Engineering Bacillus Subtilis J46 for Efficient Utilization of Galactose Through Adaptive Laboratory Evolution

Overview

Journal AMB Express

Date 2024 Jan 28

PMID 38282124

Authors

Jae Woong Choi

Nho-Eul Song

Sang-Pil Hong

Young Kyoung Rhee

Hee-Do Hong

Chang-Won Cho

Affiliations

Soon will be listed here.

Abstract

Efficient utilization of galactose by microorganisms can lead to the production of valuable bio-products and improved metabolic processes. While Bacillus subtilis has inherent pathways for galactose metabolism, there is potential for enhancement via evolutionary strategies. This study aimed to boost galactose utilization in B. subtilis using adaptive laboratory evolution (ALE) and to elucidate the genetic and metabolic changes underlying the observed enhancements. The strains of B. subtilis underwent multiple rounds of adaptive laboratory evolution (approximately 5000 generations) in an environment that favored the use of galactose. This process resulted in an enhanced specific growth rate of 0.319 ± 0.005 h, a significant increase from the 0.03 ± 0.008 h observed in the wild-type strains. Upon selecting the evolved strain BSGA14, a comprehensive whole-genome sequencing revealed the presence of 63 single nucleotide polymorphisms (SNPs). Two of them, located in the coding sequences of the genes araR and glcR, were found to be the advantageous mutations after reverse engineering. The strain with these two accumulated mutations, BSGALE4, exhibited similar specific growth rate on galactose to the evolved strain BSGA14 (0.296 ± 0.01 h). Furthermore, evolved strain showed higher productivity of protease and β-galactosidase in mock soybean biomass medium. ALE proved to be a potent tool for enhancing galactose metabolism in B. subtilis. The findings offer valuable insights into the potential of evolutionary strategies in microbial engineering and pave the way for industrial applications harnessing enhanced galactose conversion.

References

yzturk S, yalik P, yzdamar T . Fed-Batch Biomolecule Production by Bacillus subtilis: A State of the Art Review. Trends Biotechnol. 2016; 34(4):329-345. DOI: 10.1016/j.tibtech.2015.12.008. View

Niu T, Lv X, Liu Y, Li J, Du G, Ledesma-Amaro R . The elucidation of phosphosugar stress response in Bacillus subtilis guides strain engineering for high N-acetylglucosamine production. Biotechnol Bioeng. 2020; 118(1):383-396. DOI: 10.1002/bit.27577. View

Chang S, Cohen S . High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979; 168(1):111-5. DOI: 10.1007/BF00267940. View

Schallmey M, Singh A, Ward O . Developments in the use of Bacillus species for industrial production. Can J Microbiol. 2004; 50(1):1-17. DOI: 10.1139/w03-076. View

Prasad C, Freese E . Cell lysis of Bacillus subtilis caused by intracellular accumulation of glucose-1-phosphate. J Bacteriol. 1974; 118(3):1111-22. PMC: 246862. DOI: 10.1128/jb.118.3.1111-1122.1974. View

Ferreira M, de Sa-Nogueira I . A multitask ATPase serving different ABC-type sugar importers in Bacillus subtilis. J Bacteriol. 2010; 192(20):5312-8. PMC: 2950484. DOI: 10.1128/JB.00832-10. View

Harwood C, Mouillon J, Pohl S, Arnau J . Secondary metabolite production and the safety of industrially important members of the Bacillus subtilis group. FEMS Microbiol Rev. 2018; 42(6):721-738. PMC: 6199538. DOI: 10.1093/femsre/fuy028. View

Kuge T, Teramoto H, Inui M . AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions. J Bacteriol. 2015; 197(24):3788-96. PMC: 4652040. DOI: 10.1128/JB.00314-15. View

Lee K, Hong M, Jung S, Ha S, Yu B, Koo H . Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering. Biotechnol Bioeng. 2011; 108(3):621-31. DOI: 10.1002/bit.22988. View

10.

Cui W, Han L, Suo F, Liu Z, Zhou L, Zhou Z . Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond. World J Microbiol Biotechnol. 2018; 34(10):145. DOI: 10.1007/s11274-018-2531-7. View

11.

Sandberg T, Salazar M, Weng L, Palsson B, Feist A . The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology. Metab Eng. 2019; 56:1-16. PMC: 6944292. DOI: 10.1016/j.ymben.2019.08.004. View

12.

Overbeek R, Begley T, Butler R, Choudhuri J, Chuang H, Cohoon M . The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 2005; 33(17):5691-702. PMC: 1251668. DOI: 10.1093/nar/gki866. View

13.

Yoon S, Ha S, Kwon S, Lim J, Kim Y, Seo H . Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol. 2016; 67(5):1613-1617. PMC: 5563544. DOI: 10.1099/ijsem.0.001755. View

14.

Lim H, Seo S, Jung G . Engineered Escherichia coli for simultaneous utilization of galactose and glucose. Bioresour Technol. 2012; 135:564-7. DOI: 10.1016/j.biortech.2012.10.124. View

15.

Mota L, Tavares P . Mode of action of AraR, the key regulator of L-arabinose metabolism in Bacillus subtilis. Mol Microbiol. 1999; 33(3):476-89. DOI: 10.1046/j.1365-2958.1999.01484.x. View

16.

Mans R, Daran J, Pronk J . Under pressure: evolutionary engineering of yeast strains for improved performance in fuels and chemicals production. Curr Opin Biotechnol. 2017; 50:47-56. DOI: 10.1016/j.copbio.2017.10.011. View

17.

Sar T, Harirchi S, Ramezani M, Bulkan G, Akbas M, Pandey A . Potential utilization of dairy industries by-products and wastes through microbial processes: A critical review. Sci Total Environ. 2021; 810:152253. DOI: 10.1016/j.scitotenv.2021.152253. View

18.

Yuan K, Song P, Li S, Gao S, Wen J, Huang H . Combining metabolic flux analysis and adaptive evolution to enhance lipase production in Bacillus subtilis. J Ind Microbiol Biotechnol. 2019; 46(8):1091-1101. DOI: 10.1007/s10295-019-02205-2. View

19.

Krispin O, Allmansberger R . The Bacillus subtilis AraE protein displays a broad substrate specificity for several different sugars. J Bacteriol. 1998; 180(12):3250-2. PMC: 107832. DOI: 10.1128/JB.180.12.3250-3252.1998. View

20.

Stulke J, Martin-Verstraete I, Glaser P, Rapoport G . Characterization of glucose-repression-resistant mutants of Bacillus subtilis: identification of the glcR gene. Arch Microbiol. 2001; 175(6):441-9. DOI: 10.1007/s002030100288. View