Mannitol production in Lactococcus Lactis

Mannitol is a six-carbon polyol with many applications in food and therapeutic area, due to its low glycemic index, low insulin response, low calorie, osmotic and anti-oxidative traits. Microbial mannitol production, particularly using Lactic acid bacteria (LAB) has been extensively studied and most of these have relied on reducing fructose via mannitol dehydrogenase (MDH). Lactococcus lactis (L. lactis), a homofermentative LAB with a long history of safe use in the food industry, is equipped with all the necessary enzymes for mannitol production by reducing fructose-6-phosphate (F6P), which means it could produce mannitol from various sugars, and is therefore considered as a new promising host for mannitol production. Nevertheless, the low expression level of the relevant genes for producing mannitol renders L. lactis a low efficient mannitol producer. This dilemma has been bypassed by introducing heterologous genes; however, such strains are not food-grade and are limited for their applications.

In this study, we found that L. lactis has an inherent capacity to produce mannitol from glucose. By adaptively evolving L. lactis, or derivatives blocked in NAD⁺ regenerating pathways, we managed to accelerate growth on mannitol. When cells of such adapted strains are resuspended in buffer containing glucose, 4-58% of the glucose metabolized is converted into mannitol, in contrast to non-adapted strains. Whole genome sequencing of these mutants revealed one critical mutation, C-39T, in the promoter region of mtlA gene, which appear to enhance the promoter strength.

Bioinformatics analysis of the mannitol operon and reporter gene assay showed the expression of mtlD gene, encoding mannitol-1-phosphate dehydrogenase (M1PDH), which is a key enzyme for mannitol production, is tightly controlled by transcriptional regulator MtlR and CcpA together. In addition, we found that the expression of the mannitol operon could be enhanced by shifting cells to stationary phase and by deleting the mtlF gene.

Production of mannitol in L. lactis will indirectly cost ATP due to the phosphoenolpyruvate (PEP) consumption during glucose transportation, and indeed, increasing the energy supply by adding arginine to the growth medium or activating the acetate-producing pathway also resulted in a stimulation of mannitol production. By applying these combined findings, we developed a two-step fermentation setup, i.e., growing cells to stationary phase, then incubating them with limited aeration. Using this method and glucose as a substrate, we could achieve 6.1 g/L mannitol with 60% yield using a food-grade strain. 9.8 g/L mannitol and 57% yield was obtained using an mtlD-overexpressed strain, which to our knowledge are the highest yield and titers reported to date, respectively. Furthermore, we showed that L. lactis could produce mannitol solely from fructose, galactose, or maltose, which demonstrates the great potential of using L. lactis as a mannitol producer.

Our studies have resulted in a deep insight into the mechanism for mannitol production in L. lactis, as well as provided clues to further develop a food-grade L. lactis that could efficiently produce mannitol from various sugars.