Data_Sheet_1_Further Elucidation of Galactose Utilization in Lactococcus lactis MG1363.docx

<p>Since the 1970s, galactose metabolism in Lactococcus lactis has been in debate. Different studies led to diverse outcomes making it difficult to conclude whether galactose uptake was PEP- or ATP- dependent and decide what the exact connection was between galactose and lactose uptake and metabolism. It was shown that some Lactococcus strains possess two galactose-specific systems – a permease and a PTS, even if they lack the lactose utilization plasmid, proving that a lactose-independent PTS^Gal exists. However, the PTS^Gal transporter was never identified. Here, with the help of transcriptome analyses and genetic knock-out mutants, we reveal the identities of two low-affinity galactose PTSs. A novel plant-niche-related PTS component Llmg_0963 forming a hybrid transporter Llmg_0963PtcBA and a glucose/mannose-specific PTS are shown to be involved in galactose transport in L. lactis MG1363.</p

Table_2_Further Elucidation of Galactose Utilization in Lactococcus lactis MG1363.DOCX

<p>Since the 1970s, galactose metabolism in Lactococcus lactis has been in debate. Different studies led to diverse outcomes making it difficult to conclude whether galactose uptake was PEP- or ATP- dependent and decide what the exact connection was between galactose and lactose uptake and metabolism. It was shown that some Lactococcus strains possess two galactose-specific systems – a permease and a PTS, even if they lack the lactose utilization plasmid, proving that a lactose-independent PTS^Gal exists. However, the PTS^Gal transporter was never identified. Here, with the help of transcriptome analyses and genetic knock-out mutants, we reveal the identities of two low-affinity galactose PTSs. A novel plant-niche-related PTS component Llmg_0963 forming a hybrid transporter Llmg_0963PtcBA and a glucose/mannose-specific PTS are shown to be involved in galactose transport in L. lactis MG1363.</p

Table_1_Further Elucidation of Galactose Utilization in Lactococcus lactis MG1363.DOCX

<p>Since the 1970s, galactose metabolism in Lactococcus lactis has been in debate. Different studies led to diverse outcomes making it difficult to conclude whether galactose uptake was PEP- or ATP- dependent and decide what the exact connection was between galactose and lactose uptake and metabolism. It was shown that some Lactococcus strains possess two galactose-specific systems – a permease and a PTS, even if they lack the lactose utilization plasmid, proving that a lactose-independent PTS^Gal exists. However, the PTS^Gal transporter was never identified. Here, with the help of transcriptome analyses and genetic knock-out mutants, we reveal the identities of two low-affinity galactose PTSs. A novel plant-niche-related PTS component Llmg_0963 forming a hybrid transporter Llmg_0963PtcBA and a glucose/mannose-specific PTS are shown to be involved in galactose transport in L. lactis MG1363.</p

Nonhierarchical Flux Regulation Exposes the Fitness Burden Associated with Lactate Production in <i>Synechocystis</i> sp. PCC6803

Cyanobacteria are mostly engineered to be sustainable cell-factories by genetic manipulations alone. Here, by modulating the concentration of allosteric effectors, we focus on increasing product formation without further burdening the cells with increased expression of enzymes. Resorting to a novel 96-well microplate cultivation system for cyanobacteria, and using lactate-producing strains of <i>Synechocystis</i> PCC6803 expressing different l-lactate dehydrogenases (LDH), we titrated the effect of 2,5-anhydro-mannitol supplementation. The latter acts in cells as a nonmetabolizable analogue of fructose 1,6-bisphosphate, a known allosteric regulator of one of the tested LDHs. In this strain (SAA023), we achieved over 2-fold increase of lactate productivity. Furthermore, we observed that as carbon is increasingly deviated during growth toward product formation, there is an increased fixation rate in the population of spontaneous mutants harboring an impaired production pathway. This is a challenge in the development of green cell factories, which may be countered by the incorporation in biotechnological processes of strategies such as the one pioneered here

Schematic representation of the pilus gene cluster on pSH74 and the suggested functions of its constituting genes (not to scale).

<p>The internal deletion of 1451 bases in <i>spaCB</i> is indicated in red.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Plasmid maps of pSH73, pSH74, and pNZ712.

<p>Plasmid pNZ712 includes genes encoding functional nisin immunity (<i>nisCIP</i>) and copper resistance (<i>lcoRSABC</i>). The 16-kb plasmid pSH74 contains a novel 8-kb pilus gene cluster <i>spaCB-spaA-srtC1-srtC2</i>. Plasmid pSH73 harbors <i>repX</i>, <i>repB</i> and <i>cadCA</i> genes. The latter genes were annotated as a cadmium resistance regulatory protein and a cadmium efflux ATPase.</p

Scanning electron microscopy at 50.000 x magnification of <i>L</i>. <i>lactis</i> strains over-expressing <i>spaCB</i>-<i>spaA</i>-<i>srtC1</i>-<i>srtC2</i>.

<p>A—NCDO712, B—IL1403, C—IL1403(pIL253<i>pil</i>), D—MG1363(pIL253), E—MG1363(pIL253<i>pil</i>), F—MG1363(pIL253<i>pil</i>Δ<i>1</i>). White arrows indicate pili in panel C. Black bars: 2 μm in all panels.</p

Scanning electron microscopy of <i>spaCB</i>-<i>spaA</i>-<i>srtC1</i>-<i>srtC2</i> over-expressing <i>L</i>. <i>lactis</i> strains.

<p>A—NCDO712(pIL253<i>pil</i>), B—NCDO712, C—MG1363(pIL253<i>pil</i>), D—MG1363(pIL253), E—IL1403(pIL253<i>pil</i>), F- MG1363(pIL253<i>pilΔ1)</i>. Black bars: 1 μm in all panels.</p

Pilin gene overexpression in <i>L</i>. <i>lactis</i> MG1363 leads to increased chain length.

<p>A—MG1363(pIL253), B—MG1363(pIL253<i>pil</i>), C—MG1363(pIL253<i>pil</i>Δ<i>1</i>).</p