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Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis

  • Applied genetics and molecular biotechnology
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Abstract

The has operon genes in the hyaluronan (HA) producer, Streptococcus zooepidemicus, encode for some of the critical enzymes in the HA biosynthetic pathway. Heterologous expression of different combinations of multiple has genes has resulted in increasing HA production to varying degrees in different recombinant strains. In this work, a recombinant Lactococcus lactis strain (SJR6) was constructed, with insertion of three has operon genes (hasABD) from S. zooepidemicus. The SJR6 strain was found to be a better HA producer than two previously constructed recombinant L. lactis strains (SJR2 and SJR3), containing hasAB and hasABC genes, respectively, but exhibited lower HA production than the native HA producer S. zooepidemicus. To understand the differences in HA yield between the various strains, transcriptions of the HA biosynthesis genes (has genes and their homologues) were compared at different phases of exponential growth of the L. lactis and S. zooepidemicus cultures. The mRNA levels of all the heterologous has genes were expectedly far higher than their corresponding homologues in the L. lactis strains. The relative mRNA level of the hasB-homologue, viz. ugd (encoding UDP-glucose dehydrogenase), was found to be much lower than that of other homologues, corroborating earlier reports which indicate tight transcriptional regulation of the ugd gene in L. lactis. Interestingly, all the has gene homologues were found to be up-regulated in all the recombinant L. lactis strains, when compared with the corresponding genes in the untransformed strain, L. lactis NZ9000. A transcription analysis of S. zooepidemicus cultures revealed that the has operon was down-regulated in the mid-exponential growth phase in comparison to the early- and late-exponential growth phases. The transcription analyses in this study have provided insights for the design of recombinant strains with higher HA productivity.

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Acknowledgements

The authors would like to acknowledge the financial support provided by Department of Science and Technology, Government of India, under WOS-A scheme for this project.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Madras, Chennai, 600036, India

    Shashi Bala Prasad, K. B. Ramachandran & Guhan Jayaraman

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  1. Shashi Bala Prasad
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  2. K. B. Ramachandran
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  3. Guhan Jayaraman
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Correspondence to Guhan Jayaraman.

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ESM 1

Comparative genomic analysis of three strains of S. zooepidemicus and L. lactis MG1363 for the presence of genes of the HA biosynthetic pathway (MW molecular mass, pI isoelectric point, CDS coding sequence, bp basepair, AA amino acid residue) (DOC 96 kb)

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Prasad, S.B., Ramachandran, K.B. & Jayaraman, G. Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis . Appl Microbiol Biotechnol 94, 1593–1607 (2012). https://doi.org/10.1007/s00253-012-3944-0

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