research; Di You performed research with the help of Miao-miao Wang; Di You, Miao-miao Wang and Bang-Ce Ye contributed new reagents/analytic tools; Di You and Bang-Ce Ye analyzed data and wrote the manuscript. 17


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Notes The authors declare no competing financial interest.

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Acknowledgements

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This work was supported by grants from the National Natural Science Foundation of China (31730004, 31700058, and 21575089), the China Postdoctoral Science Foundation Funded Project (2017M610232) and the Fundamental Research Funds for the Central Universities (222201714025). The authors have no conflict of interest to declare.

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