Mariko Shimizu-Kadota

<italic>Enterococcus mundtii</italic> QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure <sc>l</sc>-lactic acid (≥ 99.9%) <italic>via</italic> homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar (usually glucose), termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of <italic>E</italic>. <italic>mundtii</italic> QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of <italic>talC</italic>, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates.

<title>Abstract</title> <sec> <title>Objective</title> <italic>Apis mellifera</italic> is a species of honeybee that has been introduced around the world as an industrial beekeeping species. Recently, urban beekeeping has attracted attention as a means of ecosystem protection and urban greening. This study aimed to investigate nectar sources of urban beekeeping in Kōtō-ku, Tokyo using pollen DNA metabarcoding. </sec> <sec> <title>Results</title> We extracted DNA from pollen collected by the honeybees of a local urban beekeeping operation. DNA metabarcoding analysis was carried out by sequencing a part of the <italic>rbc</italic>L region of the chloroplast genome. A total of 31 samples collected between mid-March, 2018 and mid-October, 2018 yielded 54 operational taxonomic units (OTUs) comprising 14 families, 32 genera, and 8 species. Whereas 5 OTUs were profiled throughout all seasons, 38 OTUs were season-specific (spring, summer, or autumn). Therefore, we were able to infer seasonal nectar sources for the beekeeping operation at the family or genus level, as well as at the species level to a lesser extent. Our pollen-sampling strategy was effective for profiling season-specific nectar sources, with the exception of a few anomalies that can be accounted for by out-of-season flowering associated with artificial gardening and/or pollen accumulation over multiple seasons. </sec>

Phosphoketolase (PK) is responsible for heterolactic fermentation; however, the PK gene of Enterococcus mundtii QU 25, xfpA, is transcribed constitutively, even under homolactic fermentation conditions. In order to deduce the regulatory mechanisms of PK activity in QU 25, XfpA levels in QU 25 cells under hetero- and homolactic fermentation conditions were tested using western blotting. The results showed that the XfpA protein expression was similar under both conditions and that the expression products formed complexes, most likely homodimers, indicating that the regulation of PK activity is downstream of translation.