Expression of recombinant proteins in Lactococcus lactis – an application in D-tagatose production

Noora Salonen

Research output: ThesisDoctoral ThesisCollection of Articles


Lactococcus lactis is a commercially attractive host for recombinant protein production with many potential applications, for example, in the food and pharmaceutical industries. Production of recombinant proteins requires a suitable expression system, and the aim of this study was to develop new inducible recombinant protein expression systems for L. lactis. In addition, a special application for a developed expression system in D-tagatose production was investigated. D-Tagatose is a functional low-calorie sweetener, but the production costs still limit its use. Two new protein expression systems were developed, of which the first was based on a phosphate starvation-inducible pstF promoter of L. lactis MG1363. High expression levels of intracellular β-galactosidase (670 µkat g-1) and secreted α-amylase (3.6 μkat l-1) were achieved using this strictly regulated expression system. The other new protein expression system was based on the salt-inducible BusA promoter and the BusR repressor gene of L. lactis MG1363. To achieve salt-inducible protein expression, the BusR expression was adjusted by generating random mutations to its promoter area. In the bioreactor, a 6.0 μkat l-1 α-amylase activity was reached, however, without strict regulation. Inducing agents were not needed with either expression system in the bioreactor. Due to strict regulation and high expression levels, the phosphate starvation-inducible expression system was chosen to overexpress Bifidobacterium longum L-arabinose isomerase. The purified enzyme was characterized, and it turned out to be active and stable at acidic pH (optimum 6.0-6.5), and optimally active at 55 °C. The Km values were 120 mM and 590 mM, and Vmax values were 42 U mg-1 and 7.7 U mg-1, for L-arabinose and D-galactose, respectively. The enzyme bound the metal cofactors (Mg2+, Ca2+) tightly, but the metal ion requirement was low for catalytic activity. Divalent metal ions, preferably Mg2+, were required for enzyme stability at higher temperatures. Because of the promising characteristics of the enzyme and high expression level, the use of resting L. lactis cells harboring B. longum L-arabinose isomerase to produce D-tagatose was investigated. Optimization analysis showed high pH, temperature, and borate concentration favored D-tagatose production. Almost quantitative conversion (92 %) was reached in a borate buffer after five days (20 g l-1 D-galactose, 37.5 °C). D-Tagatose production was also investigated with high substrate concentration (300 g l-1 D-galactose, 1.15 M borate, 41 °C) in a 10-day batch process, changing the production medium every 24 h. A D-tagatose production rate of 185 g l-1 day-1 was achieved, comparable with reported numbers in the literature. The results indicated that the use of resting cells stabilized the enzyme. There was no loss in productivity during the ten sequential batches. To further develop the D-tagatose production process, the cells could be immobilized in a packed-bed bioreactor to enable continuous production. Developing the new expression systems to be food-grade could enable more applications.
Translated title of the contributionRekombinanttiproteiinien tuotto Lactococcus lactis -bakteerissa – sovelluksena D-tagatoosin tuotto
Original languageEnglish
QualificationDoctor's degree
Awarding Institution
  • Aalto University
  • Frey, Alexander, Supervising Professor
  • Nyyssölä, Antti, Thesis Advisor
Print ISBNs978-952-64-1533-8
Electronic ISBNs978-952-64-1534-5
Publication statusPublished - 2023
MoE publication typeG5 Doctoral dissertation (article)


  • lactococcus lactis
  • recombinant protein expression
  • D-tagatose
  • bifidobacterium longum
  • L-arabinose isomerase


Dive into the research topics of 'Expression of recombinant proteins in Lactococcus lactis – an application in D-tagatose production'. Together they form a unique fingerprint.

Cite this