Human milk oligosaccharides (HMOs) have been proven to be beneficial to infants’ intestinal health and immune systems. 2′-Fucosyllactose (2′-FL) is the most abundant and thoroughly studied HMO and has been approved to be an additive of infant formula. How to construct efficient and safe microbial cell factories for the production of 2′-FL attracts increasing attention. In this work, we engineered the Bacillus subtilis as an efficient 2′-FL producer by engineering the substrate transport and cofactor guanosine 5′-triphosphate (GTP) regeneration systems. First, we constructed a synthesis pathway for the 2′-FL precursor guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) by introducing the salvage pathway gene fkp from Bacteriodes fragilis and improved the fucose importation by overexpressing the transporters. Then, the complete synthesis pathway of 2′-FL was constructed by introducing the heterologous fucosyltransferases from different sources, and it was found that the gene from Helicobacter pylori was the best one for 2′-FL synthesis. We also improved the substrate lactose importation by introducing heterologous lactose permeases and eliminated endogenous β-galactosidase (yesZ) to block the lactose degradation. Next, the production of 2′-FL and GDP-l-fucose was improved by fine-tuning the expression of cofactor guanosine 5′-triphosphate regeneration module genes gmd, ndk, guaA, guaC, ykfN, deoD, and xpt. Finally, a 3 L fed-batch fermentation was performed, and the highest 2′-FL titer reached 5.01 g/L with a yield up to 0.85 mol/mol fucose. We optimized the synthesis modules of 2′-FL in B. subtilis, and this provides a good starting point for metabolic engineering to further improve 2′-FL production in the future.

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Lactococcus lactis, a lactic acid bacterium with a typical fermentative metabolism, can also use oxygen as an extracellular electron acceptor. Here we demonstrate, for the first time, that L. lactis blocked in NAD + regeneration can use the alternative electron acceptor ferricyanide to support growth. By electrochemical analysis and characterization of strains carrying mutations in the respiratory chain, we pinpoint the essential role of the NADH dehydrogenase and 2-amino-3-carboxy-1,4-naphtoquinone in extracellular electron transfer (EET) and uncover the underlying pathway systematically. Ferricyanide respiration has unexpected effects on L. lactis, e.g., we find that morphology is altered from the normal coccoid to a more rod shaped appearance, and that acid resistance is increased. Using adaptive laboratory evolution (ALE), we successfully enhance the capacity for EET. Whole-genome sequencing reveals the underlying reason for the observed enhanced EET capacity to be a late-stage blocking of menaquinone biosynthesis. The perspectives of the study are numerous, especially within food fermentation and microbiome engineering, where EET can help relieve oxidative stress, promote growth of oxygen sensitive microorganisms and play critical roles in shaping microbial communities.

Plant-based dairy alternatives are gaining increasing interest, e.g. alternatives to yoghurt, cheese and butter. In all these products butter flavor (diacetyl + acetoin) plays an important role. We previously have reported efficient butter flavor formation from low value dairy side streams using a dairy isolate of Lactococcus lactis deficient in lactate dehydrogenase. Here we have tested the ability of this strain, RD1M5, to form butter flavor in plant milks based on oat and soy. We found that oat milk, with its high sugar content, supported more efficient production of butter aroma, when compared to soy milk. When supplemented with glucose, efficient butter aroma production was achieved in soy milk as well. We also carried out an extended adaptive laboratory evolution of the dairy strain in oat milk. After two months of adaptation, we obtained a strain with enhanced capacity for producing butter aroma. Despite of its high sugar content, RD1M5 and its adapted version only metabolized approximately 10% of the fermentable sugars available in the oat milk, which we found was due to amino acid starvation and partly starvation for vitamins. The study demonstrates that dairy cultures have great potential for use in plant-based fermentations.

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