PccD Regulates Branched-Chain Amino Acid Degradation and Exerts a Negative Effect on Erythromycin Production in Saccharopolyspora erythraea
PccD Regulates Branched-Chain Amino Acid Degradation and Exerts a Negative Effect on Erythromycin Production in Saccharopolyspora erythraea
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PccD 调节支链氨基酸降解并对红糖多孢菌中的红霉素产生产生负面影响
DOI：
10.1128/aem.00049-18
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发表时间：
2018-02
影响因子：
4.4
通讯作者：
Ye Bang-Ce
中科院分区：
文献类型：
--
作者：
Xu Zhen;Liu Yong;Ye Bang-Ce
摘要 支链氨基酸 (BCAA) 降解是丙酰辅酶 A (丙酰-CoA) 的主要来源，丙酰辅酶 A 是红霉素生物合成的关键前体。在这项研究中，我们发现负责 BCAA 降解的 bkd 操纵子直接受到 PccD（丙酰辅酶 A 羧化酶基因的转录调节因子）的调节。 bkd 操纵子的转录水平在 pccD 基因缺失菌株（ΔpccD 菌株）中上调 5 倍，在 pccD 过表达菌株（WT/pIB-pccD）中降低 3 倍，表明 PccD 是操纵子的负转录调节因子。在以 30 mM 缬氨酸作为唯一碳源和氮源的基础埃文斯培养基中，pccD 的缺失显着提高了 ΔpccD 菌株的生长速率，而 pccD 过表达抑制了 WT/pIB-pccD 的生长速率。 gdhA1 和 BcdhE1 基因（bkd 操纵子中的基因）的缺失分别导致 ΔgdhA1 和 ΔBcdhE1 菌株在 30 mM 缬氨酸上的生长速率降低，进一步表明 bkd 操纵子参与 BCAA 降解。 bkd 过表达（WT/pIB-bkd）和 pccD 失活（ΔpccD 菌株）均提高了红霉素产量（分别为 38% 和 64%），而菌株 WT/pIB-pccD 的红霉素产量减少了 48%。最后，我们探讨了工程化pccD和bkd在工业高产红霉素菌株中的应用。与工业发酵培养基中的红霉素 S. erythraea E3 相比，工业菌株 S. erythraea E3 (E3pccD) 中的 pccD 缺失使红霉素产量提高了 20%，而 E3ΔpccD (E3ΔpccD/pIB-bkd) 中 bkd 的过表达使红霉素产量提高了 39%。在工业发酵培养基中添加30 mM缬氨酸进一步将红霉素产量提高了23%，比初始菌株S. erythraea E3提高了72%。重要性 我们描述了参与 S. erythraea 中 BCAA 降解的 bkd 操纵子。操纵子的基因受到 TetR 调节因子 PccD 的抑制。结果表明，PccD通过调节BCAA降解和丙酰辅酶A同化来控制红霉素生物合成前体的供应，并对红霉素生产产生负面影响。这些发现揭示了饲养途径中的调节机制，并为设计代谢工程以提高红霉素产量提供了新策略。
ABSTRACT Branched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis in Saccharopolyspora erythraea. In this study, we found that the bkd operon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of the bkd operon was upregulated 5-fold in a pccD gene deletion strain (ΔpccD strain) and decreased 3-fold in a pccD overexpression strain (WT/pIB-pccD), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion of pccD significantly improved the ΔpccD strain's growth rate, whereas pccD overexpression repressed WT/pIB-pccD growth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion of gdhA1 and the BcdhE1 gene (genes in the bkd operon) resulted in lower growth rates of ΔgdhA1 and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that the bkd operon is involved in BCAA degradation. Both bkd overexpression (WT/pIB-bkd) and pccD inactivation (ΔpccD strain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB-pccD was decreased by 48%. Lastly, we explored the applications of engineering pccD and bkd in an industrial high-erythromycin-producing strain. pccD deletion in industrial strain S. erythraea E3 (E3pccD) improved erythromycin production by 20%, and the overexpression of bkd in E3ΔpccD (E3ΔpccD/pIB-bkd) increased erythromycin production by 39% compared with S. erythraea E3 in an industrial fermentation medium. Addition of 30 mM valine to industrial fermentation medium further improved the erythromycin production by 23%, a 72% increase from the initial strain S. erythraea E3. IMPORTANCE We describe a bkd operon involved in BCAA degradation in S. erythraea. The genes of the operon are repressed by a TetR regulator, PccD. The results demonstrated that PccD controlled the supply of precursors for biosynthesis of erythromycin via regulating the BCAA degradation and propionyl-CoA assimilation and exerted a negative effect on erythromycin production. The findings reveal a regulatory mechanism in feeder pathways and provide new strategies for designing metabolic engineering to increase erythromycin yield.
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影响因子：
3.2
作者：
K. T. Madhusudhan;G. Huang;G. Burns;J. R. Sokatch
通讯作者：
K. T. Madhusudhan;G. Huang;G. Burns;J. R. Sokatch
DOI：
10.7164/antibiotics.39.1304
发表时间：
1986-09
期刊：
The Journal of antibiotics
影响因子：
--
作者：
H. Yamamoto;K. Maurer;C. Hutchinson
通讯作者：
H. Yamamoto;K. Maurer;C. Hutchinson
影响因子：
1.2
作者：
C. Wilkinson;Z. Hughes-Thomas;Christine J. Martin;I. Böhm;T. Mironenko;M. Deacon;M. Wheatcroft;G. Wirtz;J. Staunton;P. Leadlay
通讯作者：
C. Wilkinson;Z. Hughes-Thomas;Christine J. Martin;I. Böhm;T. Mironenko;M. Deacon;M. Wheatcroft;G. Wirtz;J. Staunton;P. Leadlay
DOI：
10.1016/0006-2944(81)90026-0
发表时间：
1981-01-01
期刊：
BIOCHEMICAL MEDICINE
影响因子：
--
作者：
GREGERSEN, N
通讯作者：
GREGERSEN, N
影响因子：
3.2
作者：
DENOYA, CD;FEDECHKO, RW;WERNAU, WC
通讯作者：
WERNAU, WC