Supplementary Material

Ammonium acrylate biomanufacturing by an engineered Rhodococcus ruber with nitrilase overexpression and double-knockout of nitrile hydratase and amidase

Jizhe Sun1,2, Huimin Yu1,2, 3*, Jie Chen1,2, Hui Luo4 and Zhongyao Shen1,2

1 Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

2 Key Laboratory of Industrial Biocatalysis (Tsinghua University), the Ministry of Education, Beijing 100084, China

3 Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China

4 Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China

Table S1 Primers used in current study

Primers / Sequences（5’-3’） / Restriction
sites
A-sense-EcoRI / GGAATTCCAGGGCTATCAGGGCACCGGCATCG’ / EcoR I
A-anti-XbaI / GCTCTAGATCCTTTCATCGGAGCTGGGCTCAAA’ / Xba I
B-sense-XbaI / GCTCTAGATGAAGACACACTCACTGATCGGCTC / BamH I
B-anti-HindIII / CCCAAGCTTAGTTGCGAGGTCGGTATCGGTTAGA / Hind III
testA / CGGAGTCTTCATCCTGTTTTG
testB / GGTGCGTACTGCCGTTATTC

Fig. S1 Colony morphotype of B. subtilis THY-7 and transcriptome analyses of THY-7 cells cultured for 12 h, 24h and 36h, respectively.

Fig. S2 Sketch map of the construction procedure of the Amidase-NHase (amiE-nbBA) double knockout mutant, R. ruber THdAdN. The sacB is the levansucrase gene. The nptII is the kanamycin resistance gene. Gene A and B is upstream and downstream fragment of nbBA, respectively. R1 and R2 indicate two different recombination pathways during the homologous recombination.

Fig. S3 Cloning of the up-stream gene fragment (A) and down-stream gene fragment (B) of NHase to construct the plasmid p18B-TH.

Fig. S4 Cloning of promoter Pa2 and nitrilase gene Surfactin synthase cluster in length of 26.2 kb in B. subtilis THY-7.

Fig. S5 The second batch hydration reaction of acrylonitrile to ammonium acrylate catalyzed by the engineered R.ruber THdAdN(Nit) and THdA(Nit) versus the control, R. rhodochrous tg1-A6 at 25oC, pH7.5. Equal density of cells (THdAdN(Nit), THdA(Nit) and tg1-A6) was added in the reaction system, and the initial nitrilase activity was 115, 53 and 35 U/mL, respectively, as quantified by GC.

Fig. S6 The third batch hydration reaction of acrylonitrile to ammonium acrylate catalyzed by the engineered R.ruber THdAdN(Nit) and THdA(Nit) versus the control, R. rhodochrous tg1-A6 at 25oC, pH7.5. Equal density of cells (THdAdN(Nit), THdA(Nit) and tg1-A6) was added in the reaction system, and the initial nitrilase activity was 109, 50 and 39 U/mL, respectively, as quantified by GC.

Fig. S7 GC results of the reaction solution during biomanufacturing of ammonium acrylate with acrylonitrile as substrate and THdA(Nit) cells as catalysts. The three peaks represent substrate acrylonitrile (AN), product ammonium acrylate (AA) and the extra-supplemented internal standard, acetamide (Aa) for GC. The sample was loaded after 20-folds dilution, and the measured concentration of AN and AA was 0.974 g/L and 25.73 g/L, respectively.