Fine Tuning of the Lactate and Diacetyl Production through Promoter Engineering in Lactococcus lactis

Tingting Guo,Jian Kong,Li Zhang,Chenchen Zhang,Shumin Hu,Paul Jaak Janssen

doi:10.1371/journal.pone.0036296

Abstract

Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H2O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H2O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15±0.08 mM to 9.94±0.07 mM, and the corresponding diacetyl production increased from 1.07±0.03 mM to 4.16±0.06 mM with the intracellular NADH/NAD+ ratios varying from 0.711±0.005 to 0.383±0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD+ ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H2O-forming NADH oxidase activity led to 76.95% lower H2O2 concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H2O2 accumulation and prolong cell survival.

Highlights

Lactococcus lactis has long been used in dairy fermentation processes and considered as one of the most important starter cultures
MG1363 genome with the consensus promoter sequences of L. lactis revealed that the 235 region had high identity with the TTGACA sequence, whereas the 210 region was less conserved with the sequence TAAAAT deviating from the canonical TATAAT sequence in the 3rd position
The partial redistribution of the pyruvate flux from lactate to diacetyl was achieved by controlling the NADH oxidase (noxE) gene expression through a constitutive promoter library in L. lactis

Summary

Introduction

Lactococcus lactis has long been used in dairy fermentation processes and considered as one of the most important starter cultures. It produces multifarious end metabolites during dairy fermentation, such as lactate, diacetyl, acetoin, vitamins and extracellular exopolysaccharides which contribute to the organoleptic and health-promoting properties of the fermented products [1,2,3,4]. Considering that lactate is an important metabolite that prevents fermented products from spoilage and contributes to the texture of dairy products, the strong overexpression or complete deletion of target genes limits the application of genetically modified lactic acid bacteria as starter cultures for industrial production of dairy products. Strategies for the fine-tuning of gene expression are required to control the aimed metabolic fluxes

Methods

Results

Conclusion