isible spectrophotometer (Eppendorf, Hamburg, Germany). Biomass concentration was determined by the correlation of dry cell weight (DCW) with OD~600~ as described in a previous report^[@CR18]^. The concentration of formate was measured using high-performance liquid chromatography (HPLC) equipped with a UV detector and an RSpak KC-811 column (Shodex, Tokyo, Japan) at a flow rate of 1.0 ml min^−1^ with a mobile phase of 0.1% (vol/vol) H~3~PO~4~. The concentration of H~2~ in the headspace was measured by sampling the headspace gas (100 μl) using gas-tight syringes using an YL6100GC gas chromatograph (GC) (YL Instrument Co., Anyang, Republic of Korea) equipped with a Molsieve 5A column (Supelco, Bellefonte, PA, USA), a Porapak N column (Supelco), a thermal conductivity detector and a flame ionization detector. Then, the production rate of hydrogen (mmol per liter of medium) was calculated. Argon was used as a carrier gas at a flow rate of 30 ml min^−1^. The total volume of outlet gas in a bioreactor was measured using a wet gas meter (Shinagawa, Tokyo, Japan)

Genome sequencing {#Sec12}
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Genomic DNA was extracted from cultures of the WTF-156T strain without single-colony isolation. Whole-genome sequencing of the strain was carried out using PacBio Single Molecule Real-Time (SMRT) sequencing (Pacific Biosciences, Menlo Park, CA, USA) with a 10-kb insert library approximately 100X coverage^[@CR36]^. Assembly and consensus polishing were performed using SMRTpipe HGAP and SMRTpipe Quiver, respectively^[@CR37]^. The sequence was compared with that of the parent strain and variants were identified using SAMtools v0.1.18. All the mutations were verified by polymerase chain reaction (PCR) and Sanger sequencing. All the primers for PCR confirmation are listed in Supplementary Table [4](#MOESM1){ref-type="media"}.

Construction of mutants {#Sec13}
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All the mutants were made by applying the gene disruption system^[@CR38]^. Briefly, we designed primer pairs for single base-