Metabolic Engineering of Acetaldehyde Production by Streptococcus thermophilus

Abstract

The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type S. thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine. Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption. Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an S. thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P LacA ). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in S. thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S. thermophilus as starter culture.Yogurt is a product obtained through milk fermentation with a specific yogurt starter culture consisting of a mixture of two species of lactic acid bacteria (LAB), Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (23). The main roles of this mixed starter in the production of yogurt are (i) acidification through the conversion of lactose into lactic acid, (ii) creation of the viscous texture by the production of exopolysaccharides, and (iii) development of the typical yogurt flavor (29).The typical yogurt flavor is caused by lactic acid, which imparts an acidic and refreshing taste, and a mixture of various carbonyl compounds like acetone, diacetyl, and acetaldehyde, the latter of which is considered the major flavor component (10,11,21,29). The relatively high concentration of acetaldehyde (in the range of 5 to 21 mg/liter) found in yogurt must be due to a low utilization rate of this metabolite since the yogurt bacteria lack the main enzyme for acetaldehyde conversion into ethanol, alcohol dehydrogenase (12).The production of acetaldehyde by LAB seems to be strain dependent. L. delbrueckii subsp. bulgaricus has been reported by some authors to be a greater acetaldehyde producer than S. thermophilus, whereas other authors have reported the contrary (21, 27). Although it is presently unclear what the major pathway for acetaldehyde production by LAB is, several metabolic pathways have been shown to lead to its formation (5, 24) and it is possible that more th...

Excess of threonine compared with serine promotes threonine aldolase activity in Lactococcus lactis IL1403

“…SHMT might have TA activity, as shown by Chaves et al (2002), although it is not a common feature in LAB (Ogawa et al, 2000). Similarly, this activity of SHMT may be present in Lc.…”

“…In fact, acetaldehyde can be produced by several other biochemical pathways in LAB (e.g. by pyruvate decarboxylation), which can operate simultaneously (Ott et al, 2000;Chaves et al, 2002). However, the yield of acetaldehyde during fermentation of Lactococcus lactis is low (Bongers et al, 2005), as opposed to species used in yogurt starter culture, Lactobacillus delbrueckii subsp.…”

“…However, the yield of acetaldehyde during fermentation of Lactococcus lactis is low (Bongers et al, 2005), as opposed to species used in yogurt starter culture, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (Chaves et al, 2002). Acetaldehyde can be further metabolized into ethanol by alcohol dehydrogenase, and the gene encoding this enzyme (adhE) is present in Lc.…”

“…Interestingly, some SHMTs have a secondary activity as TA, e.g. in S. thermophilus (Chaves et al, 2002), although this is not a ubiquitous property amongst LAB (Ogawa et al, 2000). A low-specificity TA (encoded by ltaE), which catalyses the cleavage of Lthreonine or L-allo-threonine to glycine and acetaldehyde, has been found in Escherichia coli (Liu et al, 1998).…”

“…TA activity in bacteria can be associated with two enzymes: serine hydroxymethyltransferase (SHMT), encoded by glyA, and a low-specificity TA (Liu et al, 1998;Chaves et al, 2002;Simic et al, 2002;Fernández & Zú ñiga, 2006). The principal role of SHMT is catalysing the reversible formation of glycine and 5,10-methylenetetrahydrofolate from serine (Fernández & Zú ñiga, 2006).…”