Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

capsular polysaccharide, capsule degrading depolymerase, Klebsiella phage

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3	aDGalp	95.8	68.5	78.1	72.3	67.9	61.9
3	aDGalpA	101.5	67.9	75.0	67.9	72.8	176.2
	aLFucp	101.5	68.4	78.3	72.7	67.8	16.1


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3	aDGalp	5.25	4.09	4.04	3.92	4.20	3.74
3	aDGalpA	5.31	4.00	4.14	4.55	4.37	-
	aLFucp	5.21	3.96	4.06	3.90	4.18	1.18


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3	aDGalp	95.8/5.25	68.5/4.09	78.1/4.04	72.3/3.92	67.9/4.20	61.9/3.74
3	aDGalpA	101.5/5.31	67.9/4.00	75.0/4.14	67.9/4.55	72.8/4.37	
	aLFucp	101.5/5.21	68.4/3.96	78.3/4.06	72.7/3.90	67.8/4.18	16.1/1.18

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@@H](O)[C@@H](CO)O[C@H](O[C@@H]2[C@@H](O)[C@@H](O[C@H]3[C@H](O)[C@H]([*])O[C@@H](C)[C@H]3O)O[C@H](C(=O)O)[C@@H]2O)[C@@H]1O

484.407 g/mol (C₁₈H₂₈R₂O₁₅, R = next and previous repeats, not counted in mol weight)

Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

capsular polysaccharide, capsule degrading depolymerase, Klebsiella phage

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3,3,3	aDGalp	95.8	68.1	80.0	70.0	71.3	61.9
3,3,3,3	bDGalp	105.2	70.5	77.7	65.6	75.6	61.9
3,3,3	bDGalf	110.5	81.4	85.3	80.5	71.0	63.8
3,3,4	aDGalp	101.2	69.9	70.0	69.5	71.4	60.9
3,3	aDGalp	100.8	68.7	77.7	79.1	71.4	60.9
3	bDGalf
4	aDGalp
	aDGalp


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3,3,3	aDGalp	5.19	4.05	4.16	4.29	4.24	3.76
3,3,3,3	bDGalp	4.69	3.75	3.81	4.19	3.68	3.76
3,3,3	bDGalf	5.22	4.35	4.09	4.31	3.86	3.69
3,3,4	aDGalp	5.01	3.83	3.92	4.07	4.24	3.79
3,3	aDGalp	5.10	4.10	3.92	4.18	4.24	3.79
3	bDGalf
4	aDGalp
	aDGalp


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3,3,3	aDGalp	95.8/5.19	68.1/4.05	80.0/4.16	70.0/4.29	71.3/4.24	61.9/3.76
3,3,3,3	bDGalp	105.2/4.69	70.5/3.75	77.7/3.81	65.6/4.19	75.6/3.68	61.9/3.76
3,3,3	bDGalf	110.5/5.22	81.4/4.35	85.3/4.09	80.5/4.31	71.0/3.86	63.8/3.69
3,3,4	aDGalp	101.2/5.01	69.9/3.83	70.0/3.92	69.5/4.07	71.4/4.24	60.9/3.79
3,3	aDGalp	100.8/5.10	68.7/4.10	77.7/3.92	79.1/4.18	71.4/4.24	60.9/3.79
3	bDGalf
4	aDGalp
	aDGalp

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

Warning = sub-repeat ending at aDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at bDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at aDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at bDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at aDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at bDGalp: unknown unit count was assumed to be 3
OC[C@@H](O)[C@@H]1O[C@@H](O[C@H]2[C@@H](O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@@H](CO)O[C@H](O[C@H]3[C@H]([C@H](O)CO)O[C@@H](O[C@H]4[C@@H](O[C@H]5O[C@H](CO)[C@H](O)[C@H](O)[C@H]5O)[C@@H](CO)O[C@H](O[C@H]5[C@H]([C@H](O)CO)O[C@@H](O[C@H]6[C@@H](O[C@H]7O[C@H](CO)[C@H](O)[C@H](O)[C@H]7O)[C@@H](CO)O[C@H](O[C@H]7[C@H]([C@H](O)CO)O[C@@H](O[C@H]8[C@@H](O[C@H]9O[C@H](CO)[C@H](O)[C@H](O)[C@H]9O)[C@@H](CO)O[C@H](O)[C@@H]8O)[C@@H]7O)[C@@H]6O)[C@@H]5O)[C@@H]4O)[C@@H]3O)[C@@H]2O)[C@H](O)[C@H]1O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O[C@H]2O[C@H](CO)[C@H](O)[C@H](O[C@@H]3O[C@H](CO)[C@H](O)[C@H](O[C@H]4O[C@H](CO)[C@H](O)[C@H](O[C@@H]5O[C@H](CO)[C@H](O)[C@H](O[C@H]6O[C@H](CO)[C@H](O)[C@H](O)[C@H]6O)[C@H]5O)[C@H]4O)[C@H]3O)[C@H]2O)[C@H]1O

