食品与发酵工业 >

2020 , Vol. 46 >Issue 19: 17 - 22

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.023524

研究报告

强化乙醛酸和TCA循环对克雷伯氏菌合成1,3-丙二醇的影响

  • 谢梦梦 ,
  • 胡会涛 ,
  • 陆信曜 ,
  • 宗红 ,
  • 诸葛斌
展开
  • 1(糖化学与生物技术教育部重点实验室(江南大学),江苏 无锡,214122);
    2(工业生物技术教育部重点实验室(江南大学),江苏 无锡,214122);
    3(江南大学,工业微生物研究中心,江苏 无锡,214122)
第一作者:硕士研究生(诸葛斌教授为通讯作者,E-mail:bzhuge@163.com)

收稿日期: 2020-02-05

  修回日期: 2020-02-29

  网络出版日期: 2020-11-02

收起

Strengthening glyoxylate bypass and the TCA cycle improved 1,3-propanediol production in Klebsiella pneumoniae

  • XIE Mengmeng ,
  • HU Huitao ,
  • LU Xinyao ,
  • ZONG Hong ,
  • ZHUGE Bin
Expand
  • 1(Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China);
    2(The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, School of Biotechnology,Wuxi 214122, China);
    3(Research Centre of Industrial Microbiology, Jiangnan University, Wuxi 214122, China)

Received date: 2020-02-05

  Revised date: 2020-02-29

  Online published: 2020-11-02

Fold

摘要

克雷伯氏菌(Klebsiella pneumoniae)代谢甘油合成1,3-丙二醇(1,3-propanediol,1,3-PDO)的过程中,存在乙酸溢流、TCA循环活性低的问题,影响1,3-PDO合成。该研究对K. pneumoniae中乙醛酸循环抑制因子iclR进行敲除,并过表达TCA循环中琥珀酸脱氢酶基因sdhC和苹果酸脱氢酶基因mdh,缓解乙酰辅酶A节点的碳流溢出。结果表明,敲除iclR后乙酸积累量降低了41%,1,3-PDO产量提高了8%。在K. p Lric中单独过表达sdhC或mdh基因,2,3-丁二醇产量分别降低了47%和52%,1,3-PDO产量分别提高了7%和8%。共表达sdhC和mdh基因后,菌株的甘油利用能力增强,1,3-PDO产量比K. p Lric提高了11%。5 L发酵罐分批补料发酵结果表明,K. p Lric-sdhC-mdh的生物量明显提高,1,3-PDO产量达77.2 g/L,摩尔转化率为0.69 mol/mol。以上结果表明,敲除iclR激活乙醛酸循环、过表达sdhC和mdh强化TCA循环可以弱化乙酸溢流,增强菌株的甘油利用能力,促进合成1,3-PDO。

关键词： iclR; sdhC; mdh; 乙醛酸循环; TCA循环; 克雷伯氏菌; 1-3-丙二醇

本文引用格式

谢梦梦 , 胡会涛 , 陆信曜 , 宗红 , 诸葛斌 . 强化乙醛酸和TCA循环对克雷伯氏菌合成1,3-丙二醇的影响[J]. 食品与发酵工业, 2020 , 46(19) : 17 -22 . DOI: 10.13995/j.cnki.11-1802/ts.023524

Abstract

Acetate overflow and low tricarboxylic acid cycle (TCA cycle) activity inhibit the bioproduction of 1,3-propanediol from glycerol by Klebsiella pneumoniae. To relieve the overflow in acetyl-CoA node, iclR encoding isocitrate lyase regulator was deleted, sdhC encoding succinate dehydrogenase and mdh encoding malate dehydrogenase were overexpressed. Deletion of iclR decreased the accumulation of acetate by 41% and increased the 1,3-propanediol titer by 8%. Further overexpression of sdhC or mdh decreased the accumulation of 2,3-butanediol by 47% or 52% and improved the yield of 1,3-propanediol by 7% or 8%. Co-expression of sdhC and mdh improved the glycerol consumption and increased the 1,3-propanediol titer by 11%. The biomass of K. pneumoniae Lric-sdhC-mdh was significantly improved and the 1,3-propanediol titer reached 77.2 g/L with a molar yield of 0.69 mol/moL in fed-batch fermentation. These results indicated that activating glyoxylate bypass by disruption of iclR and reinforcement of the TCA cycle by overexpression of sdhC and mdh decrease acetate accumulation and increase glycerol consumption, which are favorable for the synthesis of 1,3-propanediol.

Key words： iclR; sdhC; mdh; glyoxylate bypass; TCA cycle; Klebsiella pneumoniae; 1,3-propanediol

