Abstract
Hyaluronic acid (HA) is a biopolymer used in several industries. There is increasing global demand. HA is normally produced on a large scale using attenuated strains of group C streptococci that are pathogenic and fastidious. Accordingly, it is of interest to use a “generally recognized as safe” (GRAS) organism such as Bacillus subtilis for HA production. Here, we report an engineered B. subtilis strain named WmB that produces different molecular weights (MW) and titers of HA at different temperatures. The faster the bacteria grew, the lower the MW of HA produced and the higher the titer. The MW of HA obtained ranged from 6.937 MDa at 47 °C to 0.392 MDa at 32 °C. At 32 °C, the HA titer reached 3.65 ± 0.13 g/L. We have engineered a strain that can produce high-molecular-weight and medium-molecular-weight HA at different growth temperatures. This GRAS B. subtilis strain can be applied in industry and provides a new strategy for production of HA with different molecular weights.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bitter T, Muir HM (1962) A modified uronic acid carbazole reaction. Anal Biochem 4:330–334
Cheng F, Luozhong S, Guo Z, Yu H, Stephanopoulos G (2017) Enhanced biosynthesis of hyaluronic acid using engineered Corynebacterium glutamicum via metabolic pathway regulation. Biotechnol J. https://doi.org/10.1002/biot.201700191
Chien LJ, Lee CK (2007a) Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin. Biotechnol Prog 23(5):1017–1022
Chien LJ, Lee CK (2007b) Hyaluronic acid production by recombinant Lactococcus lactis. Appl Microbiol Biotechnol 77(2):339–346
Chong B, Blank L, McLaughlin R, Nielsen L (2005) Microbial hyaluronic acid production. Appl Microbiol Biotechnol 66(4):341–351
Dowhan W (1997) Molecular basis for membrane phospholipid diversity: why are there so many lipids? Annu Rev Biochem 66(1):199–232
Errington J (2003) Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol 1:117–126
Fabret C, Ehrlich SD, Noirot P (2002) A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46(1):25–36
Fu J, Ma XW, Huang CF, Zhao YH, Li M (2012) Folding and convoluting manipulation for the treatment of distal radius fractures: a report of 78 cases. Zhongguo Gu Shang 25(9):755–756
Hilbert DW, Piggot PJ (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68(2):234–262
Ishiwa H, Tsuchida N (1986) New shuttle vectors for Escherichia coli and Bacillus subtilis. The nucleotide sequence of pHY300PLK and some properties in relation to transformation. Jpn J Genet 61:515–528
Jia Y, Zhu J, Chen X, Tang D, Su D, Yao W, Gao X (2013) Metabolic engineering of Bacillus subtilis for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights. Bioresour Technol 132:427–431
Jin P, Kang Z, Yuan P, Du G, Chen J (2016) Production of specific molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168. Metab Eng 35:21–30
Kawai F, Hara H, Takamatsu H, Watabe K, Matsumoto K (2006) Cardiolipin enrichment in spore membranes and its involvement in germination of Bacillus subtilis Marburg. Genes Genet Syst 81(2):69–76
Keasling JD (2010) Manufacturing molecules through metabolic engineering. Science 330:1355–1358
Lee JW, Na D, Park JM, Lee J, Choi S, Lee SY (2012) Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat Chem Biol 8:536–546
Liu L, Liu Y, Li J, Du G, Chen J (2011) Microbial production of hyaluronic acid: current state, challenges, and perspectives. Microb Cell Fact 10(1):99
Mao ZC, Chen RR (2007) Recombinant synthesis of hyaluronan by Agrobacterium sp. Biotechnol Prog 23(5):1038–1042
Overkamp W, Kuipers OP (2015) Transcriptional profile of Bacillus subtilis sigF-mutant during vegetative growth. PLoS One 10(10):e0141553
Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37:e45
Salzberg LI, Helmann JD (2008) Phenotypic and transcriptomic characterization of Bacillus subtilis mutants with grossly altered membrane composition. J Bacteriol 190(23):7797–7807
Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Harbor
Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50(1):1–17
Tlapak-Simmons VL, Kempner ES, Baggenstoss BA, Weigel PH (1998) The active streptococcal hyaluronan synthases (HASs) contain a single HAS monomer and multiple cardiolipin molecules. J Biol Chem 273(40):26100–26109
Tlapak-Simmons VL, Baggenstoss BA, Clyne T, Weigel PH (1999) Purification and lipid dependence of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis. J Biol Chem 274(7):4239–4245
Triscott MX, van de Rijn I (1986) Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids. J Biol Chem 261(13):6004–6009
Weigel PH, Baggenstoss BA (2012) Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines. Glycobiology 22(10):1302–1310
Westbrook AW, Moo-Young M, Chou CP (2016) Development of a CRISPR-Cas9 tool kit for comprehensive engineering of Bacillus subtilis. Appl Environ Microbiol 82(16):4876–4895
Westbrook AW, Ren X, Moo-Young M, Chou CP (2018) Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis. Biotechnol Bioeng 115(1):216–231
Widner B, Behr R, Von Dollen S, Tang M, Heu T, Sloma A, Sternberg D, Deangelis PL, Weigel PH, Brown S (2005) Hyaluronic acid production in Bacillus subtilis. Appl Environ Microbiol 71(7):3747–3752
Wu XC, Lee W, Tran L, Wong SL (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol 173(16):4952–4958
Yang G, Guo X, Luan Y (2005) The application on different molecular weight of sodium hyaluronate. Food Drug 12:1–3
Yu H, Stephanopoulos G (2008) Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid. Metab Eng 10(1):24–32
Acknowledgements
This work was supported by the Tianjin Research Program of Application Foundation (16JCTPJC50100).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Li, Y., Li, G., Zhao, X. et al. Regulation of hyaluronic acid molecular weight and titer by temperature in engineered Bacillus subtilis. 3 Biotech 9, 225 (2019). https://doi.org/10.1007/s13205-019-1749-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-019-1749-x