Applied Microbiology and Biotechnology

, Volume 103, Issue 5, pp 2155–2170 | Cite as

Evolutionary engineering of Escherichia coli for improved anaerobic growth in minimal medium accelerated lactate production

  • Baowei Wang
  • Xiaoxia Zhang
  • Xinlei Yu
  • Zhenzhen Cui
  • Zhiwen Wang
  • Tao ChenEmail author
  • Xueming Zhao
Biotechnological products and process engineering


Anaerobic fermentation is a favorable process for microbial production of bulk chemicals like ethanol and organic acids. Low productivity is the bottleneck of several anaerobic processes which has significant impact on the technique competitiveness of production strain. Improving growth rate of production strain can speed up the total production cycle and may finally increase productivity of anaerobic processes. In this work, evolutionary engineering of wild-type strain Escherichia coli W3110 was adopted to improve anaerobic growth in mineral medium. Significant increases in exponential growth rate and stationary cell density were achieved in evolved strain WE269, and a 96.5% increase in lactate productivity has also been observed in batch fermentation of this strain with M9 minimal medium. Then, an engineered strain for lactate production (BW100) was constructed by using WE269 as a platform and 98.3 g/L lactate (with an optical purity of D-lactate above 95%) was produced in a 5-L bioreactor after 48 h with a productivity of 2.05 g/(L·h). Finally, preliminary investigation demonstrated that mutation in sucD (sucD M245I) (encoding succinyl-CoA synthetase); ilvG (ilvG Δ1bp) (encoding acetolactate synthase 2 catalytic subunit), and rpoB (rpoB T1037P) (encoding RNA polymerase β subunit) significantly improved anaerobic growth of E. coli. Double-gene mutation in ilvG and sucD resumed most of the growth potential of evolved strain WE269. This work suggested that improving anaerobic growth of production host can increase productivity of organic acids like lactate, and specific mutation-enabled improved growth may also be applied to metabolic engineering for production of other bulk chemicals.


Evolutionary engineering Escherichia coli Anaerobic growth Productivity sucD rpoB Lactate 



We thank Prof. Qinghong Wang (TIB, CAS) for his kind gift of wild-type strain of this study. We also thank Dr. Zhubo Dai (TIB, CAS) and Yufeng Mao (TJU, China) for discussion on whole genome resequencing analysis.

Funding information

This work was supported by the National Natural Science Foundation of China (NSFC-21776208, NSFC- 21621004, NSFC-21776209 and NSFC-21390201).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9588_MOESM1_ESM.pdf (801 kb)
ESM 1 (PDF 800 kb)


