Supporting information for

Contributory roles of two L-lactate dehydrogenases for L- lactic acid production in thermotolerant Bacillus coagulans

Lifan Suna,b†, Caili Zhanga†, Pengcheng Lvc, Yanping Wangb, Limin Wanga* , Bo Yua,* a CAS Key Laboratory of Microbial Physiological and Metabolic Engineering,

Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China b School of Food Engineering and Biological Technology, Tianjin University of

Science & Technology, Tianjin 300457, China c College of Life Science and Bioengineering, Beijing University of Technology,

Beijing 100124, China

† L.S. and C.Z. contributed equally to this study.

* Corresponding author.

Correspondence and requests for materials should be addressed to L. W.

([email protected]) or B. Y. ([email protected]) .

Phone/Fax: +86-10-6480 6132

1 Lifan Sun, E-mail: [email protected]

Caili Zhang, E-mail: [email protected]

Yanping Wang, E-mail: [email protected]

Pengcheng Lv, , E-mail: [email protected]

2 Table S1 Strains and plasmids used in this study.

Strain or plasmid Relevant features a Reference or source Strains B. coagulans strains DSM1 Wild type DSMZ, Germany DSM1ΔldhL1 ldhL1 null mutant This study DSM1ΔldhL1- ldhL1 Complementation of DSM1ΔldhL1 with This study ldhL1 DSM1ΔldhL2 ldhL2 null mutant This study DSM1ΔldhL1ΔldhL2 ldhL1and ldhL2 null mutant This study DSM1ΔldhL1ΔldhL2- Complementation of DSM1ΔldhL1ΔldhL2 This study ldhL1 with ldhL1 This study DSM1ΔldhL1ΔldhL2- Complementation of DSM1ΔldhL1ΔldhL2 ldhL2 with ldhL2

E. coli strains Host for protein expression Tiangen Co., China BL21(DE3) Host for gene cloning Tiangen Co., China DH5α

Plasmids r pET-28a Protein expression vector, Kan Merck Co., Germany pET-28a-ldhL1 N-terminal His-tagged ldhL1 in pET28a, Kanr This study pET-28a-ldhL2 N-terminal His-tagged ldhL2 in pET28a, Kanr This study pET-28a-ldhD N-terminal His-tagged ldhD in pET28a, Kanr This study pUC19 Cloning vector, Apr Tiangen Co., China pMH77 pSH71 replication containing temperature 25 sensitive vector, Cmr pNW33n E. coli-Bacillus shuttle vector, cloning vector, BGSC, USA Cmr pNW33n-ldhL1 ldhL1-restoration vector, pNW33n harboring This study ldhL1gene with its native promoter, Cmr pNW33n-ldhL2 ldhL2-restoration vector, pNW33n harboring This study ldhL2 gene with its native promoter, Cmr pMH77-ΔldhL1 ldhL1 gene deletion vector, Cmr This study pMH77-ΔldhL2 ldhL2 gene deletion vector, Cmr This study a Cmr chloramphenicol resistant, Kanr kanamycin resistant, Apr ampicillin resistant.

3 Table S2 Primers used in this study.

Primer Sequence 5′→ 3′a Primers used for gene expression in pET-28a DSM1ldhL1F CCGGAATTCATGAAAAAAGTCAATCGTATTGC DSM1ldhL1R CCGCTCGAGTTACAATATCGGTGCCATTGTTTC DSM1ldhL2F TTCCATATGATGAGAAAGACGAAATTGGTGGTTG DSM1ldhL2R CCGCTCGAGAGCCCGAATATACGATTTTCCGG DSM1ldhDF CGCGGATCCATGAGAAAAGTTGTTGCC DSM1ldhDR CCGCTCGAGTACTTTTATCTCCCACCTG

Primers used for gene deletion L1 up-For CCGGAATTCGCTCCTTTCATTTGGTCAGAAAAATG L1 up-Rev AGCCCGGCCGGCACAAATGCATATAATCTTCCTCCCCATC L1 down-For GATGGGGAGGAAGATTATATGCATTTGTGCCGGCCGGGC L1 down-rev CCGCTCGAGGATCAACCGGGTCAGTGCAGTC L1 For GGGGGGCTTTCTTTTCATCAATTTG L1 Rev TGATTGAAACGATTTTAAACGCGG L2 up-For CCGGAATTCTGAAGGAGGGATACATATTTG L2 up-Rev GCCCTTTTACACCTTTCAGGTGTTGTCTCCCCTCCTTGTTTTC L2 down-For AACAAGGAGGGGAGACAACACCTGAAAGGTGTAAAAGGGC L2 down-Rev CCGCTCGAGCGGAGCACTGACATCGCAATACGAT L2 For AAACCGTCAACCGGGTTGTATTCCAGG L2 Rev GATGATGATCATCAAAATAAACATCAAACCGGC

Primers used for gene complementation ldhL1-For CCCAAGCTTAGCCTCATCGCCGGTTTCCCTCGC ldhL1-Rev CGCGAGCTCTTACAATATCGGTGCCATTGTTTCT ldhL2-For CCCAAGCTTTGGGCGATGCCAATCTGGCTTTATG ldhL2-Rev CGAGCTCTCAAGCCCGAATATACGATTTTCCG a Restriction sites in the primer sequences are underlined.

4 5 Figure S1. HPLC analysis of fermentation products in Bacillus coagulans DSM1 wild-type strain and mutants. DSM1 at 24-h (a), DSM1ΔldhL1 at 0-h (b), DSM1ΔldhL1 at 24-h (c), DSM1ΔldhL2

at 24-h (d), DSM1△ldhL1△ldhL2 at 0 h (e), DSM1△ldhL1△ldhL2 at 24-h (f), DSM1△ldhL1△ldhL2-ldhL1 complementation at 24-h (g), DSM1△ldhL1△ldhL2- ldhL2 complementation at 0-h (h), DSM1△ldhL1△ldhL2-ldhL2 complementation at 24-h (i).

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