Rewiring the respiratory pathway of Lactococcus lactis to enhance extracellular electron transfer

Abstract

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.

Thiamine-Starved Lactococcus lactis for Producing Food-Grade Pyruvate

“…RD1M5 has a mutation in its main lactate dehydrogenase, and thus, there is a low residual lactate dehydrogenase activity, which becomes more apparent when alternative product formation pathways are shut down, i.e., those involving PDHc and ALS when thiamine is limiting. At high cell densities, the carryover of thiamine becomes significant, thereby funneling more pyruvate through PDHc and ALS and less through the residual lactate dehydrogenase whereby less lactate is formed. , When the initial cell density was high (OD 600nm = 1, and 2) in SN –thiamine medium, more acetate was formed (Figure (e)), which was reflected in a boost in biomass yield due to the additional ATP formed …”

“…At high cell densities, the carryover of thiamine becomes significant, thereby funneling more pyruvate through PDHc and ALS and less through the residual lactate dehydrogenase whereby less lactate is formed. 34,35 When the initial cell density was high (OD 600nm = 1, and 2) in SN −thiamine medium, more acetate was formed (Figure 2(e)), which was reflected in a boost in biomass yield due to the additional ATP formed. 36 In summary, in the defined medium, the highest pyruvate titer obtained was 21.4 mM (1.9 g/L), which was reached in 6 h when the RD1M5 cell density was 0.5 (OD 600nm ) in the SN −thiamine medium.…”

Burning questions: Exploring the limits of microbial electrochemical technology for industrial biotechnological applications

“…Most of these strains showed lower electrogenic activity compared to the model electrogen Shewanella and Geobacter, where productivity was also too low for practical application. Recently, however, a surprising but significant article was published in which the Lactococcus was applied for anodic fermentation (Gu et al, 2023). A recombined mutant that was incapable of anaerobic growth was able to use ferricyanide and anode as the only electron sink to grow anaerobically.…”

Unraveling the Electron Transfer in Cupriavidus necator – Insights Into Mediator Reduction Mechanics

“…[14] In Lactococcus lactis, the endogenous mediator 2-amino-3-carboxy-1,4-naphthoquinone accepts electrons from the type II NADH dehydrogenase (NoxAB) which then reduces ferricyanide in the periplasm or extracellular. [15] Adding to the complexity, various mediators may interact with different interaction sites. As an example, the lipophilic compound 2,6dichlorphenolindophenol (DCIP) can accept electrons directly from complex I of the respiration chain in Staphylococcus aureus, [16] while neutral red can penetrate both membranes and most likely reduces NAD + directly in the cytosol.…”