Tài liệu Báo cáo khoa học: Control analysis as a tool to understand the formation of the las operon

Control analysis as a tool to understand the formation

of the las operon in Lactococcus lactis

Brian Koebmann, Christian Solem and Peter Ruhdal Jensen

Microbial Physiology and Genetics, BioCentrum-DTU, Technical University of Denmark, Kgs Lyngby, Denmark

Over the last three decades increasing attention has

been paid to how metabolic pathways are controlled.

Metabolic control analysis [1,2] has been applied suc￾cessfully to determine the flux control of many single

enzymes [3–7], but much less attention has been paid

to determine flux control by individual enzymes

cotranscribed in prokaryotic operons.

In Lactococcus lactis, an industrially important

organism used extensively in the fermentation of dairy

products, the three glycolytic enzymes phosphofructo￾kinase (PFK), pyruvate kinase (PK) and lactate dehy￾drogenase (LDH) are clustered in the so-called las

operon [8]. This organization of glycolytic genes is

unique and has given rise to speculation that the three

enzymes might play an important role in the control

and regulation of lactic acid production by this organ￾ism. We have previously shown that small changes

in the activity of PFK result in pronounced changes in

metabolite pools, glycolytic flux and growth rate in

L. lactis, but control by PFK has not been quantified

[9]. LDH was shown to have no control over either

growth or glycolytic flux at wild-type levels, but a

strong negative control over the minor flux to mixed

acids via pyruvate formate lyase (PFL) [10].

In this study, the activities of PFK and PK were

modulated individually by changing expression of the

Keywords

glycolysis; Lactococcus; las; metabolic

control analysis; operon

Correspondence

P. R. Jensen, Microbial Physiology and

Genetics, BioCentrum-DTU, Technical

University of Denmark, Building 301,

DK-2800 Kgs. Lyngby, Denmark

Tel: +45 4525 2510

Fax: +45 4593 2809

E-mail: [email protected]

(Received 23 December 2004, revised 28

February 2005, accepted 9 March 2005)

doi:10.1111/j.1742-4658.2005.04656.x

In Lactococcus lactis the enzymes phosphofructokinase (PFK), pyruvate

kinase (PK) and lactate dehydrogenase (LDH) are uniquely encoded in the

las operon. We used metabolic control analysis to study the role of this

organization. Earlier studies have shown that, at wild-type levels, LDH has

no control over glycolysis and growth rate, but high negative control over

formate production (CJformate

LDH ¼
1:3). We found that PFK and PK exert no

control over glycolysis and growth rate at wild-type enzyme levels but both

enzymes exert strong positive control on the glycolytic flux at reduced

activities. PK exerts high positive control over formate (CJformate

PK ¼ 0:9
1:1)

and acetate production (CJacetate

PK ¼ 0:8
1:0), whereas PFK exerts no control

over these fluxes at increased expression. Decreased expression of the entire

las operon resulted in a strong decrease in the growth rate and glycolytic

flux; at 53% expression of the las operon glycolytic flux was reduced to

44% and the flux control coefficient increased towards 3. Increased las

expression resulted in a slight decrease in the glycolytic flux. At wild-type

levels, control was close to zero on both glycolysis and the pyruvate bran￾ches. The sum of control coefficients for the three enzymes individually

was comparable with the control coefficient found for the entire operon;

the strong positive control exerted by PK almost cancels out the negative

control exerted by LDH on formate production. Our analysis suggests that

coregulation of PFK and PK provides a very efficient way to regulate gly￾colysis, and coregulating PK and LDH allows cells to maintain homolactic

fermentation during glycolysis regulation.

Abbreviations

LDH, lactate dehydrogenase; PFK, phosphofructokinase; PFL, pyruvate formate lyase; PK, pyruvate kinase.

2292 FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS