Strain improvement for fermentation and biocatalysis processes by genetic engineering technology

  • Shu-Jen ChiangEmail author
Review Paper


Twenty years ago, the first complete gene cluster encoding the actinorhodin biosynthetic pathway was cloned and characterized. Subsequently, the gene clusters encoding the biosynthetic pathways for many antibiotics were isolated. In the past decade, breakthroughs in technology brought that generation of rationally designed or new hybrid metabolites to fruition. Now, the development of high-throughput DNA sequencing and DNA microarray techniques enables researchers to identify the regulatory mechanisms for the overproduction of secondary metabolites and to monitor gene expression during the fermentation cycle, accelerating the rational application of metabolic pathway engineering. How are the new tools of biotechnology currently being applied to improve the production of secondary metabolites? Where will this progress lead us tomorrow? The use of whole cells or partially purified enzymes as catalysts has been increased significantly for chemical synthesis in pharmaceutical and fine-chemical industries. The development of PCR technologies for protein engineering and DNA shuffling is leading to the generation of new enzymes with increased stability to a wide range of pHs, temperatures and solvents and with increased substrate specificity, reaction rate and enantioselectivity. Where will this emerging technology lead us in the twenty-first century?


Biocatalysis Enzyme engineering Fermentation Pathway engineering Secondary metabolites 