参考文献

[1] 祃栋猛,陆信曜,陈文强,等.发酵后期补加2种氮源对克雷伯氏菌合成1,3-丙二醇的影响[J].食品与发酵工业, 2016, 42(8): 8-12.
[2] VIVEK N, CHRISTOPHER M, KUMAR M K, et al.Pentose rich acid pretreated liquor as co-substrate for 1,3-propanediol production[J]. Renewable Energy, 2018, 129:794-799.
[3] TANG T, QI F, LIU H J, et al.Recent developments in the microbial production of 1,3-propanediol[J]. Biofuels, 2013, 4(6): 651-667.
[4] RHIE M N, KIM H T, JO S Y, et al.Recent advances in the metabolic engineering of Klebsiella pneumoniae: a potential platform microorganism for biorefineries[J]. Biotechnology and Bioprocess Engineering, 2019, 31(6): 945-961.
[5] KUMAR V, SANKARANARAYANAN M, DURGAPAL M, et al.Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of the lactate dehydrogenase-deficient recombinant Klebsiella pneumoniae overexpressing an aldehyde dehydrogenase[J]. Bioresource Technology, 2013, 135: 555-563.
[6] XU Y Z, GUO N N, ZHENG Z M, et al.Metabolism in 1,3-propanediol fed-batch fermentation by a D-lactate deficient mutant of Klebsiella pneumoniae[J]. Biotechnology & Bioengineering, 2009, 104(5): 965-972.
[7] CELINSKA E.Klebsiella spp as a 1, 3-propanediol producer: the metabolic engineering approach[J]. Critical Reviews in Biotechnology, 2012, 32(3):274-288.
[8] ZHOU S, LI L L, PERSEKE M, et al.Isolation and characterization of a Klebsiella pneumoniae strain from mangrove sediment for efficient biosynthesis of 1,3-propanediol[J]. Science Bulletin, 2015, 60(5): 511-521.
[9] KO Y, SEOL E, SUNDARA S B, et al.Metabolic engineering of Klebsiella pneumoniae J2B for co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol: Reduction of acetate and other by-products[J]. Bioresource Technology, 2017, 244(1):1 096-1 103.
[10] PEEBO K, VALGEPEA K, NAHKU R, et al.Coordinated activation of PTA-ACS and TCA cycles strongly reduces overflow metabolism of acetate in Escherichia coli[J]. Applied Microbiology and Biotechnology, 2014, 98(11): 5 131-5 143.
[11] 梁川,王洁茹,诸葛斌,等.基于回补途径的TCA循环改造对克雷伯氏菌生长和甘油代谢的影响[J].应用与环境生物学报, 2019, 25(4):972-976.
[12] ANAND P,SAXEAN R K,MARWAH R G.A novel downstream process for 1,3-propanediol from glycerol-based fermentation[J].Applied Microbiology and Biotechnology,2011,90(4):1 267-1 276.
[13] LIU M, DING Y M, CHEN H L, et al.Improving the production of acetyl-CoA-derived chemicals in Escherichia coli BL21(DE3) through iclR and arcA deletion[J]. BMC Microbiology, 2017, 17(1):10.
[14] DING Z X, FANG Y, ZHU L F, et al.Deletion of arcA, iclR, and tdcC in Escherichia coli to improve L-threonine production[J]. Biotechnology and Applied Biochemistry, 2019, 66(5): 794-807.
[15] LU X Y, REN S L, LU J Z, et al.Enhanced 1,3-propanediol production in Klebsiella pneumoniae by a combined strategy of strengthening the TCA cycle and weakening the glucose effect[J]. Journal of Applied Microbiology, 2018, 124(3): 682-690.
[16] KUMAR V, PARK S.Potential and limitations of Klebsiella pneumoniae as a microbial cell factory utilizing glycerol as the carbon source[J]. Biotechnology Advances, 2018, 36(1): 150-167.
[17] JIANG Y, CHEN B, DUAN C L, et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system[J]. Applied and Environmental Microbiology, 2015, 81(7): 2 506-2 514.
[18] 刘情,王小婉,诸葛斌,等.基因敲除弱化产 1,3-丙二醇 Klebsiella pneumoniae 荚膜多糖的合成[J].化工进展, 2017, 36(9): 3 447-3 452.
[19] MOLINA-HENARES A J, KRELL T, GUAZZARONI M E, et al. Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors[J]. FEMS Microbiology Reviews, 2006, 30(2):157-186.
[20] YAMAMOTO K, ISHIHAMA A.Two different modes of transcription repression of the Escherichia coli acetate operon by IcIR[J]. Molecular Microbiology, 2003, 47(1):183-194.
[21] WAEGEMAN H, BEAUPREZ J, MOENS H, et al.Effect of iclR and arcA deletions on physiology and metabolic fluxes in Escherichia coli BL21(DE3)[J]. Biotechnology Letters, 2012, 34(2): 329-337.
[22] YANG T H, RATHANSINGH C, LEE H J, et al.Identification of acetoin reductases involved in 2,3-butanediol pathway in Klebsiella oxytoca[J]. Journal of Biotechnology, 2014, 172(1): 59-66.
[23] LIN J, ZHANG Y Q, XU D F, et al.Deletion of poxB, pta, and ackA improves 1,3-propanediol production by Klebsiella pneumoniae[J]. Applied Microbiology and Biotechnology, 2016, 100(6):2 775-2 784.
[24] LEE J H, JUNG H M, JUNG M Y, et al.Effects of gltA and arcA mutations on biomass and 1,3-propanediol production in Klebsiella pneumoniae[J]. Biotechnology and Bioprocess Engineering, 2019, 24(1): 95-102.
[25] BAO W, WEI R, LIU X, et al.Regulation of pyruvate formate lyase-deficient Klebsiella pneumoniae for efficient 1,3-propanediol bioproduction[J]. Current Microbiology, 2020, 77(1): 55-61.
[26] ZHOU J D, WANG D X, WANG C H, et al.The role of the pyruvate acetyl-CoA switch in the production of 1,3-propanediol by Klebsiella pneumoniae[J]. Applied Biochemistry and Biotechnology, 2017, 181(3): 1 199-1 210.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司