Research Article: Heterologous expression of Oenococcus oeni malolactic enzyme in Lactobacillus plantarum for improved malolactic fermentation

Date Published: March 27, 2012

Publisher: Springer

Author(s): Christina Schümann, Herbert Michlmayr, Reinhard Eder, Andrés M del Hierro, Klaus D Kulbe, Geir Mathiesen, Thu-Ha Nguyen.


Lactobacillus plantarum is involved in a multitude of food related industrial fermentation processes including the malolactic fermentation (MLF) of wine. This work is the first report on a recombinant L. plantarum strain successfully conducting MLF. The malolactic enzyme (MLE) from Oenococcus oeni was cloned into the lactobacillal expression vector pSIP409 which is based on the sakacin P operon of Lactobacillus sakei and expressed in the host strain L. plantarum WCFS1. Both recombinant and wild-type L. plantarum strains were tested for MLF using a buffered malic acid solution in absence of glucose. Under the conditions with L-malic acid as the only energy source and in presence of Mn2+ and NAD+, the recombinant L. plantarum and the wild-type strain converted 85% (2.5 g/l) and 51% (1.5 g/l), respectively, of L-malic acid in 3.5 days. Furthermore, the recombinant L. plantarum cells converted in a modified wine 15% (0.4 g/l) of initial L-malic acid concentration in 2 days. In conclusion, recombinant L. plantarum cells expressing MLE accelerate the malolactic fermentation.

Partial Text

Lactic acid bacteria (LAB) contribute to taste and texture of a wide range of fermented products and inhibit the growth of spoilage bacteria (Mozzi et al. 2010). This includes the application of LAB in winemaking to increase the stability of wines that undergo barrel or bottle-ageing. This process, the malolactic fermentation (MLF), normally occurs after the alcoholic fermentation (AF). Apart from the decarboxylation of L-malic into L-lactic acid, MLF removes carbon sources of other microorganisms and bestows sensory changes to the wine (Bartowsky 2005). The genera mainly found during MLF are Lactobacillus, Leuconostoc, Pediococcus and Oenococcus. Due to its high tolerance to low pH and higher amounts of SO2 and ethanol, Oenococcos oeni is the primary species encountered during spontaneous MLF (Capozzi et al. 2010). Furthermore, O. oeni is the preferred organism for malolactic starter cultures, since the presence of Lactobacillus sp. and Pediococcus sp. during MLF can lead to development of spoilage aroma and off-flavours (Moreno-Arribas and Polo 2005). However, also O. oeni is able to generate for example acetic acid, diacetyl (buttery flavour), mannitol (viscous, sweet) and mousy off-flavour (Bartowsky 2009). Additionally, O. oeni needs several weeks to degrade malic acid completely, and growth of LAB is often delayed or can even fail (Zhang and Lovitt 2005). Consequently, innovations are desirable to reduce malic acid in a faster and more efficient way.

At present, genetically modified organisms are already used in winemaking. Recombinant Saccharomyces strains conduct simultaneous alcoholic and malolactic fermentation, however, if the distinct aroma produced during MLF is desired, the use of LAB is necessary. Although O. oeni is the preferred microorganism to conduct MLF, recent research showed that L. plantarum is a promising candidate to reduce L-malic acid in wine as well (Pozo-Bayon et al. 2005,
du Toit et al. 2010). Not only that L. plantarum occurs naturally at different stages during wine making, it is also of interest as efficient antimicrobial agent to control spoilage microorganisms in winemaking (Navarro et al. 2008). Furthermore, wine related Lactobacillus species, including L. plantarum, are as efficient as O. oeni with excellent potential as starter cultures (du Toit et al. 2010). In this study we present a L. plantarum strain which produces recombinant MLE from O. oeni for enhanced MLF. The induced recombinant strain converted more L-malic acid and adapted better to wine condition than the wild-type L. plantarum.

The authors declare that they have no competing interests.