hba titer  (Urogen Pharma)

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Assembly of the artificial HBA synthetic operon. a, Schematic illustration of natural HBA biosynthetic pathway. The natural 5-ALA biosynthetic route originating from TCA cycle is showed in gray; the aerobic HBA biosynthetic pathway investigated in this study is highlighted with pink shading; natural anaerobic HBA biosynthetic pathway is showed in purple; the downstream vitamin B12 biosynthetic pathway originating from HBA is in red. Enzymes are depicted in navy and blue, while cofactors, cosubstrates and by-products are indicated in brown. Abbreviation: 5-ALA, 5-Aminolevulinate; PBG, porphobilinogen; HMB, hydroxymethylbilane; PC, precorrin; HBA, hydrogenobyrinic acid; HBAD, hydrogenobyrinate a, c-diamide; CBAD, cob (II)yrinate a, c-diamide; SAM, S-adenosyl- l -methionine; SAH, S-adenosyl- l -homocysteine; L-Gln, l -Glutamine; L-Glu, l -Glutamate; Pi, phosphate. b, Illustration of natural HBA operons from R. capsulatus and S. meliloti , and schematic representation of two basic artificial HBA operons composed of enzymes from different microorganisms. c, Comparison of HBA production between the two artificial HBA operons in E. coli . H1: E. coli MG1655 (DE3), pET28a-1wRcHBA. H2: E. coli MG1655 (DE3), pET28a-1wSmHBA. All results were performed in triplicate (n = 3 biologically independent samples) and data are presented as mean values ± SD. Two-sided unpaired t -test is carried out between H1 and H2. Unpaired t -test of data: HBA/biomass, ∗∗∗∗, P < 0.0001 (t = 18.14); biomass, ns, P = 0.1226 (t = 1.953). d, SDS–PAGE analysis of heterologous expression of Cob enzymes from S. meliloti in E. coli BL21 (DE3) harboring the pET28a plasmid. M, prestained protein ladder; C, pET28a plasmid control; I, pET28a-1w-SmCobI; G, pET28a-1w-SmCobG; J, pET28a-1w-SmCobJ; M, pET28a-1w-SmCobM; F, pET28a-1w-SmCobF; K, pET28a-1w-SmCobK; L, pET28a-1w-SmCobL; H, pET28a-1w-SmCobH; GST-K, pET28a-GST-CobK; K opti , pET28a-CobK opti . Target protein bands are highlighted with red or navy arrows. e, Quantitative analysis of SDS–PAGE results in (d). Target protein bands were quantified using ImageJ based on pixel intensity. f, Comparison of HBA production by the two artificial HBA operons in E. coli . H45: E. coli MG1655 (DE3), pET28a-1wSmHBA-GST-CobK. All results were performed in triplicate (n = 3 biologically independent samples) and data are presented as mean values ± SD. Two-sided unpaired t -test is carried out between H2 and H45. Unpaired t -test of data: HBA/biomass, ∗∗, P = 0.0016 (t = 7.562); biomass, ∗∗, P = 0.0029 (t = 6.501).

Article Snippet: After 12 h of reaction, CCE derived from strain H2 produced a higher HBA titer (3.16 ± 0.14 μM) than those from H1 and LvH0, consistent with the trends observed in vivo ( b, MS identification of synthetic HBA and Urogen III are showed in ).

