Rhodopseudomonas palustris is a purple non-sulfide bacterium (PNSB), and some strains have been proven to promote plant growth. However, the mechanism underlying the effect of these PNSBs remains limited. Based on genetic information, R. palustris possesses the ability to produce pyrroloquinoline quinone (PQQ). PQQ is known to play a crucial role in stimulating plant growth, facilitating phosphorous solubilization, and acting as a reactive oxygen species scavenger. However, it is still uncertain whether growth conditions influence R. palustris’s production of PQQ and other characteristics. In the present study, it was found that R. palustris exhibited a higher expression of genes related to PQQ synthesis under autotrophic culture conditions as compared to acetate culture conditions. Moreover, similar patterns were observed for phosphorous solubilization and siderophore activity, both of which are recognized to contribute to plant-growth benefits. However, these PNSB culture conditions did not show differences in Arabidopsis growth experiments, indicating that there may be other factors influencing plant growth in addition to PQQ content. Furthermore, the endophytic bacterial strains isolated from Arabidopsis exhibited differences according to the PNSB culture conditions. These findings imply that, depending on the PNSB’s growing conditions, it may interact with various soil bacteria and facilitate their infiltration into plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO₂ fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C₅ and C₆ sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks.

Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC–MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future.

The enzymatic mechanisms of carbon fixation by autotrophs, such as the reductive tricarboxylic acid cycle (rTCA), have inspired biotechnological approaches to producing bio-based chemicals directly through CO₂. To explore the possibility of constructing an rTCA cycle in Escherichia coli and to investigate their potential for CO₂ assimilation, a total of ten genes encoding the key rTCA cycle enzymes, including α-ketoglutarate:ferredoxin oxidoreductase, ATP-dependent citrate lyase, and fumarate reductase/succinate dehydrogenase, were cloned into E. coli. The transgenic E. coli strain exhibited enhanced growth and the ability to assimilate external inorganic carbon with a gaseous CO₂ supply. Further experiments conducted in sugar-free medium containing hydrogen as the electron donor and dimethyl sulfoxide (DMSO) as the electron acceptor proved that the strain is able to undergo anaerobic respiration, using CO₂ as the major carbon source. The transgenic stain demonstrated CO₂-enhanced growth, whereas the genes involved in chemotaxis, flagellar assembly, and acid-resistance were upregulated under the anaerobic respiration. Furthermore, metabolomic analysis demonstrated that the total concentrations of ATP, ADP, and AMP in the transgenic strain were higher than those in the vector control strain and these results coincided with the enhanced growth. Our approach offers a novel strategy to engineer E. coli for assimilating external gaseous CO₂.

We report the mixotrophic growth of Escherichia coli based on recombinant 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) to assimilate CO₂ using malate as an auxiliary carbon source and hydrogen as an energy source. We employ a long-term (~184 days) two-stage adaptive evolution to convert heterotrophic E. coli into mixotrophic E. coli. In the first stage of evolution with serine, diauxic growth emerges as a prominent feature. At the end of the second stage of evolution with malate, the strain exhibits mixotrophy with CO₂ as an essential substrate for growth. We expect this work will open new possibilities in the utilization of OGOR for microbial CO₂ assimilation and future hydrogen-based electro-microbial conversion.

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden–Mayerhof–Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage.