Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea
ABSTRACT Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly underst...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-12-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02149-22 |
| _version_ | 1811195056504176640 |
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| author | Haibo Yang Junyu Chen Ruchira Mitra Qiong Xue Hua Xiang Jing Han |
| author_facet | Haibo Yang Junyu Chen Ruchira Mitra Qiong Xue Hua Xiang Jing Han |
| author_sort | Haibo Yang |
| collection | DOAJ |
| description | ABSTRACT Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly understood. Here, we demonstrate that deletion of the three active chromosomal origins in Haloferax mediterranei remarkably reduces its DNA amounts and ploidy numbers. Consequently, the mutant strain H. mediterranei Δ123 is more sensitive to UV and mitomycin C. Surprisingly, the cell size increases by 21.2%, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in shake flask culture enhances from 7.23 to 8.11 g/L in ΔEPSΔ123, although there is also a decrease in cell growth. In this mutant, the chromosomal copy number decreases, whereas the pha-encoding pHM300 megaplasmid copy number increases. Moreover, our transcriptome analysis reveals that the genes involved in primary metabolisms are significantly downregulated in ΔEPSΔ123, whereas those responsible for starch utilization and precursor supplying for PHBV monomers are upregulated. This indicates that more energy and carbon flux is redirected from primary metabolism to PHBV synthesis, thereby enhancing its PHBV accumulation. These findings may therefore provide a rational design to enhance PHBV synthesis by simply tuning the replication origins to modulate the chromosome/megaplasmid copy number ratio and subsequently influence cellular metabolism and physiological functions. IMPORTANCE The haloarchaeon Haloferax mediterranei is a potential producer of PHBV (100% biodegradable plastic) from inexpensive carbon sources. We previously reported that H. mediterranei possessed three active chromosomal origins and, when these origins were deleted, a dormant origin was activated to initiate the replication of chromosome. In this context, in the present study, we first found a close connection between replication initiation and PHBV accumulation. We describe the potential industrial advantages of the strain H. mediterranei ΔEPSΔ123, which includes the enlargement of cell volume by 21.2% and enhancement of PHBV production by 11.2%. We further reveal the possible mechanism that contributes to the greater PHBV production in the ΔEPSΔ123 strain. Overall, we provide here a conceptual advance in the field of synthetic biology by modulating chromosome replication to improve the production of bio-based chemicals. |
| first_indexed | 2024-04-12T00:36:09Z |
| format | Article |
| id | doaj.art-60f5cff193a74a18984ca681e35df457 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-12T00:36:09Z |
| publishDate | 2022-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-60f5cff193a74a18984ca681e35df4572022-12-22T03:55:08ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-12-0110610.1128/spectrum.02149-22Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in HaloarchaeaHaibo Yang0Junyu Chen1Ruchira Mitra2Qiong Xue3Hua Xiang4Jing Han5State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaABSTRACT Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly understood. Here, we demonstrate that deletion of the three active chromosomal origins in Haloferax mediterranei remarkably reduces its DNA amounts and ploidy numbers. Consequently, the mutant strain H. mediterranei Δ123 is more sensitive to UV and mitomycin C. Surprisingly, the cell size increases by 21.2%, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in shake flask culture enhances from 7.23 to 8.11 g/L in ΔEPSΔ123, although there is also a decrease in cell growth. In this mutant, the chromosomal copy number decreases, whereas the pha-encoding pHM300 megaplasmid copy number increases. Moreover, our transcriptome analysis reveals that the genes involved in primary metabolisms are significantly downregulated in ΔEPSΔ123, whereas those responsible for starch utilization and precursor supplying for PHBV monomers are upregulated. This indicates that more energy and carbon flux is redirected from primary metabolism to PHBV synthesis, thereby enhancing its PHBV accumulation. These findings may therefore provide a rational design to enhance PHBV synthesis by simply tuning the replication origins to modulate the chromosome/megaplasmid copy number ratio and subsequently influence cellular metabolism and physiological functions. IMPORTANCE The haloarchaeon Haloferax mediterranei is a potential producer of PHBV (100% biodegradable plastic) from inexpensive carbon sources. We previously reported that H. mediterranei possessed three active chromosomal origins and, when these origins were deleted, a dormant origin was activated to initiate the replication of chromosome. In this context, in the present study, we first found a close connection between replication initiation and PHBV accumulation. We describe the potential industrial advantages of the strain H. mediterranei ΔEPSΔ123, which includes the enlargement of cell volume by 21.2% and enhancement of PHBV production by 11.2%. We further reveal the possible mechanism that contributes to the greater PHBV production in the ΔEPSΔ123 strain. Overall, we provide here a conceptual advance in the field of synthetic biology by modulating chromosome replication to improve the production of bio-based chemicals.https://journals.asm.org/doi/10.1128/spectrum.02149-22haloarchaeaactive replication originpolyploidycopy numberchromosomemegaplasmid |
| spellingShingle | Haibo Yang Junyu Chen Ruchira Mitra Qiong Xue Hua Xiang Jing Han Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea Microbiology Spectrum haloarchaea active replication origin polyploidy copy number chromosome megaplasmid |
| title | Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea |
| title_full | Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea |
| title_fullStr | Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea |
| title_full_unstemmed | Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea |
| title_short | Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea |
| title_sort | replication origin deletion enhances poly 3 hydroxybutyrate co 3 hydroxyvalerate synthesis in haloarchaea |
| topic | haloarchaea active replication origin polyploidy copy number chromosome megaplasmid |
| url | https://journals.asm.org/doi/10.1128/spectrum.02149-22 |
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