  1. 1.
    Cohen SN, Chang A, Boyer H, Helling R (1973) Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci USA 70:3240–3244Google Scholar
  2. 2.
    Jackson DA, Symons RH, Berg P (1972) Biochemical method for inserting new genetic information into DNA of simian virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc Natl Acad Sci USA 69:2904–2909PubMedGoogle Scholar
  3. 3.
    Hopwood DA, Malpartida F, Kieser HM, Ikeda H, Duncan J, Fujii I, Rudd BAM, Floss HG, Omura S (1985) Production of ‘hybrid’ antibiotics by genetic engineering. Nature 314:642–644PubMedGoogle Scholar
  4. 4.
    Miller JH, Reznikoff WS (1980) The operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  5. 5.
    Fujii T, Gramajo HC, Takano E, Bibb MJ (1996) redD and actII-ORF4, pathway specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing σhrdD. J Bacteriol 178:3402–3405PubMedGoogle Scholar
  6. 6.
    Wezel GP van, White J, Hoogvliet G, Bibb MJ (2000) Application of redD, the transcriptional activator gene of the undecylprodigiosin biosynthesis pathway, as reporter for transcriptional activity in Streptomyces coelicolor A3(2) and Streptomyces lividans. J Mol Biotechnol 2:551–556Google Scholar
  7. 7.
    Demain L, Aharonowitz Y, Martin J-F (1984) Metabolic control of secondary biosynthetic pathways. In: Vining LC (ed) Biochemistry and genetic regulation of commercially important antibiotics. Addison–Wesley, Cambridge, Mass., pp 49–72Google Scholar
  8. 8.
    Basch J, Chiang S-J (1998) Genetic engineering approach to reduce undesirable by-products in cephalosporin C fermentation. J Ind Microbiol Biotechnol 20:344–353Google Scholar
  9. 9.
    Skatrud PL, Tietz AJ, Ingolia TD, Cantwell CA, Fisher, DL, Chapman JL, Queener SW (1989) Use of recombinant DNA to improve production of cephalosporin C by Cephalosporium acremonium. Bio/Technology 7:477–485Google Scholar
  10. 10.
    Usher JJ, Hughes DW, Lewis MA, Chiang S-J (1992) Determination of the rate-limiting step(s) in the biosynthetic pathways leading to penicillin and cephalosporin. J Ind Microbiol 10:157–163PubMedGoogle Scholar
  11. 11.
    Ikeda H, Omura S (1995) Control of avermectin biosynthesis in Streptomyces avermitilis for the selective production of a useful component. J Antibiot 48:549–562Google Scholar
  12. 12.
    Stutzman-Engwall K, Conlon S, Fedechko R, Kaczmarek F, McArthur H, Krebber A, Chen Y, Minshull J, Raillard SA, Gustafasson C (2003) Engineering the aveC gene to enhance the ratio of doramectin to its CHC-B2 analogue produced in Streptomyces avermitilis. Biotechnol Bioeng 82:359–369CrossRefPubMedGoogle Scholar
  13. 13.
    Cantwell CA, Beckman RJ, Dotzlaf JE, Fisher DL, Skatrud PL, Yeh WH, Queener SW (1990) Cloning and expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum. Curr Genet 17:213–221Google Scholar
  14. 14.
    Crawford L, Stepan AM, McAda PC, Rambosek JA, Conder MJ, Vinci VA, Reeves CD (1995) Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Biotechnology 13:58–62PubMedGoogle Scholar
  15. 15.
    Sutherland J, Bovenberg R, Lann J van der (1997) Improved process for the production of semi-synthetic cephalosporins via expandase activity on penicillin G. World patent WO 97/20,053Google Scholar
  16. 16.
    Pfeifer BA, Khosla C (2001) Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev 65:106–118PubMedGoogle Scholar
  17. 17.
    Chotani G, Dodge T, Hsu A, Kumar M, LaDuca R, Trimbur D, Weyler D, Sanford K (2000) The commercial production of chemicals using pathway engineering. Biochim Biophys Acta 1543:434–455PubMedGoogle Scholar
  18. 18.
    Gatenby AA, Haynie SL, Nagara-Jan V, Nair RV, Nakamura CE, Payne MS, Picataggio SK, Dias-Torres M, Hsu AK-H, Lareau RD, Trimbur DE, Whited GM (1998) Methods for the production of 1,3-propanediol by recombinant organisms. World patent WO 98/21,339Google Scholar
  19. 19.
    Hopwood DA (1997) Genetic contributions to understanding polyketide synthases. Chem Rev 97:2465–2497PubMedGoogle Scholar
  20. 20.
    Stassi DL, Kakavas SJ, Reynolds KA, Gunawardana G, Swanson S, Zeidner D, Jackson M, Liu H, Buko A, Katz L (1998) Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering. Proc Natl Acad Sci USA 95:7305–7309CrossRefPubMedGoogle Scholar
  21. 21.
    Chiang S-J, Chang LT, Chen YS, Hou HH, Elander RP (1991) Strain improvement in Penicillium chrysogenum, from classical genetics to genetic engineering. In: Kleinkauf H, Dohren H von (eds) 50 years of penicillin applications—history and trends. Public Press, Bratislava, pp 245–257Google Scholar
  22. 22.
    Chiang S-J, Basch J (1999) Cephalosporins. In: Flickinger MC, Drew SW (eds) Bioprocess technology: fermentation, biocatalysis and bioseparation, vol 1. Wiley, New York, pp 560–570Google Scholar
  23. 23.
    Baldwin JE, Goh K-C, Schofield CJ (1992) Oxidation of deacetylcephalosporin C by deacetoxycephalosporin C/deacetylcephalosporin C synthase. J Antibiot 45:1378–1381PubMedGoogle Scholar
  24. 24.
    Chiang S-J, Tonzi S, Burnett WB (1996) Penicillin V amidohydrolase gene from Fusarium oxysporum. US patent 5,516,679Google Scholar
  25. 25.
    Reetz MT, Zonta A, Schimossek K, Liebeton K, Jaeger K-E (1997) Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution. Angew Chem Int Ed Engl 36:2830–2832Google Scholar
  26. 26.
    Liebeton K, Zonta A, Schimossek K, Nardini M, Lang D, Dijkstra BW, Reetz MT, Jaeger K-E (2000) Directed evolution of an enantioselective lipase. Chem Biol 7:709–718PubMedGoogle Scholar
  27. 27.
    Stemmer WPC (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91:10747–10751PubMedGoogle Scholar
  28. 28.
    Crameri A, Raillard S-A, Bermudez E, Stemmer WPC (1998) DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391:288–291PubMedGoogle Scholar
  29. 29.
    Coco WM, Levinson WE, Crist MJ, Kektor HJ, Darzins A, Pienkos PT, Squires CH, Monticello DJ (2001) DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat Biotechnol 19:354–359PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2004

Authors and Affiliations

  1. 1.Fermentation and Biocatalysis Development, Technical OperationsBristol-Myers Squibb CompanySyracuseUSA

Personalised recommendations