2936.553 g/mol (C₁₀₈H₁₈₂O₉₁)

Free fatty acids are basic oleochemicals implemented in a range of applications including surfactants, lubricants, paints, plastics, and cosmetics. Microbial fatty acid biosynthesis has gained much attention as it provides a sustainable alternative for petrol- and plant oil-derived chemicals. The yeast Starmerella bombicola is a microbial cell factory that naturally employs its powerful lipid metabolism for the production of the biodetergents sophorolipids (> 300 g/L). However, in this study we exploit the lipidic potential of S. bombicola and convert it from the glycolipid production platform into a free fatty acid cell factory. We used several metabolic engineering strategies to promote extracellular fatty acid accumulation which include blocking competing pathways (sophorolipid biosynthesis and β-oxidation) and preventing free fatty acid activation. The best producing mutant (Δcyp52m1Δfaa1Δmfe2) secreted 0.933 g/L (± 0.04) free fatty acids with a majority of C18:1 (43.8%) followed by C18:0 and C16:0 (40.0 and 13.2%, respectively). Interestingly, deletion of SbFaa1 in a strain still producing sophorolipids also resulted in 25% increased de novo sophorolipid synthesis (P = 0.0089) and when oil was supplemented to the same strain, a 50% increase in sophorolipid production was observed compared to the wild type (P = 0.03). We believe that our work is pivotal for the further development and exploration of S. bombicola as a platform for synthesis of environmentally friendly oleochemicals.

yeast, sophorolipid, Starmerella bombicola, free fatty acid, lipid metabolism

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)OC[C@H]1O[C@@H](O[C@@H]2[C@@H](O)[C@H](O)[C@@H](COC(C)=O)O[C@H]2OC(C)CCCCCC/C=C\CCCCCCCC(=O)O)[C@H](O)[C@@H](O)[C@@H]1O

706.823 g/mol (C₃₄H₅₈O₁₅)

Free fatty acids are basic oleochemicals implemented in a range of applications including surfactants, lubricants, paints, plastics, and cosmetics. Microbial fatty acid biosynthesis has gained much attention as it provides a sustainable alternative for petrol- and plant oil-derived chemicals. The yeast Starmerella bombicola is a microbial cell factory that naturally employs its powerful lipid metabolism for the production of the biodetergents sophorolipids (> 300 g/L). However, in this study we exploit the lipidic potential of S. bombicola and convert it from the glycolipid production platform into a free fatty acid cell factory. We used several metabolic engineering strategies to promote extracellular fatty acid accumulation which include blocking competing pathways (sophorolipid biosynthesis and β-oxidation) and preventing free fatty acid activation. The best producing mutant (Δcyp52m1Δfaa1Δmfe2) secreted 0.933 g/L (± 0.04) free fatty acids with a majority of C18:1 (43.8%) followed by C18:0 and C16:0 (40.0 and 13.2%, respectively). Interestingly, deletion of SbFaa1 in a strain still producing sophorolipids also resulted in 25% increased de novo sophorolipid synthesis (P = 0.0089) and when oil was supplemented to the same strain, a 50% increase in sophorolipid production was observed compared to the wild type (P = 0.03). We believe that our work is pivotal for the further development and exploration of S. bombicola as a platform for synthesis of environmentally friendly oleochemicals.

yeast, sophorolipid, Starmerella bombicola, free fatty acid, lipid metabolism

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@@H]1[C@@H](COC(C)=O)O[C@@H](O[C@@H]2[C@@H](O)[C@H](O)[C@@H](COC(C)=O)O[C@H]2OC(C)CCCCCC/C=C\CCCCCCCC([*])=O)[C@H](O)[C@H]1O

688.808 g/mol (C₃₄H₅₆R₂O₁₄, R = next and previous repeats, not counted in mol weight)