Additional file 1 of Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria
Creators: name: Kobayashi, Shunichi
name: Atsumi, Shota
name: Ikebukuro, Kazunori
name: Sode, Koji
name: Asano, Ryutaro
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1 Design of vectors used in this study. (a) pKT-GSS-KDC-ADH for the application of CcaS/CcaR system to produce alcohol in cyanobacteria. pKT-GSS-KDC-ADH was constructed by inserting kdc and adh into the downstream of PcpcG2 on pKT-GSS. (b) pKT-GSS-KDC-ADH-CcaR to tune the CcaS/CcaR system up for the enhancement of alcohol production. pKT-GSS-KDC-ADHCcaR was constructed by inserting the structural genes of kdc, adh, and ccaR into the downstream of PcpcG2 on pKT-GSS. (c) pKT-Ptrc-core-KDC-ADH for the constitutive activation of alcohol production in cyanobacteria. The oriV origin is replicated in Synechocystis sp. PCC 6803. Gene abbreviations are as follows: PccaS, ccaS promoter; PccaR, ccaR promoter; PcpcG2, cpcG2 promoter; kdc, keto-acid decarboxylase; adh, alcohol dehydrogenase; SmR, streptomycin-resistance gene; SDcpcB, Shine Dalgarno-like sequence of cpcB derived from Synechococcus sp. PCC 7002. Fig. S2 Growth curves of each strain used in this study. Growth curves of (a) the PCC 6803ΔGSS strain with pKT-GSS-KDC-ADH, (b) the PCC 6803ΔGSS strain, and (c) the PCC 6803ΔGSS strain with pKT-GSS-KDC-ADH-CcaR. All strains were cultivated under the red light illumination alone (black markers) and under the red and green light illumination (white markers). All analyses were performed in triplicate. Error bars represent the standard deviation (SD) calculated from triplicate experiments using one clone. Fig. S3 Transcriptional analysis of kdc gene in the Synechocystis sp. PCC 6803ΔGSS strain. The relative gene transcription levels of kdc in the transformants of Synechocystis sp. PCC 6803ΔGSS integrated with pKT-GSS-KDC-ADH cultivated under the red light illumination alone (black bar) and under the red and green light illumination (white bar) are shown. All analyses were performed in triplicate. Error bars represent the SD calculated from triplicate experiments using one clone. Fig. S4 Evaluation of alcohol production utilizing the Ptrc-core system in Synechocystis sp, PCC 6803ΔGSS. (a) Production titer of isobutanol in the culture supernatant of Synechocystis sp. PCC 6803ΔGSS transformed with pKT-Ptrc-core-KDC-ADH cultivated under the red and green light illumination. (b) Specific titer of isobutanol under the red and green light illumination. (c) Growth curve of the transformant cultivated under the red and green light illumination. All analyses were performed in triplicate. Error bars represent the SD calculated from triplicate experiments using one clone. Fig. S5 Comparison of transcription level of kdc gene between the Ptrc-core and the PcpcG2. Transcription analysis was conducted after 24 h of cultivation of the PCC 6803ΔGSS strain transformed with pKT-Ptrc-core-KDC-ADH or pKT-GSS-KDC-ADH under red and green light illumination. All analyses were performed in triplicate. Error bars represent the SD calculated from triplicate experiments using one clone. Statistical analysis was performed using Student’s t-test: * p &lt; 0.05. Fig. S6 Comparison of transcription level of kdc gene from the PcpcG2 under the various intensities of green light. Transcription level of kdc in the PCC 6803ΔGSS strain transformed with pKT-GSSKDC-ADH under green light illumination (0 µmol m-2 s-1, 1 µmol m-2 s-1, 5 µmol m-2 s-1, 30 µmol m-2 s-1, 50 µmol m-2 s-1, or 100 µmol m-2 s-1) with red light illumination (30 µmol m-2 s-1) was shown. All analyses were performed in triplicate. Error bars represent the SD calculated from triplicate experiments using one clone. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test: * p &lt; 0.05, ** p &lt; 0.005. Table S1 Comparison of the isobutanol production titer in cyanobacteria expressing keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) in this study with that in previous studies. Table S2 Comparison of the isobutanol production in this study with the highest titer and productivity achieved in previous studies. Table S3 Comparison of the 3-methyl-1-butanol titer in cyanobacteria obtained in this study with that in previous studies. Table S4 Sequences of the vector inserts used in this study. pKT-GSS-KDC-ADH. Table S5 Primer sequences used in this study.