Techniques: Comparison, SDS Page, Expressing, Plasmid Preparation, Control

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Screening of the bottleneck reaction step in the 1w–SmHBA operon. a, Schematic illustration of the one-plasmid and two-plasmid assemblies of the 1w–SmHBA operon and the corresponding engineered strains. pET28a plasmid was showed in black, pACYC-Duet-1 plasmid was showed in red. b, Comparison of HBA production in strains H2, LvH0, and LvH15. Two-sided unpaired t -test is carried out between H2, LvH0 and LvH15. Unpaired t -test of data: H2 to LvH0, ∗∗∗∗, P < 0.0001 (t = 15.94); H2 to LvH15, ∗∗∗∗, P < 0.0001 (t = 17.87); LvH0 to LvH15, ∗∗∗, P = 0.0003 (t = 12.02). c, Comparison of HBA production between the LvH0 strain and LvH1–LvH8 strains, in which the RBS strength of individual cob genes was enhanced. Two-sided unpaired t -test is carried out between LvH0 and LvH1 to 15. Unpaired t -test of data: LvH1, ns, P = 0.9088 (t = 0.1220); LvH2, ∗∗, P = 0.0022 (t = 6.960); LvH3; ∗∗∗, P = 0.0002 (t = 14.02); LvH4, ∗∗∗, P = 0.0004 (t = 10.78); LvH5, ∗∗∗∗, P < 0.0001 (t = 38.56); LvH6, ∗∗∗, P = 0.0003 (t = 11.46); LvH7, ∗∗, P = 0.0013 (t = 8.015); LvH8, ∗∗∗, P = 0.0004 (t = 11.15); d, Schematic illustration of screening bottleneck enzymes by either introducing additional Cob enzymes from R. capsulatus (Rc) or replacing S. meliloti (Sm) Cob enzymes with the corresponding RcCob enzymes. e, Comparison of HBA production between the H2 strain and H21–H28 strains, each harboring an additional RcCob enzyme. Two-sided unpaired t -test is carried out between H2 and H21 to 28. Unpaired t -test of data:H21, ns, P = 0.0940 (t = 2.187); H22, ns, P = 0.0522 (t = 2.734); H23, ns, P = 0.5933 (t = 0.5795); H24, ∗, P = 0.0121 (t = 4.359); H25, ns, P = 0.0964 (t = 2.164); H26, ∗∗∗, P = 0.0003 (t = 11.48); H27, ns, P = 0.7136 (t = 0.3941); H28, ns, P = 0.1479 (t = 1.790). f, Comparison of HBA production between the H2 strain and H37–H44 strains, in which SmCob enzymes were replaced with the corresponding RcCob enzymes. Two-sided unpaired t -test is carried out between H2 and H37 to 44. Unpaired t -test of data: H37, ∗∗∗∗, P < 0.0001 (t = 17.94); H38, ∗∗∗, P = 0.0001 (t = 14.14); H39, ∗∗∗∗, P < 0.0001 (t = 28.35); H40, ∗∗∗∗, P < 0.0001 (t = 21.08); H41, ns, P = 0.2829 (t = 1.240); H42, ∗∗∗∗, P < 0.0001 (t = 89.48); H43, ∗, P = 0.0252 (t = 3.488); H44, ∗∗∗∗, P < 0.0001 (t = 120.7).

Article Snippet: After 12 h of reaction, CCE derived from strain H2 produced a higher HBA titer (3.16 ± 0.14 μM) than those from H1 and LvH0, consistent with the trends observed in vivo ( b, MS identification of synthetic HBA and Urogen III are showed in ).

