Recombinant Lactobacillus plantarum Probable phosphoketolase 2 (lp_3551), partial

Basic Research Questions

• What is phosphoketolase 2 (lp_3551) in Lactobacillus plantarum and how does it function?

Phosphoketolase 2 (lp_3551) is one of two phosphoketolase enzymes present in Lactobacillus plantarum that plays a key role in the phosphoketolase pathway for carbohydrate metabolism. This enzyme catalyzes the phosphate-consuming conversion of xylulose-5-phosphate to glyceraldehyde-3-phosphate and acetylphosphate with the release of one water molecule . L. plantarum contains two phosphoketolase genes, designated as xpk1 and xpk2, with phosphoketolase 2 (xpk2) functioning alongside xpk1 in the heterofermentative metabolism of pentose sugars .

The activity of phosphoketolase 2 can be measured using methods described by Goldberg et al. (Methods Enzymol., 9, 515-520, 1966) or L. Meile (J. Bacteriol., 183, 2929-2936, 2001) . Under standard experimental conditions, the enzyme demonstrates specific activity with its substrates, contributing to the production of lactic acid and acetic acid from pentose sugars.

• What are the differences between phosphoketolase 1 and phosphoketolase 2 in L. plantarum?

In L. plantarum, phosphoketolase 1 (xpk1) and phosphoketolase 2 (xpk2) have distinct but complementary roles in carbohydrate metabolism:

• Substrate preference: While both enzymes can act on xylulose-5-phosphate, they may have different affinities for secondary substrates like fructose-6-phosphate .

• Impact on metabolism: Experimental evidence shows that disruption of xpk1 causes a more significant impact on xylose metabolism than disruption of xpk2. When xpk2 was deleted in an L. plantarum ΔldhL1-xpk1::tkt strain, there was only a slight decrease in cell growth and fermentation rates compared to the more dramatic effects seen with xpk1 deletion .

• Contribution to heterolactic fermentation: Both enzymes participate in the phosphoketolase pathway, but xpk1 appears to be the predominant enzyme, as indicated by metabolic engineering studies that focused on replacing xpk1 with transketolase (tkt) to shift from heterolactic to homolactic fermentation .

• How can phosphoketolase activity be measured in L. plantarum?

Phosphoketolase activity in L. plantarum can be measured using several established methods:

• For D-xylulose-5-phosphate phosphoketolase activity: The method described by Goldberg et al. (Methods Enzymol., 9, 515-520, 1966) measures the phosphate-consuming conversion of xylulose-5-phosphate to glyceraldehyde-3-phosphate and acetylphosphate .

• For fructose-6-phosphate phosphoketolase activity: The method by Racker, E. (Methods Enzymol., 5, 276-280, 1962) can be used to measure the conversion of fructose-6-phosphate to erythrose-4-phosphate and acetylphosphate .

• Modern adaptations: L. Meile's method (J. Bacteriol., 2001, 183; 2929-2936) offers an updated approach for measuring both activities .

• Colorimetric quantification: Measurement at 505 nm allows differentiation between microbial strains with low activity and those without F6PPK activity .

These assays typically involve cell extracts prepared from L. plantarum cultures, with specific substrate reactions followed by detection of reaction products.

• What is the role of phosphoketolase 2 in the metabolism of L. plantarum?

Phosphoketolase 2 contributes to the following metabolic processes in L. plantarum:

• Pentose sugar metabolism: It participates in the phosphoketolase pathway where pentoses are converted to pentose-5-phosphate, which is subsequently split into glyceraldehyde-3-phosphate and acetyl phosphate .

• Carbon flux regulation: It influences the distribution of carbon between lactic acid and acetic acid production, particularly during growth on pentose sugars .

• Complementary function: It works alongside phosphoketolase 1, providing a level of metabolic redundancy. Experimental evidence shows that when xpk2 was disrupted in a strain already lacking xpk1, there was only a slight additional decrease in fermentation performance, suggesting a secondary but still meaningful role .

• Heterolactic fermentation: Both phosphoketolases contribute to the heterolactic fermentation characteristic of L. plantarum when metabolizing pentoses, producing equimolar amounts of lactic acid and acetic acid .