Autotrophic lactate production from H2 + CO2 using recombinant and fluorescent FAST-tagged Acetobacterium woodii strains

Abstract

Lactate has various uses as industrial platform chemical, poly-lactic acid precursor or feedstock for anaerobic co-cultivations. The aim of this study was to construct and characterise Acetobacterium woodii strains capable of autotrophic lactate production. Therefore, the lctBCD genes, encoding the native Lct dehydrogenase complex, responsible for lactate consumption, were knocked out. Subsequently, a gene encoding a d-lactate dehydrogenase (LDHD) originating from Leuconostoc mesenteroides was expressed in A. woodii, either under the control of the anhydrotetracycline-inducible promoter Ptet or under the lactose-inducible promoter PbgaL. Moreover, LDHD was N-terminally fused to the oxygen-independent fluorescence-activating and absorption-shifting tag (FAST) and expressed in respective A. woodii strains. Cells that produced the LDHD fusion protein were capable of lactate production of up to 18.8 mM in autotrophic batch experiments using H2 + CO2 as energy and carbon source. Furthermore, cells showed a clear and bright fluorescence during exponential growth, as well as in the stationary phase after induction, mediated by the N-terminal FAST. Flow cytometry at the single-cell level revealed phenotypic heterogeneities for cells expressing the FAST-tagged LDHD fusion protein. This study shows that FAST provides a new reporter tool to quickly analyze gene expression over the course of growth experiments of A. woodii. Consequently, fluorescence-based reporters allow for faster and more targeted optimization of production strains.Key points •Autotrophic lactate production was achieved with A. woodii. •FAST functions as fluorescent marker protein in A. woodii. •Fluorescence measurements on single-cell level revealed population heterogeneity.

Model-driven approach for the production of butyrate from CO2/H2 by a novel co-culture of C. autoethanogenum and C. beijerinckii

“…Lactate is a minor fermentation product of acetogens grown on syngas or CO 2 /H 2 (Valgepea et al, 2018 ; Im et al, 2022 ), but a major fermentation product of acetogens grown on sugars (Drake and Daniel, 2004 ). Acetogens could be engineered toward autotrophic lactate production from CO 2 /H 2 (Mook et al, 2022 ) or syngas, thereby facilitating butyrate production in co-cultivation with C. beijerinckii without the need of adding an additional carbon source. Alternatively, lactate can be obtained from other sources, such as side-streams from the dairy industry (Sar et al, 2022 ), spoiled agri-food products (Xu et al, 2020 ), ensiled agricultural biomass (Chen et al, 2007 ), and fermented grass (Sakarika et al, 2022 ).…”

Model-driven approach for the production of butyrate from CO2/H2by a novel co-culture ofC. autoethanogenumandC. beijerinckii

“…Lactate has been reported to be a minor fermentation product by acetogens grown on syngas or CO 2 /H 2 (9, 48), but a major fermentation product by acetogens grown on sugars (49). Therefore, acetogens can be engineered to increase lactate production on CO 2 /H 2 (50), or engineered to grow on CO 2 , instead of sugars, producing lactate, and thus, produce butyrate in co-cultivation with C. beijerinckii . Furthermore, lactate can be obtained as side-stream in dairy industry, in almost all stored AgriFood as a result of a rotting (spontaneous fermentation) process, from storage using ensiling (51), or from the fermentation of grass (52), which could be alternative sources when establishment this co-culture.…”

Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing

“…This work has opened the door for applications in other anaerobes. FAST was used for instance to monitor the levels of proteins involved in (i) the production of the biocommodities butanol and acetone from methanol in Eubacterium limosum and (ii) the autotrophic production of lactate from dihydrogen and carbon dioxide in Acetobacterium woodii . More recently, FAST was shown to allow the monitoring of protein dynamics in live cells of Methanococcus maripaludis , opening great prospects for future studies focused on the metabolism and physiology of methanogenic archaea.…”

“…FAST was used for instance to monitor the levels of proteins involved in (i) the production of the biocommodities butanol and acetone from methanol in Eubacterium limosum 24 and (ii) the autotrophic production of lactate from dihydrogen and carbon dioxide in Acetobacterium woodii. 31 More recently, FAST was shown to allow the monitoring of protein dynamics in live cells of Methanococcus maripaludis, 32 opening great prospects for future studies focused on the metabolism and physiology of methanogenic archaea. Finally, because of its molecular oxygen-independent fluorescence, FAST was shown to be usable to study the vitality of bacteria transplanted to the gut macrobiota of mice.…”