Techniques: Plasmid Preparation, Comparison

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Screening of bottleneck reaction steps in the 1w–SmHBA operon by multienzyme catalysis. a, Schematic illustration of the reaction components. The in vitro reaction system consisted of crude cell extracts (CCE) from strains harboring the 1w–SmHBA plasmid as the basic catalytic source, supplemented with multienzyme-expressing strains carrying plasmids encoding 4–5, 2–3, or single heterologously expressed Cob enzymes to alter the enzyme composition in each reaction. b, Comparison of in vitro HBA production using CCE from strains H1, H2, and LvH0. Two-sided unpaired t -test is carried out between H1, H2, and LvH0. Unpaired t -test of data: H1 to H2, ∗∗∗∗, P < 0.0001 (t = 35.12); LvH0 to H2, ∗∗∗∗, P < 0.0001 (t = 30.84). c, In vitro HBA production using CCE from H21–H28 strains, each harboring an additional RcCob enzyme. Red bars indicate values higher than those of the H2 reactant, whereas blue bars indicate values lower than those of H2 reactant. Two-sided unpaired t -test is carried out between H1 to H21-28. Unpaired t -test of data:H2 to H21, ∗, P = 0.0227 (t = 3.605); H24 to H2, ∗∗∗∗, P < 0.0001 (t = 24.60); H27 to H2, ∗, P = 0.5363 (t = 0.6757). d, In vitro HBA production using CCE from H37–H44 strains, in which SmCob enzymes were replaced with the corresponding RcCob enzymes. Two-sided unpaired t -test is carried out between H1 to H37-44. Unpaired t -test of data:H37 to H2, ∗, P = 0.0145 (t = 4.129); H41 to H2, ∗, P = 0.0315 (t = 3.246); H42 to H2, ∗∗∗, P = 0.0003 (t = 11.66); H43 to H2, ∗, P = 0.0229 (t = 3.592); H44 to H2, ∗∗∗∗, P < 0.0001 (t = 15.76). e, Screening of reactions supplemented with crude cell extracts from strains heterologously expressing 4–5 Cob enzymes. HBA-A: CCE with pET28a–CobAIGJM; HBA-B: CCE with pACYCDuet-1–CobFKLH. Unpaired t -test of data: HBA-A 45 OD 600 to Control in HBA titer, ns, P = 0.0729 (t = 2.418); HBA-B 45 OD 600 to Control in HBA titer, ∗∗, P = 0.0029 (t = 6.502); HBA-A 45 OD 600 to Control in Urogen III titer, ∗∗, P = 0.0022 (t = 7.002); HBA-B 45 OD 600 to Control in Urogen III titer, ∗∗, P = 0.0011 (t = 8.309); f, Screening of reactions supplemented with crude cell extracts from strains heterologously expressing 2–3 Cob enzymes. AIG: CCE with pet28a-CobAIG; JM: CCE with pet28a-CobJM; FK: CCE with pet28a-CobFK; LH: CCE with pet28a-CobLH. Unpaired t -test of data: AIG 30 OD600 to Control in Urogen III titer, ∗∗∗, P = 0.0009 (t = 8.801); JM 30 OD600 to Control in Urogen III titer, ∗∗∗∗, P < 0.0001 (t = 24.39); FK 30 OD600 to Control in Urogen III titer, ∗, P = 0.0304 (t = 3.285); LH 30 OD600 to Control in Urogen III titer, ∗∗∗∗, P < 0.0001 (t = 15.93); g, Screening of reactions supplemented with CCE from strains heterologously expressing a single Cob enzyme. Unpaired t -test of data: CobA + to ori, ∗∗∗, P = 0.0002 (t = 13.95); CobI + to ori, ∗∗∗, P = 0.0005 (t = 10.57); CobG + to ori, ∗∗∗, P = 0.0003 (t = 12.00); CobJ + to ori, ∗∗∗, P = 0.0001 (t = 15.48); CobM + to ori, ∗∗∗, P = 0.0005 (t = 10.46); CobF + to ori, ∗∗∗∗, P < 0.0001 (t = 16.42); CobK + to ori, ∗∗∗, P = 0.0006 (t = 9.873); CobL + to ori, ∗∗, P = 0.0042 (t = 5.882); CobH + to ori, ∗∗∗, P = 0.001 (t = 8.675).

Article Snippet: After 12 h of reaction, CCE derived from strain H2 produced a higher HBA titer (3.16 ± 0.14 μM) than those from H1 and LvH0, consistent with the trends observed in vivo ( b, MS identification of synthetic HBA and Urogen III are showed in ).