  1. Assavasirijinda N, Ge D, Yu B, Xue Y, Ma Y (2016) Efficient fermentative production of polymer-grade D-lactate by an engineered alkaliphilic Bacillus sp. strain under non-sterile conditions. Microb Cell Factories 15(1):3. Google Scholar
  2. Baek SH, Kwon EY, Kim YH, Hahn JS (2016) Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 100(6):2737–2748. Google Scholar
  3. Barrick JE, Yu DS, Yoon SH, Jeong H, Oh TK, Schneider D, Lenski RE, Kim JF (2009) Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 461(7268):1243–1247. Google Scholar
  4. Barrick JE, Colburn G, Deatherage DE, Traverse CC, Strand MD, Borges JJ, Knoester DB, Reba A, Meyer AG (2014) Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq. BMC Genomics 15:1039. Google Scholar
  5. Clark DP (1989) The fermentation pathways of Escherichia coli. FEMS Microbiol Rev 5(3):223–234Google Scholar
  6. Conrad TM, Joyce AR, Applebee MK, Barrett CL, Xie B, Gao Y, Palsson BO (2009) Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations. Genome Biol 10(10):R118. Google Scholar
  7. Conrad TM, Frazier M, Joyce AR, Cho BK, Knight EM, Lewis NE, Landick R, Palsson BO (2010) RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media. Proc Natl Acad Sci U S A 107(47):20500–20505. Google Scholar
  8. Deatherage DE, Barrick JE (2014) Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol Biol (Clifton, NJ) 1151:165–188. Google Scholar
  9. Feng X, Ding Y, Xian M, Xu X, Zhang R, Zhao G (2014) Production of optically pure d-lactate from glycerol by engineered Klebsiella pneumoniae strain. Bioresour Technol 172:269–275. Google Scholar
  10. Finn TJ, Shewaramani S, Leahy SC, Janssen PH, Moon CD (2017) Dynamics and genetic diversification of Escherichia coli during experimental adaptation to an anaerobic environment. PeerJ 5:e3244. Google Scholar
  11. Fong SS, Burgard AP, Herring CD, Knight EM, Blattner FR, Maranas CD, Palsson BO (2005) In silico design and adaptive evolution of Escherichia coli for production of lactic acid. Biotechnol Bioeng 91(5):643–648. Google Scholar
  12. Forster AH, Gescher J (2014) Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products. Front Bioeng Biotechnol 2:16. Google Scholar
  13. Fu X, Wang Y, Wang J, Garza E, Manow R, Zhou S (2017) Semi-industrial scale (30 m(3)) fed-batch fermentation for the production of D-lactate by Escherichia coli strain HBUT-D15. J Ind Microbiol Biotechnol 44(2):221–228. Google Scholar
  14. Grabar TB, Zhou S, Shanmugam KT, Yomano LP, Ingram LO (2006) Methylglyoxal bypass identified as source of chiral contamination in l(+) and d(−)-lactate fermentations by recombinant Escherichia coli. Biotechnol Lett 28(19):1527–1535. Google Scholar
  15. Guzman GI, Utrilla J, Nurk S, Brunk E, Monk JM, Ebrahim A, Palsson BO, Feist AM (2015) Model-driven discovery of underground metabolic functions in Escherichia coli. Proc Natl Acad Sci U S A 112(3):929–934. Google Scholar
  16. Harden MM, He A, Creamer K, Clark MW, Hamdallah I, Martinez KA 2nd, Kresslein RL, Bush SP, Slonczewski JL (2015) Acid-adapted strains of Escherichia coli K-12 obtained by experimental evolution. Appl Environ Microbiol 81(6):1932–1941. Google Scholar
  17. He A, Penix SR, Basting PJ, Griffith JM, Creamer KE, Camperchioli D, Clark MW, Gonzales AS, Chavez Erazo JS, George NS, Bhagwat AA, Slonczewski JL (2017) Acid evolution of Escherichia coli K-12 eliminates amino acid decarboxylases and reregulates catabolism. Appl Environ Microbiol 83(12).
  18. Herring CD, Raghunathan A, Honisch C, Patel T, Applebee MK, Joyce AR, Albert TJ, Blattner FR, van den Boom D, Cantor CR, Palsson BO (2006) Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale. Nat Genet 38(12):1406–1412. Google Scholar
  19. Horinouchi T, Sakai A, Kotani H, Tanabe K, Furusawa C (2017) Improvement of isopropanol tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies. J Biotechnol 255:47–56. Google Scholar
  20. Hua Q, Joyce AR, Fong SS, Palsson BO (2006) Metabolic analysis of adaptive evolution for in silico-designed lactate-producing strains. Biotechnol Bioeng 95(5):992–1002. Google Scholar
  21. Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF (1987) Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol 53(10):2420–2425Google Scholar
  22. Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res 38(6):e92–e92. Google Scholar
  23. LaCroix RA, Sandberg TE, O'Brien EJ, Utrilla J, Ebrahim A, Guzman GI, Szubin R, Palsson BO, Feist AM (2015) Use of adaptive laboratory evolution to discover key mutations enabling rapid growth of Escherichia coli K-12 MG1655 on glucose minimal medium. Appl Environ Microbiol 81(1):17–30. Google Scholar
  24. Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang YJ, Chen T, Zhao X (2015) Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab Eng 31:13–21. Google Scholar
  25. Liang S, Gao D, Liu H, Wang C, Wen J (2018) Metabolomic and proteomic analysis of D-lactate-producing Lactobacillus delbrueckii under various fermentation conditions. J Ind Microbiol Biotechnol
  26. Liu M, Han X, Xian M, Ding Y, Liu H, Zhao G (2016) Development of a 3-hydroxypropionate resistant Escherichia coli strain. Bioengineered 7(1):21–27. Google Scholar
  27. Lu H, Zhao X, Wang Y, Ding X, Wang J, Garza E, Manow R, Iverson A, Zhou S (2016) Enhancement of D-lactic acid production from a mixed glucose and xylose substrate by the Escherichia coli strain JH15 devoid of the glucose effect. BMC Biotechnol 16:19. Google Scholar
  28. Maddamsetti R, Hatcher PJ, Green AG, Williams BL, Marks DS, Lenski RE (2017) Core genes evolve rapidly in the long-term evolution experiment with Escherichia coli. Genome Biol Evol 9:1072–1083. Google Scholar
  29. Morris JG (1983) Anaerobic fermentations—some new possibilities. Biochem Soc Symp 48:147–172Google Scholar
  30. Niu D, Tian K, Prior BA, Wang M, Wang Z, Lu F, Singh S (2014) Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth. Microb Cell Factories 13:78. Google Scholar
  31. Okino S, Suda M, Fujikura K, Inui M, Yukawa H (2008) Production of D-lactic acid by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 78(3):449–454. Google Scholar
  32. Portnoy VA, Bezdan D, Zengler K (2011) Adaptive laboratory evolution—harnessing the power of biology for metabolic engineering. Curr Opin Biotechnol 22(4):590–594. Google Scholar
  33. Qi X, Zha J, Liu GG, Zhang W, Li BZ, Yuan YJ (2015) Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae. Front Microbiol 6:1165. Google Scholar
  34. Sauer U (2001) Evolutionary engineering of industrially important microbial phenotypes. Adv Biochem Eng Biotechnol 73:129–169Google Scholar
  35. Shui ZX, Qin H, Wu B, Ruan ZY, Wang LS, Tan FR, Wang JL, Tang XY, Dai LC, Hu GQ, He MX (2015) Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors. Appl Microbiol Biotechnol 99(13):5739–5748. Google Scholar
  36. Tan Z, Chen J, Zhang X (2016) Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production. Biotechnol Biofuels 9:262. Google Scholar
  37. Thompson AD, Bernard SM, Skiniotis G, Gestwicki JE (2012) Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK). Cell Stress Chaperones 17(3):313–327. Google Scholar
  38. Tian K, Niu D, Liu X, Prior BA, Zhou L, Lu F, Singh S, Wang Z (2016) Limitation of thiamine pyrophosphate supply to growing Escherichia coli switches metabolism to efficient d-lactate formation. Biotechnol Bioeng 113(1):182–188. Google Scholar
  39. Valgepea K, Adamberg K, Seiman A, Vilu R (2013) Escherichia coli achieves faster growth by increasing catalytic and translation rates of proteins. Mol BioSyst 9(9):2344–2358. Google Scholar
  40. Wang Y, Tian T, Zhao J, Wang J, Yan T, Xu L, Liu Z, Garza E, Iverson A, Manow R, Finan C, Zhou S (2012) Homofermentative production of D-lactic acid from sucrose by a metabolically engineered Escherichia coli. Biotechnol Lett 34(11):2069–2075. Google Scholar
  41. Wang ZW, Saini M, Lin LJ, Chiang CJ, Chao YP (2015) Systematic engineering of Escherichia coli for d-lactate production from crude glycerol. J Agric Food Chem 63(43):9583–9589. Google Scholar
  42. Wang J, Lin M, Xu M, Yang ST (2016) Anaerobic fermentation for production of carboxylic acids as bulk chemicals from renewable biomass. Adv Biochem Eng Biotechnol 156:323–361. Google Scholar
  43. Weusthuis RA, Lamot I, van der Oost J, Sanders JP (2011) Microbial production of bulk chemicals: development of anaerobic processes. Trends Biotechnol 29(4):153–158. Google Scholar
  44. Winkler JD, Kao KC (2014) Recent advances in the evolutionary engineering of industrial biocatalysts. Genomics 104(6 Pt A):406–411. Google Scholar
  45. Wu SG, He L, Wang Q, Tang YJ (2015) An ancient Chinese wisdom for metabolic engineering: Yin-Yang. Microb Cell Factories 14(1):39. Google Scholar
  46. Xu K, Lv B, Huo YX, Li C (2018) Toward the lowest energy consumption and emission in biofuel production: combination of ideal reactors and robust hosts. Curr Opin Biotechnol 50:19–24. Google Scholar
  47. Zeikus JG (1980) Chemical and fuel production by anaerobic bacteria. Annu Rev Microbiol 34:423–464. Google Scholar
  48. Zhang X, Jantama K, Moore JC, Jarboe LR, Shanmugam KT, Ingram LO (2009) Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli. Proc Natl Acad Sci U S A 106(48):20180–20185. Google Scholar
  49. Zhang H, Yang J, Wu S, Gong W, Chen C, Perrett S (2016) Glutathionylation of the bacterial Hsp70 chaperone DnaK provides a link between oxidative stress and the heat shock response. J Biol Chem 291(13):6967–6981.
  50. Zhao T, Liu D, Ren H, Shi X, Zhao N, Chen Y, Ying H (2014) D-lactic acid production by Sporolactobacillus inulinus Y2-8 immobilized in fibrous bed bioreactor using corn flour hydrolyzate. J Microbiol Biotechnol 24(12):1664–1672Google Scholar
  51. Zhou S, Causey TB, Hasona A, Shanmugam KT, Ingram LO (2003) Production of optically pure d-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl Environ Microbiol 69(1):399–407. Google Scholar
  52. Zhou S, Grabar TB, Shanmugam KT, Ingram LO (2006) Betaine tripled the volumetric productivity of D(−)-lactate by Escherichia coli strain SZ132 in mineral salts medium. Biotechnol Lett 28(9):671–676. Google Scholar
  53. Zhu J, Shimizu K (2004) The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. Appl Microbiol Biotechnol 64(3):367–375. Google Scholar
  54. Zhu J, Shimizu K (2005) Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition. Metab Eng 7(2):104–115. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biochemical Engineering, School of Chemical Engineering and TechnologyTianjin UniversityTianjinPeople’s Republic of China
  2. 2.SynBio Research PlatformCollaborative Innovation Center of Chemical Science and Engineering (Tianjin)TianjinPeople’s Republic of China
  3. 3.Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and TechnologyTianjin UniversityTianjinPeople’s Republic of China

Personalised recommendations