Techniques: In Vitro, Plasmid Preparation, Expressing, Comparison, Control

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Optimization of the artificial HBA operon. a, In vitro HBA production using CCE from various strains under different modifications. promoter replacement (H2 vs. H46 and H47, trc or tac promoters): Two-sided unpaired t -test is carried out between H2 to H46 and H47. Unpaired t -test of data: H46 to H2, ∗∗∗∗, P < 0.0001 (t = 23.77); H47 to H2, ∗∗∗∗, P < 0.0001 (t = 33.93); chassis variation (H2 vs. H48–H52, different E. coli strains harboring the SmHBA plasmid): Two-sided unpaired t -test is carried out between H2 to H48-52, but no significant differences were observed; culture medium effects (H2 grown in different media): Unpaired t -test of data: 2YT to LB, ∗, P = 0.0168 (t = 3.954); and operon composition (H2 vs. H53 and H54, carrying the FKLHIGJM or FKLH + operon): Two-sided unpaired t -test is carried out between H2 to H53 and H54. Unpaired t -test of data: H53 to H2, ∗∗∗∗, P < 0.0001 (t = 44.32). b, Schematic illustration of the ASmHBA (H2 strain), FKLHIGJM (H53 strain), and FKLH + (H54 strain) operons. c, In vitro HBA synthesis using CCEs from H57 cultivated in 2YT medium, supplemented with gradient amounts of either CCE or CobA enzyme. d. Chromatogram of synthetic HBA in CCE3 group, CCE1 group in (c) with standards. UroIII-STD, uroporphyrinogen III standard; HBA-STD, hydrogenobyrinate acid standard; CCE3 and CCE1, reactants using 1-fold H57 CCE and 3-fold H57 CCE in (c).

Article Snippet: After 12 h of reaction, CCE derived from strain H2 produced a higher HBA titer (3.16 ± 0.14 μM) than those from H1 and LvH0, consistent with the trends observed in vivo ( b, MS identification of synthetic HBA and Urogen III are showed in ).

Techniques: In Vitro, Plasmid Preparation

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Optimization of the HBA synthetic system. a, HBA titers obtained from screening single Cob enzyme supplementation in the H53∗ CCE reaction: the control H53 CCE reaction, H53–CobA + (H53 CCE with 4 g/L CobA added), and H53–CCE + (H53 CCE with threefold CCE input). ∗ indicates that H53 CCE was prepared from cultures grown in LB medium, whereas unmarked H53 CCE was prepared from cultures grown in 2YT medium. Statistical Significance without bracket denotes unpaired t -test comparisons between each supplemented group (A+, I+, G+, J+, M+, F+, K+, L+, H+) and the corresponding Ori group under the same CCE introduction condition. Unpaired t -test of data: H53-CobA + to H53∗, ∗∗∗, P = 0.0004 (t = 10.67), H53-CCE + to H53∗, ∗∗∗∗, P < 0.0001 (t = 31.05). b, Urogen III accumulation under the same conditions as in (a). Unpaired t -test of data: H53-CobA + to H53∗, ns, P = 0.2509 (t = 1.341), H53-CCE + to H53∗, ∗∗∗∗, P < 0.0001 (t = 61.82). c, Schematic illustration of HBA biosynthesis with SAM supplementation. Abbreviation: 5-ALA, 5-Aminolevulinate; PBG, porphobilinogen; HMB, hydroxymethylbilane; L-Met, l -methionine; SAM, S-adenosyl- l -methionine; SAH, S-adenosyl- l -homocysteine; SRH, S-ribosyl- l -homocysteine. d, Orthogonal combinations of PpK, MetK, and MtnN enzymes used to enhance HBA production via SAM supplementation. The color intensity of the bar corresponds to the magnitude of the values for enhanced visual clarity. e, HBA titers in the optimized SAM supplementation system by adjusting ATP synthesis through varying AMP and SHMP inputs.

Article Snippet: After 12 h of reaction, CCE derived from strain H2 produced a higher HBA titer (3.16 ± 0.14 μM) than those from H1 and LvH0, consistent with the trends observed in vivo ( b, MS identification of synthetic HBA and Urogen III are showed in ).

Techniques: Control